WorldWideScience

Sample records for tissue culture methods

  1. Culture methods of allograft musculoskeletal tissue samples in Australian bacteriology laboratories.

    Science.gov (United States)

    Varettas, Kerry

    2013-12-01

    Samples of allograft musculoskeletal tissue are cultured by bacteriology laboratories to determine the presence of bacteria and fungi. In Australia, this testing is performed by 6 TGA-licensed clinical bacteriology laboratories with samples received from 10 tissue banks. Culture methods of swab and tissue samples employ a combination of solid agar and/or broth media to enhance micro-organism growth and maximise recovery. All six Australian laboratories receive Amies transport swabs and, except for one laboratory, a corresponding biopsy sample for testing. Three of the 6 laboratories culture at least one allograft sample directly onto solid agar. Only one laboratory did not use a broth culture for any sample received. An international literature review found that a similar combination of musculoskeletal tissue samples were cultured onto solid agar and/or broth media. Although variations of allograft musculoskeletal tissue samples, culture media and methods are used in Australian and international bacteriology laboratories, validation studies and method evaluations have challenged and supported their use in recovering fungi and aerobic and anaerobic bacteria.

  2. Plant Tissue Culture

    Indian Academy of Sciences (India)

    Admin

    Plant tissue culture is a technique of culturing plant cells, tissues and organs on ... working methods (Box 2) and discovery of the need for B vita- mins and auxins for ... Kotte (Germany) reported some success with growing isolated root tips.

  3. Optimization of an Efficient Non-Tissue Culture Transformation Method for Brassica Juncea

    International Nuclear Information System (INIS)

    Naeem, I.; Munir, I.; Iqbal, A.; Ullah, F.

    2016-01-01

    The major hurdles in successful in vitro transformation of Brassica juncea through standard tissue culture (STC) method are: culture contamination, somaclonal variations, and lack of expertise. Moreover, the current STC method is time consuming and needs continuous electricity. In the present study, the in planta transformation method through floral dip with or without vacuum infiltration was optimized for successful transformation of B. juncea. The B. juncea CV RAYA Anmol was used for transformation through Agrobacterium tumefaciens strain GV3101 harboring the binary vector plasmid pBinGlyBar4-EADcT. Based on the resistance reaction to the herbicide Basta, 20 and 40 resistant seedlings were obtained from 2000 seed germinated from the plants transformed through floral dip and vacuum infiltration methods, respectively. The PCR analyses further confirmed the presence of transgene in 3 floral dipped plants without vacuum infiltration and 17 floral dipped plants with vacuum infiltration, giving the transformation frequencies of 1.5*10/sup -3/ and 8.5*10/sup -3/, respectively. This method, which avoids tissue culture, will reduce the somaclonal variation accompanying prolonged culture of cells in a dedifferentiated state, will facilitate functional genomics and improvement of Brassica juncea with novel desirable traits while reducing time and expense. (author)

  4. Progress in planta transformation without tissue culture

    International Nuclear Information System (INIS)

    Gu Yunhong; Chinese Academy of Sciences, Hefei; Qin Guangyong; Huo Yuping; Yu Zengliang

    2004-01-01

    With the development of planta genetic engineering, more emphases have been laid on convenient and high efficient genetic transformation methods. And transformation without tissue culture is a prospective direction of it. In this paper, traditional transformation methods and the methods of non-tissue culture were summarized. With the exploration and application of Arabidopsis transformation mechanism, with the use of ion beam-mediated transformation invented by Chinese scientists and the development of other transformation methods, transformation methods without tissue culture and planta genetic engineering could be improved rapidly. (authors)

  5. A method for establishing human primary gastric epithelial cell culture from fresh surgical gastric tissues.

    Science.gov (United States)

    Aziz, Faisal; Yang, Xuesong; Wen, Qingping; Yan, Qiu

    2015-08-01

    At present, biopsy specimens, cancer cell lines and tissues obtained by gastric surgery are used in the study and analysis of gastric cancer, including the molecular mechanisms and proteomics. However, fibroblasts and other tissue components may interfere with these techniques. Therefore, the present study aimed to develop a procedure for the isolation of viable human gastric epithelial cells from gastric surgical tissues. A method was developed to culture human gastric epithelial cells using fresh, surgically excised tissues and was evaluated using immunocytochemistry, periodic acid-Schiff (PAS) staining and cell viability assays. Low cell growth was observed surrounding the gastric tissue on the seventh day of tissue explant culture. Cell growth subsequently increased, and at 12 days post-explant a high number of pure epithelial cells were detected. The gastric cancer cells exhibited rapid growth with a doubling time of 13-52 h, as compared to normal cells, which had a doubling time of 20-53 h. Immunocytochemical analyses of primary gastric cells revealed positive staining for cytokeratin 18 and 19, which indicated that the culture was comprised of pure epithelial cells and contained no fibroblasts. Furthermore, PAS staining demonstrated that the cultured gastric cells produced neutral mucin. Granulin and carbohydrate antigen 724 staining confirmed the purity of gastric cancer and normal cells in culture. This method of cell culture indicated that the gastric cells in primary culture consisted of mucin-secreting gastric epithelial cells, which may be useful for the study of gastric infection with Helicobacter pylori and gastric cancer.

  6. Application of Hanging Drop Technique for Kidney Tissue Culture

    Directory of Open Access Journals (Sweden)

    Shaohui Wang

    2017-05-01

    Full Text Available Background/Aims: The hanging drop technique is a well-established method used in culture of animal tissues. However, this method has not been used in adult kidney tissue culture yet. This study was to explore the feasibility of using this technique for culturing adult kidney cortex to study the time course of RNA viability in the tubules and vasculature, as well as the tissue structural integrity. Methods: In each Petri dish with the plate covered with sterile buffer, a section of mouse renal cortex was cultured within a drop of DMEM culture medium on the inner surface of the lip facing downward. The tissue were then harvested at each specific time points for Real-time PCR analysis and histological studies. Results: The results showed that the mRNA level of most Na+ related transporters and cotransporters were stably maintained within 6 hours in culture, and that the mRNA level of most receptors found in the vasculature and glomeruli were stably maintained for up to 9 days in culture. Paraffin sections of the cultured renal cortex indicated that the tubules began to lose tubular integrity after 6 hours, but the glomeruli and vasculatures were still recognizable up to 9 days in culture. Conclusions: We concluded that adult kidney tissue culture by hanging drop method can be used to study gene expressions in vasculature and glomeruli.

  7. Simple and high yielding method for preparing tissue specific extracellular matrix coatings for cell culture.

    Science.gov (United States)

    DeQuach, Jessica A; Mezzano, Valeria; Miglani, Amar; Lange, Stephan; Keller, Gordon M; Sheikh, Farah; Christman, Karen L

    2010-09-27

    The native extracellular matrix (ECM) consists of a highly complex, tissue-specific network of proteins and polysaccharides, which help regulate many cellular functions. Despite the complex nature of the ECM, in vitro cell-based studies traditionally assess cell behavior on single ECM component substrates, which do not adequately mimic the in vivo extracellular milieu. We present a simple approach for developing naturally derived ECM coatings for cell culture that provide important tissue-specific cues unlike traditional cell culture coatings, thereby enabling the maturation of committed C2C12 skeletal myoblast progenitors and human embryonic stem cells differentiated into cardiomyocytes. Here we show that natural muscle-specific coatings can (i) be derived from decellularized, solubilized adult porcine muscle, (ii) contain a complex mixture of ECM components including polysaccharides, (iii) adsorb onto tissue culture plastic and (iv) promote cell maturation of committed muscle progenitor and stem cells. This versatile method can create tissue-specific ECM coatings, which offer a promising platform for cell culture to more closely mimic the mature in vivo ECM microenvironment.

  8. Application of Hanging Drop Technique for Kidney Tissue Culture.

    Science.gov (United States)

    Wang, Shaohui; Wang, Ximing; Boone, Jasmine; Wie, Jin; Yip, Kay-Pong; Zhang, Jie; Wang, Lei; Liu, Ruisheng

    2017-01-01

    The hanging drop technique is a well-established method used in culture of animal tissues. However, this method has not been used in adult kidney tissue culture yet. This study was to explore the feasibility of using this technique for culturing adult kidney cortex to study the time course of RNA viability in the tubules and vasculature, as well as the tissue structural integrity. In each Petri dish with the plate covered with sterile buffer, a section of mouse renal cortex was cultured within a drop of DMEM culture medium on the inner surface of the lip facing downward. The tissue were then harvested at each specific time points for Real-time PCR analysis and histological studies. The results showed that the mRNA level of most Na+ related transporters and cotransporters were stably maintained within 6 hours in culture, and that the mRNA level of most receptors found in the vasculature and glomeruli were stably maintained for up to 9 days in culture. Paraffin sections of the cultured renal cortex indicated that the tubules began to lose tubular integrity after 6 hours, but the glomeruli and vasculatures were still recognizable up to 9 days in culture. We concluded that adult kidney tissue culture by hanging drop method can be used to study gene expressions in vasculature and glomeruli. © 2017 The Author(s). Published by S. Karger AG, Basel.

  9. Comparing culture and molecular methods for the identification of microorganisms involved in necrotizing soft tissue infections

    DEFF Research Database (Denmark)

    Rudkjøbing, Vibeke Børsholt; Thomsen, Trine Rolighed; Xu, Yijuan

    2016-01-01

    BACKGROUND: Necrotizing soft tissue infections (NSTIs) are a group of infections affecting all soft tissues. NSTI involves necrosis of the afflicted tissue and is potentially life threatening due to major and rapid destruction of tissue, which often leads to septic shock and organ failure. The gold...... to culture. Although the molecular methods generally gave concordant results, our results indicate that Microseq may misidentify or overlook microorganisms that can be detected by other molecular methods. Half of the patients were found to be infected with S. pyogenes, but several atypical findings were also...... that clinicians should be prepared to diagnose and treat any combination of microbial pathogens. Some of the tested molecular methods offer a faster turnaround time combined with a high specificity, which makes supplemental use of such methods attractive for identification of microorganisms, especially...

  10. Laboratory Workflow Analysis of Culture of Periprosthetic Tissues in Blood Culture Bottles.

    Science.gov (United States)

    Peel, Trisha N; Sedarski, John A; Dylla, Brenda L; Shannon, Samantha K; Amirahmadi, Fazlollaah; Hughes, John G; Cheng, Allen C; Patel, Robin

    2017-09-01

    Culture of periprosthetic tissue specimens in blood culture bottles is more sensitive than conventional techniques, but the impact on laboratory workflow has yet to be addressed. Herein, we examined the impact of culture of periprosthetic tissues in blood culture bottles on laboratory workflow and cost. The workflow was process mapped, decision tree models were constructed using probabilities of positive and negative cultures drawn from our published study (T. N. Peel, B. L. Dylla, J. G. Hughes, D. T. Lynch, K. E. Greenwood-Quaintance, A. C. Cheng, J. N. Mandrekar, and R. Patel, mBio 7:e01776-15, 2016, https://doi.org/10.1128/mBio.01776-15), and the processing times and resource costs from the laboratory staff time viewpoint were used to compare periprosthetic tissues culture processes using conventional techniques with culture in blood culture bottles. Sensitivity analysis was performed using various rates of positive cultures. Annualized labor savings were estimated based on salary costs from the U.S. Labor Bureau for Laboratory staff. The model demonstrated a 60.1% reduction in mean total staff time with the adoption of tissue inoculation into blood culture bottles compared to conventional techniques (mean ± standard deviation, 30.7 ± 27.6 versus 77.0 ± 35.3 h per month, respectively; P < 0.001). The estimated annualized labor cost savings of culture using blood culture bottles was $10,876.83 (±$337.16). Sensitivity analysis was performed using various rates of culture positivity (5 to 50%). Culture in blood culture bottles was cost-effective, based on the estimated labor cost savings of $2,132.71 for each percent increase in test accuracy. In conclusion, culture of periprosthetic tissue in blood culture bottles is not only more accurate than but is also cost-saving compared to conventional culture methods. Copyright © 2017 American Society for Microbiology.

  11. Plant tissue culture techniques

    Directory of Open Access Journals (Sweden)

    Rolf Dieter Illg

    1991-01-01

    Full Text Available Plant cell and tissue culture in a simple fashion refers to techniques which utilize either single plant cells, groups of unorganized cells (callus or organized tissues or organs put in culture, under controlled sterile conditions.

  12. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Tissue culture media for human ex vivo tissue and cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture...

  13. A Method to Preclude Moisture Condensation in Plated Tissue Cultures

    Science.gov (United States)

    Alex M. Diner

    1992-01-01

    Excessive condensate normally accumulates in in vitro-illuminated petri dishes containing plant tissue cultures, causing avariety of problems. A dark-colored rubber net-mesh placed over the petri dishes prevented such condensation, even when charcoal-supplemented media are used under high light intensity in a growth chamber.

  14. The autologus graft of epithelial tissue culture

    Directory of Open Access Journals (Sweden)

    Minaee B

    1999-08-01

    Full Text Available With the intention of research about culture and autologus graft of epithelial tissue we used 4 french Albino Rabbits with an average age of 2 months. After reproduction on the support in EMEM (Eagle's Minimum Essential Medium we used this for graft after 4 weeks. This region which grafted total replaced. After fixation of this sample and passing them through various process, histological sections were prepared. These sections were stained with H & E and masson's trichrome and studied by light microscope. We succeeded in graft. We hope in the near future by using the method of epithelium tissue culture improving to treat burned patients.

  15. Co-culture systems-based strategies for articular cartilage tissue engineering.

    Science.gov (United States)

    Zhang, Yu; Guo, Weimin; Wang, Mingjie; Hao, Chunxiang; Lu, Liang; Gao, Shuang; Zhang, Xueliang; Li, Xu; Chen, Mingxue; Li, Penghao; Jiang, Peng; Lu, Shibi; Liu, Shuyun; Guo, Quanyi

    2018-03-01

    Cartilage engineering facilitates repair and regeneration of damaged cartilage using engineered tissue that restores the functional properties of the impaired joint. The seed cells used most frequently in tissue engineering, are chondrocytes and mesenchymal stem cells. Seed cells activity plays a key role in the regeneration of functional cartilage tissue. However, seed cells undergo undesirable changes after in vitro processing procedures, such as degeneration of cartilage cells and induced hypertrophy of mesenchymal stem cells, which hinder cartilage tissue engineering. Compared to monoculture, which does not mimic the in vivo cellular environment, co-culture technology provides a more realistic microenvironment in terms of various physical, chemical, and biological factors. Co-culture technology is used in cartilage tissue engineering to overcome obstacles related to the degeneration of seed cells, and shows promise for cartilage regeneration and repair. In this review, we focus first on existing co-culture systems for cartilage tissue engineering and related fields, and discuss the conditions and mechanisms thereof. This is followed by methods for optimizing seed cell co-culture conditions to generate functional neo-cartilage tissue, which will lead to a new era in cartilage tissue engineering. © 2017 Wiley Periodicals, Inc.

  16. Micro fluidic System for Culturing and Monitoring of Neuronal Cells and Tissue

    DEFF Research Database (Denmark)

    Bakmand, Tanya; Waagepetersen, Helle S.

    The aim of this Ph.D. project was to combine experience within cell and tissue culturing, electrochemistry and microfabrication in order to develop an in vivo-like fluidic culturing platform, challenging the traditional culturing methods. The first goal was to develope a fluidic system for cultur...... with mass production. The last part of this thesis also includes perspectives on how to expand the latest designed device to facilitate culturing of tissue and co-culturing of cells....

  17. Umbilical cord Wharton's jelly repeated culture system: a new device and method for obtaining abundant mesenchymal stem cells for bone tissue engineering.

    Directory of Open Access Journals (Sweden)

    Zhengqi Chang

    Full Text Available To date, various types of cells for seeding regenerative scaffolds have been used for bone tissue engineering. Among seed cells, the mesenchymal stem cells derived from human umbilical cord Wharton's jelly (hUCMSCs represent a promising candidate and hold potential for bone tissue engineering due to the the lack of ethical controversies, accessibility, sourced by non-invasive procedures for donors, a reduced risk of contamination, osteogenic differentiation capacities, and higher immunomodulatory capacity. However, the current culture methods are somewhat complicated and inefficient and often fail to make the best use of the umbilical cord (UC tissues. Moreover, these culture processes cannot be performed on a large scale and under strict quality control. As a result, only a small quantity of cells can be harvested using the current culture methods. To solve these problems, we designed and evaluated an UC Wharton's jelly repeated culture device. Using this device, hUCMSCs were obtained from the repeated cultures and their quantities and biological characteristics were compared. We found that using our culture device, which retained all tissue blocks on the bottom of the dish, the total number of obtained cells increased 15-20 times, and the time required for the primary passage was reduced. Moreover, cells harvested from the repeated cultures exhibited no significant difference in their immunophenotype, potential for multilineage differentiation, or proliferative, osteoinductive capacities, and final osteogenesis. The application of the repeated culture frame (RCF not only made full use of the Wharton's jelly but also simplified and specified the culture process, and thus, the culture efficiency was significantly improved. In summary, abundant hUCMSCs of dependable quality can be acquired using the RCF.

  18. Gastrointestinal Epithelial Organoid Cultures from Postsurgical Tissues.

    Science.gov (United States)

    Hahn, Soojung; Yoo, Jongman

    2017-08-17

    An organoid is a cellular structure three-dimensionally (3D) cultured from self-organizing stem cells in vitro, which has a cell population, architectures, and organ specific functions like the originating organs. Recent advances in the 3D culture of isolated intestinal crypts or gastric glands have enabled the generation of human gastrointestinal epithelial organoids. Gastrointestinal organoids recapitulate the human in vivo physiology because of all the intestinal epithelial cell types that differentiated and proliferated from tissue resident stem cells. Thus far, gastrointestinal organoids have been extensively used for generating gastrointestinal disease models. This protocol describes the method of isolating a gland or crypt using stomach or colon tissue after surgery and establishing them into gastroids or colonoids.

  19. Tissue culture of surgically prepared temporalis fascia.

    Science.gov (United States)

    Walby, A P; Kerr, A G; Nevin, N C; Woods, G

    1982-10-01

    Temporalis fascia which is used to graft the tympanic membrane has been shown to be viable in tissue culture by a previous pilot study. This present study reports the effect on the viability of the fascia by scraping loose connective tissue from it and allowing it to dry. Pieces of fascia from 30 patients were each divided in 4 and prepared to give explants, fresh, fresh and scraped, dried, and dried and scraped. The fascia grew from 17 patients when cultured fresh, 5 when fresh and scraped, 1 when dried, and none when dried and scraped. These results are significantly different and show that the fascia is devitilized when prepared by the normal method for use in tympanoplasty.

  20. Formation of proteoglycan and collagen-rich scaffold-free stiff cartilaginous tissue using two-step culture methods with combinations of growth factors.

    Science.gov (United States)

    Miyazaki, Tatsuya; Miyauchi, Satoshi; Matsuzaka, Satoshi; Yamagishi, Chie; Kobayashi, Kohei

    2010-05-01

    Tissue-engineered cartilage may be expected to serve as an alternative to autologous chondrocyte transplantation treatment. Several methods for producing cartilaginous tissue have been reported. In this study, we describe the production of scaffold-free stiff cartilaginous tissue of pig and human, using allogeneic serum and growth factors. The tissue was formed in a mold using chondrocytes recovered from alginate bead culture and maintained in a medium with transforming growth factor-beta and several other additives. In the case of porcine tissue, the tear strength of the tissue and the contents of proteoglycan (PG) and collagen per unit of DNA increased dose-dependently with transforming growth factor-beta. The length of culture was significantly and positively correlated with thickness, tear strength, and PG and collagen contents. Tear strength showed positive high correlations with both PG and collagen contents. A positive correlation was also seen between PG content and collagen content. Similar results were obtained with human cartilaginous tissue formed from chondrocytes expanded in monolayer culture. Further, an in vivo pilot study using pig articular cartilage defect model demonstrated that the cartilaginous tissue was well integrated with surrounding tissue at 13 weeks after the implantation. In conclusion, we successfully produced implantable scaffold-free stiff cartilaginous tissue, which characterized high PG and collagen contents.

  1. Research progress in plant mutation by combining ion beam irradiations and tissue culture

    International Nuclear Information System (INIS)

    Zhou Linbin; Li Wenjian; Qu Ying; Li Ping

    2007-01-01

    About a new mutation breeding method which combines plant tissue culture technique with heavy ion beam irradiations were discussed in this paper with the principles, operation steps, molecular mechanisms, etc. The mutation method developed a few advantages coming from plant tissue culture, which can produce offspring by asexual ways. Meanwhile, using this method, the study of biological effects of high energy particles with different linear energy transfer values on plant tissues or cells can be explored and optimized in theory or practice. (authors)

  2. Establishment of primary keratinocyte culture from horse tissue biopsates

    Directory of Open Access Journals (Sweden)

    Jernej OGOREVC

    2015-12-01

    Full Text Available Primary cell lines established from skin tissue can be used in immunological, proteomic and genomic studies as in vitro skin models. The goal of our study was to establish a primary keratinocyte cell culture from tissue biopsates of two horses. The primary keratinocyte cell culture was obtained by mechanical and enzymatic dissociation and with explant culture method. The result was a heterogeneous primary culture comprised of keratinocytes and fibroblasts. To distinguish epithelial and mesenchymal cells immunofluorescent characterisation was performed, using antibodies against cytokeratin 14 and vimentin. We successfully at attained a primary cell line of keratinocytes, which could potentially be used to study equine skin diseases, as an animal model for human diseases, and for cosmetic and therapeutic product testing.

  3. Improved Diagnosis of Prosthetic Joint Infection by Culturing Periprosthetic Tissue Specimens in Blood Culture Bottles

    Directory of Open Access Journals (Sweden)

    Trisha N. Peel

    2016-01-01

    Full Text Available Despite known low sensitivity, culture of periprosthetic tissue specimens on agars and in broths is routine. Culture of periprosthetic tissue samples in blood culture bottles (BCBs is potentially more convenient, but it has been evaluated in a limited way and has not been widely adopted. The aim of this study was to compare the sensitivity and specificity of inoculation of periprosthetic tissue specimens into blood culture bottles with standard agar and thioglycolate broth culture, applying Bayesian latent class modeling (LCM in addition to applying the Infectious Diseases Society of America (IDSA criteria for prosthetic joint infection. This prospective cohort study was conducted over a 9-month period (August 2013 to April 2014 at the Mayo Clinic, Rochester, MN, and included all consecutive patients undergoing revision arthroplasty. Overall, 369 subjects were studied; 117 (32% met IDSA criteria for prosthetic joint infection, and 82% had late chronic infection. Applying LCM, inoculation of tissues into BCBs was associated with a 47% improvement in sensitivity compared to the sensitivity of conventional agar and broth cultures (92.1 versus 62.6%, respectively; this magnitude of change was similar when IDSA criteria were applied (60.7 versus 44.4%, respectively; P = 0.003. The time to microorganism detection was shorter with BCBs than with standard media (P < 0.0001, with aerobic and anaerobic BCBs yielding positive results within a median of 21 and 23 h, respectively. Results of our study demonstrate that the semiautomated method of periprosthetic tissue culture in blood culture bottles is more sensitive than and as specific as agar and thioglycolate broth cultures and yields results faster.

  4. The plant tissue culture

    International Nuclear Information System (INIS)

    Crocomo, O.J.; Sharp, W.R.

    1973-01-01

    Progress in the field of plant tissue culture at the Plant Biochemistry Sector, Centro de Energia na Agricultura (CENA), Piracicaba, S.P., Brazil, pertains to the simplification of development in 'Phaseolus vulgaris' by dividing the organism into its component organs, tissues, and cells and the maintenance of these components on defined culture media 'in vitro'. This achievement has set the stage for probing the basis for the stability of the differentiated states and/or the reentry of mature differentiated cells into the mitotic cell cycle and their subsequent redifferentiation. Data from such studies at the cytological and biochemical level have been invaluable in the elucidation of the control mechanisms responsible for expression of the cellular phenotype. Unlimited possibilities exist for the application of tissue culture in the vegetative propagation of 'Phaseolus' and other important cultivars in providing genocopies or a large scale and/or readily obtaining plantlets from haploid cell lines or from protoplast (wall-less cells) hybridization products following genetic manipulation. These tools are being applied in this laboratory for the development and selection of high protein synthesizing 'Phaseolus' cultivars

  5. A comparative study of three tissue-cultured Dendrobium species and their wild correspondences by headspace gas chromatography-mass spectrometry combined with chemometric methods.

    Science.gov (United States)

    Chen, Nai-Dong; You, Tao; Li, Jun; Bai, Li-Tao; Hao, Jing-Wen; Xu, Xiao-Yuan

    2016-10-01

    Plant tissue culture technique is widely used in the conservation and utilization of rare and endangered medicinal plants and it is crucial for tissue culture stocks to obtain the ability to produce similar bioactive components as their wild correspondences. In this paper, a headspace gas chromatography-mass spectrometry method combined with chemometric methods was applied to analyze and evaluate the volatile compounds in tissue-cultured and wild Dendrobium huoshanense Cheng and Tang, Dendrobium officinale Kimura et Migo and Dendrobium moniliforme (Linn.) Sw. In total, 63 volatile compounds were separated, with 53 being identified from the three Dendrobium spp. Different provenances of Dendrobiums had characteristic chemicals and showed remarkable quantity discrepancy of common compositions. The similarity evaluation disclosed that the accumulation of volatile compounds in Dendrobium samples might be affected by their provenance. Principal component analysis showed that the first three components explained 85.9% of data variance, demonstrating a good discrimination between samples. Gas chromatography-mass spectrometry techniques, combined with chemometrics, might be an effective strategy for identifying the species and their provenance, especially in the assessment of tissue-cultured Dendrobium quality for use in raw herbal medicines. Copyright © 2016. Published by Elsevier B.V.

  6. Yield improvement strategies for the production of secondary metabolites in plant tissue culture: silymarin from Silybum marianum tissue culture.

    Science.gov (United States)

    AbouZid, S

    2014-01-01

    Plant cell culture can be a potential source for the production of important secondary metabolites. This technology bears many advantages over conventional agricultural methods. The main problem to arrive at a cost-effective process is the low productivity. This is mainly due to lack of differentiation in the cultured cells. Many approaches have been used to maximise the yield of secondary metabolites produced by cultured plant cells. Among these approaches: choosing a plant with a high biosynthetic capacity, obtaining efficient cell line for growth and production of metabolite of interest, manipulating culture conditions, elicitation, metabolic engineering and organ culture. This article gives an overview of the various approaches used to maximise the production of pharmaceutically important secondary metabolites in plant cell cultures. Examples of using these different approaches are shown for the production of silymarin from Silybum marianum tissue culture.

  7. Propagation of Aquilaria malaccensis seedlings through tissue culture techniques

    International Nuclear Information System (INIS)

    Salahbiah Abdul Majid; Zaiton Ahmad; Mohd Rafaie Abdul Salam; Nurhayati Irwan; Affrida Abu Hassan; Rusli Ibrahim

    2010-01-01

    Aquilaria malaccensis or karas is the principal source of gaharu resin, which is used in many cultures for incense, perfumes and traditional medicines. The species is mainly propagated conventionally through seeds, cuttings and graftings. Propagation by seeds is usually a reliable method for other forest species, but for karas, this technique is inadequate to meet the current demand of seedling supplies. This is principally due to its low seed viability, low germination rate, delayed rooting of seedlings, long life-cycle and rare seed production. Tissue culture has several advantages over conventional propagation, especially for obtaining large number of uniform and high-yielding plantlets or clones. This paper presents the current progress on mass-propagation of Aquilaria malaccensis seedlings through tissue culture technique at Nuclear Malaysia. (author)

  8. Identification of Stevioside Using Tissue Culture-Derived Stevia (Stevia rebaudiana) Leaves

    Science.gov (United States)

    Karim, Md. Ziaul; Uesugi, Daisuke; Nakayama, Noriyuki; Hossain, M. Monzur; Ishihara, Kohji; Hamada, Hiroki

    2015-01-01

    Stevioside is a natural sweetener from Stevia leaf, which is 300 times sweeter than sugar. It helps to reduce blood sugar levels dramatically and thus can be of benefit to diabetic people. Tissue culture is a very potential modern technology that can be used in large-scale disease-free stevia production throughout the year. We successfully produced stevia plant through in vitro culture for identification of stevioside in this experiment. The present study describes a potential method for identification of stevioside from tissue culture-derived stevia leaf. Stevioside in the sample was identified using HPLC by measuring the retention time. The percentage of stevioside content in the leaf samples was found to be 9.6%. This identification method can be used for commercial production and industrialization of stevia through in vitro culture across the world. PMID:28008268

  9. Improved Cell Culture Method for Growing Contracting Skeletal Muscle Models

    Science.gov (United States)

    Marquette, Michele L.; Sognier, Marguerite A.

    2013-01-01

    An improved method for culturing immature muscle cells (myoblasts) into a mature skeletal muscle overcomes some of the notable limitations of prior culture methods. The development of the method is a major advance in tissue engineering in that, for the first time, a cell-based model spontaneously fuses and differentiates into masses of highly aligned, contracting myotubes. This method enables (1) the construction of improved two-dimensional (monolayer) skeletal muscle test beds; (2) development of contracting three-dimensional tissue models; and (3) improved transplantable tissues for biomedical and regenerative medicine applications. With adaptation, this method also offers potential application for production of other tissue types (i.e., bone and cardiac) from corresponding precursor cells.

  10. Culture of three-dimensional tissue model and its application in bystander-effect research

    International Nuclear Information System (INIS)

    Wu Ruqun; Xu An; Wu Lijun; Hu Burong

    2012-01-01

    Compared with the cultured monolayer (2D) cells, three-dimensional (3D) tissue could be more similar to the environment in vivo including the physical support, chemical factors, cell-cell and cell-matrix interaction and so on. With the development of three-dimensional cell culture techniques (TDCC), 3D tissue is widely used in the areas of bystander effect research. This review focuses on introducing the TDCC method and its application in bystander-effect research. First, the development process of 3D tissue culture method was introduced. Secondly, the induction of radiation induced bystander effects both in 2D cell and 3D tissue and its mechanisms were reviewed. Finally, because heavy ion (carbon ion beam) has been developed as a useful tool to cure solid cancer, and the 3D tissue model is an ideal material to study the damages on body after being irradiated and to understand the underlying mechanisms, future study about heavy ion radiation inducing bystander effect in 3D tissue was discussed. (authors)

  11. Structure and component alteration of rabbit Achilles tendon in tissue culture.

    Science.gov (United States)

    Hosaka, Yoshinao; Ueda, Hiromi; Yamasaki, Tadatsugu; Suzuki, Daisuke; Matsuda, Naoya; Takehana, Kazushige

    2005-12-01

    The aim of this study was to investigate alterations of cultured tendon tissues to determine whether tissue culture is a useful method for biological analyses of the tendon. Tendon tissues for tissue culture were isolated from Achilles tendons of rabbits. The tendon segments were placed one segment per well and incubated in growth medium consisting of Dullbecco's modified Eagle's medium supplemented with 5% fetal bovine serum at 37 degrees C in a humidified atmosphere with 5% CO(2) for various periods. The alignment of collagen fibrils was preserved for 48 h, but tendon structure has disintegrated at 96 h. Alcian blue staining and gelatine zymography revealed that proteoglycan markedly diminished and that matrix metalloproteinase (MMPs) activity was upregulated sharply at 72 and 96 h. The ratio of collagen fibrils with large diameter had increased and the mean diameter and mass average diameter value had reached maximum at 48 h. The values then decreased and mean diameters at 72 and 96 h were significantly different from that at 48 h. At 96 h, the ratio of collagen fibrils with small diameters had increased and collagen fibrils with large diameters had disappeared. These findings indicate that structural alteration is possible to be induced by disintegration of collagen fibrils and disappearance of glycosaminoglycans from extracellular matrix (ECM), subsequent of upregulation of MMPs activity. Although the study period is limited, the tissue culture method is available for investigating cell-ECM interaction in tendons.

  12. Effect of lunar materials on plant tissue culture.

    Science.gov (United States)

    Walkinshaw, C. H.; Venketeswaran, S.; Baur, P. S.; Croley, T. E.; Scholes, V. E.; Weete, J. D.; Halliwell, R. S.; Hall, R. H.

    1973-01-01

    Lunar material collected during the Apollo 11, 12, 14, and 15 missions has been used to treat 12 species of higher plant tissue cultures. Biochemical and morphological studies have been conducted on several of these species. Tobacco tissue cultures treated with 0.22 g of lunar material exhibited increased greening more complex chloroplasts, less cytoplasmic vacuolation and greater vesiculation. Pine tissue cultures reacted to treatment by an increased deposition of tannin-like materials. The percentage of dry weight and soluble protein was increased in cultures treated with either lunar or terrestrial rock materials.

  13. [Comparative study on alkaloids of tissue-culture seedling and wild plant of Dendrobium huoshanense ].

    Science.gov (United States)

    Chen, Nai-dong; Gao, Feng; Lin, Xin; Jin, Hui

    2014-06-01

    To compare the composition and content of alkaloid of Dendrobium huoshanense tissue-culture seedling and wild plant. A comparative evaluation on the quality was carried out by HPLC and TLC methods including the composition and the content of alkaloids. Remarkable variation existed in the two kinds of Dendrobium huoshanense. For the tissue-culture plant, only two alkaloids were checked out by both HPLC and TLC while four alkaloids were observed in the wild plant. The alkaloid content of tissue-culture seedling and wild plant was(0. 29 ± 0. 11)%o and(0. 43 ± 0. 15) %o,respectively. Distinguished difference is observed in both composition and content of alkaloids from the annual shoots of different provenances of Dendrobium huoshanense. It suggested that the quality of tissue-culture seedling of Dendrobium huoshanense might be inconsistent with the wild plant. Furthermore, the established alkaloids-knock-out HPLC method would provide a new research tool on quality control of Chinese medicinal materials which contain unknown alkaloids.

  14. Smallholder adoption and economic impacts of tissue culture ...

    African Journals Online (AJOL)

    This study was conducted with an objective of determining the correlates of adoption of tissue culture banana technology and its impacts on household incomes in Kenya. The results show that while some households have opted not to adopt tissue culture banana biotechnology, almost all the adopters are growing tissue ...

  15. Walnut tissue culture: research and field applications

    Science.gov (United States)

    2004-01-01

    Vitrotech Biotecnologia Vegetal began researching propagating Juglans regia (English walnut) and various Juglans hybrids by tissue culture in 1993 and has operated on a commercial scale since 1996. Since this time, more than one and a half million walnuts of different species have been propagated and field planted. Tissue cultured...

  16. Biotransformations with plant tissue cultures.

    Science.gov (United States)

    Carew, D P; Bainbridge, T

    1976-01-01

    Suspension cultures of Catharanthus roseus, Apocynum cannabinum and Conium maculatum were examined for their capacity to transform aniline, anisole, acetanilide, benzoic acid and coumarin. None of the cultures transformed acetanilide but each produced acetanilide when fed aniline. All three cultures converted benzoic acid to its para-hydroxy derivative. Coumarin was selectively hydroxylated at the 7-position by Catharanthus and Conium and anisole was O-demethylated only by older Catharanthus tissue.

  17. The basic design and requirement for plant tissue culture laboratory in MINT

    International Nuclear Information System (INIS)

    Azraf Azman; Rosli Darmawan; Rusli Ibrahim; Mohd Nazir Basiran; Azhar Mohamad; Mohamed Najli Mohamed Yasin; Shuhaimi Shamsuddin

    2005-01-01

    The production of multiple species plantlets involves a relatively complex process and it is a highly specialized operation. Tissue culture technology is rapidly becoming a commercialized method for propagating new cultivars, rare species and difficult-to-propagate plant. Not only are skills and knowledge essential but the laboratory itself also plays an important role to ensure the successful growth of the plantlets. To produce quality plantlets, plant tissue culture laboratories should fulfill the basic requirements. The laboratory should have proper building and layout which comprise of media preparation and washing room, sterilization or autoclave room, transfer room and culture or growth room. The scope of this paper is to compare these fundamental requirements with the plant tissue culture laboratory in MINT. All the basic needs and differences will be discussed and the proposal for corrective actions will be presented. (Author)

  18. Tissue culture as a plant production technique for horticultural crops ...

    African Journals Online (AJOL)

    Over 100 years ago, Haberlandt envisioned the concept of plant tissue culture and provided the groundwork for the cultivation of plant cells, tissues and organs in culture. Initially plant tissue cultures arose as a research tool and focused on attempts to culture and study the development of small, isolated cells and segments ...

  19. Mathematical modelling of tissue formation in chondrocyte filter cultures.

    Science.gov (United States)

    Catt, C J; Schuurman, W; Sengers, B G; van Weeren, P R; Dhert, W J A; Please, C P; Malda, J

    2011-12-17

    In the field of cartilage tissue engineering, filter cultures are a frequently used three-dimensional differentiation model. However, understanding of the governing processes of in vitro growth and development of tissue in these models is limited. Therefore, this study aimed to further characterise these processes by means of an approach combining both experimental and applied mathematical methods. A mathematical model was constructed, consisting of partial differential equations predicting the distribution of cells and glycosaminoglycans (GAGs), as well as the overall thickness of the tissue. Experimental data was collected to allow comparison with the predictions of the simulation and refinement of the initial models. Healthy mature equine chondrocytes were expanded and subsequently seeded on collagen-coated filters and cultured for up to 7 weeks. Resulting samples were characterised biochemically, as well as histologically. The simulations showed a good representation of the experimentally obtained cell and matrix distribution within the cultures. The mathematical results indicate that the experimental GAG and cell distribution is critically dependent on the rate at which the cell differentiation process takes place, which has important implications for interpreting experimental results. This study demonstrates that large regions of the tissue are inactive in terms of proliferation and growth of the layer. In particular, this would imply that higher seeding densities will not significantly affect the growth rate. A simple mathematical model was developed to predict the observed experimental data and enable interpretation of the principal underlying mechanisms controlling growth-related changes in tissue composition.

  20. Culture-Independent Identification of Mycobacterium avium Subspecies paratuberculosis in Ovine Tissues: Comparison with Bacterial Culture and Histopathological Lesions

    Directory of Open Access Journals (Sweden)

    Kamal R. Acharya

    2017-12-01

    Full Text Available Johne’s disease is a chronic debilitating enteropathy of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP. Current abattoir surveillance programs detect disease via examination of gross lesions and confirmation by histopathological and/or tissue culture, which is time-consuming and has relatively low sensitivity. This study aimed to investigate whether a high-throughput quantitative PCR (qPCR test is a viable alternative for tissue testing. Intestine and mesenteric lymph nodes were sourced from sheep experimentally infected with MAP and the DNA extracted using a protocol developed for tissues, comprised enzymatic digestion of the tissue homogenate, chemical and mechanical lysis, and magnetic bead-based DNA purification. The extracted DNA was tested by adapting a previously validated qPCR for fecal samples, and the results were compared with culture and histopathology results of the corresponding tissues. The MAP tissue qPCR confirmed infection in the majority of sheep with gross lesions on postmortem (37/38. Likewise, almost all tissue culture (61/64 or histopathology (52/58 positives were detected with good to moderate agreement (Cohen’s kappa statistic and no significant difference to the reference tests (McNemar’s Chi-square test. Higher MAP DNA quantities corresponded to animals with more severe histopathology (odds ratio: 1.82; 95% confidence interval: 1.60, 2.07. Culture-independent strain typing on tissue DNA was successfully performed. This MAP tissue qPCR method had a sensitivity equivalent to the reference tests and is thus a viable replacement for gross- and histopathological examination of tissue samples in abattoirs. In addition, the test could be validated for testing tissue samples intended for human consumption.

  1. Identification of Stevioside Using Tissue Culture-Derived Stevia () Leaves

    OpenAIRE

    Ziaul Karim Md.; Daisuke Uesugi; Noriyuki Nakayama; M. Monzur Hossain; Kohji Ishihara; Hiroki Hamada

    2015-01-01

    Stevioside is a natural sweetener from Stevia leaf, which is 300 times sweeter than sugar. It helps to reduce blood sugar levels dramatically and thus can be of benefit to diabetic people. Tissue culture is a very potential modern technology that can be used in large-scale disease-free stevia production throughout the year. We successfully produced stevia plant through in vitro culture for identification of stevioside in this experiment. The present study describes a potential method for iden...

  2. The use of animal tissues alongside human tissue: Cultural and ethical considerations.

    Science.gov (United States)

    Kaw, Anu; Jones, D Gareth; Zhang, Ming

    2016-01-01

    Teaching and research facilities often use cadaveric material alongside animal tissues, although there appear to be differences in the way we handle, treat, and dispose of human cadaveric material compared to animal tissue. This study sought to analyze cultural and ethical considerations and provides policy recommendations on the use of animal tissues alongside human tissue. The status of human and animal remains and the respect because of human and animal tissues were compared and analyzed from ethical, legal, and cultural perspectives. The use of animal organs and tissues is carried out within the context of understanding human anatomy and function. Consequently, the interests of human donors are to be pre-eminent in any policies that are enunciated, so that if any donors find the presence of animal remains unacceptable, the latter should not be employed. The major differences appear to lie in differences in our perceptions of their respective intrinsic and instrumental values. Animals are considered to have lesser intrinsic value and greater instrumental value than humans. These differences stem from the role played by culture and ethical considerations, and are manifested in the resulting legal frameworks. In light of this discussion, six policy recommendations are proposed, encompassing the nature of consent, respect for animal tissues as well as human remains, and appropriate separation of both sets of tissues in preparation and display. © 2015 Wiley Periodicals, Inc.

  3. HYPOLIPIDEMIC EFFECT OF ARGLABIN IN HEPATOMA TISSUE CULTURE

    Directory of Open Access Journals (Sweden)

    A. V. Ratkin

    2015-01-01

    Full Text Available Objective. Investigation of hypolipidemic effect of sesquiterpene γ-lactone Arglabin in hepatoma tissue culture (HTC.Materials and methods. In this study we’ve evaluated the effect of sesquiterpene γ-lactone Arglabin and gemfibrozil (reference drug on the lipid content in the hepatoma tissue culture (HTC which were incubated with a fat emulsion “Lipofundin” by fluorescent method with vital dye Nile Red. The cell viability was investigated using the MTT-test and staining by Trypan blue.Results. Cultivation of cell cultures of rat’s hepatoma cell line HTC with Arglabin and gemfibrozil in concentrations from 10 to 50 μmol and from 0.25 to 0.5 mmol, respectively, had no cytotoxic effect. HTC cell viability did not change compared with the corresponding rate in the control culture. Experimental hyperlipidemia in hepatoma culture was induced by the addition in the incubation medium of fat emulsion “Lipofundin” in a final concentration of 0.05 %. The fluorescence intensity of Nile Red in the cells was increased 4-fold (p < 0.05, which indicates a significant accumulation of lipids in the cytosol of cells. In these steady-state Arglabin and gemfibrozil at concentrations 75–100 μM and 0.25–1.0 mM, respectively, reduced the content of lipid in cells. Conclusion. In the model of hyperlipidemia induced by lipofundin, sesquiterpene γ-lactone Arglabin prevents the accumulation of lipids in the HTC cell line, as evidenced by a decrease in Nile Red fluorescence. However hypolipidemic effect of Arglabin is associated with cytotoxic effects, which is typical for anticancer drugs.

  4. Self-Condensation Culture Enables Vascularization of Tissue Fragments for Efficient Therapeutic Transplantation

    Directory of Open Access Journals (Sweden)

    Yoshinobu Takahashi

    2018-05-01

    Full Text Available Summary: Clinical transplantation of tissue fragments, including islets, faces a critical challenge because of a lack of effective strategies that ensure efficient engraftment through the timely integration of vascular networks. We recently developed a complex organoid engineering method by “self-condensation” culture based on mesenchymal cell-dependent contraction, thereby enabling dissociated heterotypic lineages including endothelial cells to self-organize in a spatiotemporal manner. Here, we report the successful adaptation of this method for generating complex tissues from diverse tissue fragments derived from various organs, including pancreatic islets. The self-condensation of human and mouse islets with endothelial cells not only promoted functionalization in culture but also massively improved post-transplant engraftment. Therapeutically, fulminant diabetic mice were more efficiently treated by a vascularized islet transplant compared with the conventional approach. Given the general limitations of post-transplant vascularization associated with 3D tissue-based therapy, our approach offers a promising means of enhancing efficacy in the context of therapeutic tissue transplantation. : Takahashi et al. report on generating vascularized islet tissue from humans and mice. After transplantation, vascularized islets significantly improve survival of diabetic mice, demonstrating the quick normalization of blood glucose compared with conventional islet transplantation. Keywords: tissue engineering, tissue-based therapy, vascularization, islet transplantation, organoid

  5. Advances in tissue engineering through stem cell-based co-culture.

    Science.gov (United States)

    Paschos, Nikolaos K; Brown, Wendy E; Eswaramoorthy, Rajalakshmanan; Hu, Jerry C; Athanasiou, Kyriacos A

    2015-05-01

    Stem cells are the future in tissue engineering and regeneration. In a co-culture, stem cells not only provide a target cell source with multipotent differentiation capacity, but can also act as assisting cells that promote tissue homeostasis, metabolism, growth and repair. Their incorporation into co-culture systems seems to be important in the creation of complex tissues or organs. In this review, critical aspects of stem cell use in co-culture systems are discussed. Direct and indirect co-culture methodologies used in tissue engineering are described, along with various characteristics of cellular interactions in these systems. Direct cell-cell contact, cell-extracellular matrix interaction and signalling via soluble factors are presented. The advantages of stem cell co-culture strategies and their applications in tissue engineering and regenerative medicine are portrayed through specific examples for several tissues, including orthopaedic soft tissues, bone, heart, vasculature, lung, kidney, liver and nerve. A concise review of the progress and the lessons learned are provided, with a focus on recent developments and their implications. It is hoped that knowledge developed from one tissue can be translated to other tissues. Finally, we address challenges in tissue engineering and regenerative medicine that can potentially be overcome via employing strategies for stem cell co-culture use. Copyright © 2014 John Wiley & Sons, Ltd.

  6. How-To-Do-It: Using Cauliflower to Demonstrate Plant Tissue Culture.

    Science.gov (United States)

    Haldeman, Janice H.; Ellis, Jane P.

    1988-01-01

    Presents techniques used for disinfestation of plant material, preparation of equipment and media, and laboratory procedures for tissue culture using cauliflower. Details methods for preparing solutions and plant propagation by cloning. (CW)

  7. Study on tissue culture for Gelidium seedling

    Science.gov (United States)

    Pei, Lu-Qing; Luo, Qi-Jun; Fei, Zhi-Qing; Ma, Bin

    1996-06-01

    As seedling culture is a crucial factor for successful cultivation of Gelidium, the authors researched tissue culture technology for producing seedlings. The morphogeny and experimental ecology were observed and studied fully in 2 5 mm isolated tissue fragments. Regeneration, appearance of branching creepers and attaching structure and new erect seedlings production and development were studied. Fragments were sown on bamboo slice and vinylon rope. The seedlings were cultured 20 30 days indoor, then cultured in the sea, where the density of erect seedlings was 3 19 seedlings/cm2, growth rate was 3.84% day. The frond arising from seedlings directly was up to 10 cm per year. The ecological conditions for regenerated seedlings are similar to the natural ones. The regenerated seedlings are suitable for raft culture in various sea areas.

  8. Oxygen and tissue culture affect placental gene expression.

    Science.gov (United States)

    Brew, O; Sullivan, M H F

    2017-07-01

    Placental explant culture is an important model for studying placental development and functions. We investigated the differences in placental gene expression in response to tissue culture, atmospheric and physiologic oxygen concentrations. Placental explants were collected from normal term (38-39 weeks of gestation) placentae with no previous uterine contractile activity. Placental transcriptomic expressions were evaluated with GeneChip ® Human Genome U133 Plus 2.0 arrays (Affymetrix). We uncovered sub-sets of genes that regulate response to stress, induction of apoptosis programmed cell death, mis-regulation of cell growth, proliferation, cell morphogenesis, tissue viability, and protection from apoptosis in cultured placental explants. We also identified a sub-set of genes with highly unstable pattern of expression after exposure to tissue culture. Tissue culture irrespective of oxygen concentration induced dichotomous increase in significant gene expression and increased enrichment of significant pathways and transcription factor targets (TFTs) including HIF1A. The effect was exacerbated by culture at atmospheric oxygen concentration, where further up-regulation of TFTs including PPARA, CEBPD, HOXA9 and down-regulated TFTs such as JUND/FOS suggest intrinsic heightened key biological and metabolic mechanisms such as glucose use, lipid biosynthesis, protein metabolism; apoptosis, inflammatory responses; and diminished trophoblast proliferation, differentiation, invasion, regeneration, and viability. These findings demonstrate that gene expression patterns differ between pre-culture and cultured explants, and the gene expression of explants cultured at atmospheric oxygen concentration favours stressed, pro-inflammatory and increased apoptotic transcriptomic response. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Effects of ionizing radiation on plant tissue cultures

    International Nuclear Information System (INIS)

    Hell, K.G.

    1978-01-01

    A short review is done of the biological effects of ionizing radiations on plant tissues kept in culture, from the work of Gladys King, in 1949, with X-ray irradiated tobacco. The role of plant hormones is discussed in the processes of growth inhibition and growth restoration of irradiated tissues, as well as morphogenesis. Radioresistance of cells kept in culture and the use of ionizing radiations as mutagens are also commented. Some aspects of the biological effects of ionizing radiations that need to be investigated are discussed, and the problem of genome instability of plant tissues kept in culture is pointed out. (M.A.) [pt

  10. Methods of Monitoring Cell Fate and Tissue Growth in Three-Dimensional Scaffold-Based Strategies for In Vitro Tissue Engineering.

    Science.gov (United States)

    Leferink, Anne M; van Blitterswijk, Clemens A; Moroni, Lorenzo

    2016-08-01

    In the field of tissue engineering, there is a need for methods that allow assessing the performance of tissue-engineered constructs noninvasively in vitro and in vivo. To date, histological analysis is the golden standard to retrieve information on tissue growth, cellular distribution, and cell fate on tissue-engineered constructs after in vitro cell culture or on explanted specimens after in vivo applications. Yet, many advances have been made to optimize imaging techniques for monitoring tissue-engineered constructs with a sub-mm or μm resolution. Many imaging modalities have first been developed for clinical applications, in which a high penetration depth has been often more important than lateral resolution. In this study, we have reviewed the current state of the art in several imaging approaches that have shown to be promising in monitoring cell fate and tissue growth upon in vitro culture. Depending on the aimed tissue type and scaffold properties, some imaging methods are more applicable than others. Optical methods are mostly suited for transparent materials such as hydrogels, whereas magnetic resonance-based methods are mostly applied to obtain contrast between hard and soft tissues regardless of their transparency. Overall, this review shows that the field of imaging in scaffold-based tissue engineering is developing at a fast pace and has the potential to overcome the limitations of destructive endpoint analysis.

  11. [Chromosome variability in the tissue culture of rare Gentiana species].

    Science.gov (United States)

    Tvardovs'ka, M O; Strashniuk, N M; Mel'nyk, V M; Adonin, V I; Kunakh, V A

    2008-01-01

    Cytogenetic analysis of plants and tissue culture of Gentiana lutea, G. punctata, G. acaulis has been carried out. Culturing in vitro was found to result in the changes of chromosome number in the calluses of the species involved. Species specificity for variation of the cultured cell genomes was shown. Contribution of the original plant genotypes to the cytogenetic structure of the tissue culture was established. Gentiana callus tissues (except for in vitro culture of G. punctata, derived from plant of Breskul'ska population) were found to exhibit modal class with the cells of diploid and nearly diploid chromosome sets.

  12. Analytical and diagnostic performance of a qPCR assay for Ichthyophonus spp. compared to the tissue culture 'gold standard'.

    Science.gov (United States)

    Lowe, Vanessa C; Hershberger, Paul K; Friedman, Carolyn S

    2018-06-04

    Parasites of the genus Ichthyophonus infect many fish species and have a non-uniform distribution within host tissues. Due in part to this uneven distribution, the comparative sensitivity and accuracy of using molecular-based detection methods versus culture to estimate parasite prevalence is under debate. We evaluated the analytical and diagnostic performance of an existing qPCR assay in comparison to the 'gold standard' culture method using Pacific herring Clupea pallasii with known exposure history. We determined that the assay is suitable for use in this host, and diagnostic specificity was consistently high (>98%) in both heart and liver tissues. Diagnostic sensitivity could not be fully assessed due to low infection rates, but our results suggest that qPCR is not as sensitive as culture under all circumstances. Diagnostic sensitivity of qPCR relative to culture is likely affected by the amount of sample processed. The prevalence values estimated by the 2 methods were not significantly different when sample amounts were equal (heart tissue), but when the assayed sample amounts were unequal (liver tissue), the culture method detected a significantly higher prevalence of the parasite than qPCR. Further, culture of liver also detected significantly more Ichthyophonus infections than culture of heart, suggesting that the density and distribution of parasites in tissues also plays a role in assay sensitivity. This sensitivity issue would be most problematic for fish with light infections. Although qPCR does not detect the presence of a live organism, DNA-based pathogen detection methods provide the opportunity for alternate testing strategies when culture is not possible.

  13. Lemongrass-Incorporated Tissue Conditioner Against Candida albicans Culture

    Science.gov (United States)

    Amornvit, Pokpong; Srithavaj, Theerathavaj

    2014-01-01

    Background: Tissue conditioner is applied popularly with dental prosthesis during wound healing process but it becomes a reservoir of oral microbiota, especially Candida species after long-term usage. Several antifungal drugs have been mixed with this material to control fungal level. In this study, lemongrass essential oil was added into COE-COMFORT tissue conditioner before being determined for anti-Candida efficacy. Materials and Methods: Lemongrass (Cymbopogon citratus) essential oil was primarily determined for antifungal activity against C. albicans American type culture collection (ATCC) 10231 and MIC (minimum inhibitory concentration) value by agar disk diffusion and broth microdilution methods, respectively. COE-COMFORT tissue conditioner was prepared as recommended by the manufacturer after a fixed volume of the oil at its MIC or higher concentrations were mixed thoroughly in its liquid part. Antifungal efficacy of the tissue conditioner with/without herb was finally analyzed. Results: Lemongrass essential oil displayed potent antifungal activity against C. albicans ATCC 10231and its MIC value was 0.06% (v/v). Dissimilarly, the tissue conditioner containing the oil at MIC level did not cease the growth of the tested fungus. Both reference and clinical isolates of C. albicans were completely inhibited after exposed to the tissue conditioner containing at least 0.25% (v/v) of the oil (approximately 4-time MIC). The tissue conditioner without herb or with nystatin was employed as negative or positive control, respectively. Conclusion: COE-COMFORT tissue conditioner supplemented with lemongrass essential oil obviously demonstrated another desirable property as in vitro anti-Candida efficacy to minimize the risk of getting Candidal infection. PMID:25177638

  14. Addressing the instability of DNA nanostructures in tissue culture.

    Science.gov (United States)

    Hahn, Jaeseung; Wickham, Shelley F J; Shih, William M; Perrault, Steven D

    2014-09-23

    DNA nanotechnology is an advanced technique that could contribute diagnostic, therapeutic, and biomedical research devices to nanomedicine. Although such devices are often developed and demonstrated using in vitro tissue culture models, these conditions may not be compatible with DNA nanostructure integrity and function. The purpose of this study was to characterize the sensitivity of 3D DNA nanostructures produced via the origami method to the in vitro tissue culture environment and identify solutions to prevent loss of nanostructure integrity. We examined whether the physiological cation concentrations of cell culture medium and the nucleases present in fetal bovine serum (FBS) used as a medium supplement result in denaturation and digestion, respectively. DNA nanostructure denaturation due to cation depletion was design- and time-dependent, with one of four tested designs remaining intact after 24 h at 37 °C. Adjustment of medium by addition of MgSO4 prevented denaturation. Digestion of nanostructures by FBS nucleases in Mg(2+)-adjusted medium did not appear design-dependent and became significant within 24 h and when medium was supplemented with greater than 5% FBS. We estimated that medium supplemented with 10% FBS contains greater than 256 U/L equivalent of DNase I activity in digestion of DNA nanostructures. Heat inactivation at 75 °C and inclusion of actin protein in medium inactivated and inhibited nuclease activity, respectively. We examined the impact of medium adjustments on cell growth, viability, and phenotype. Adjustment of Mg(2+) to 6 mM did not appear to have a detrimental impact on cells. Heat inactivation was found to be incompatible with in vitro tissue culture, whereas inclusion of actin had no observable effect on growth and viability. In two in vitro assays, immune cell activation and nanoparticle endocytosis, we show that using conditions compatible with cell phenotype and nanostructure integrity is critical for obtaining reliable

  15. Variations on metabolic activities of legume tissues through radiation in tissue culture

    International Nuclear Information System (INIS)

    Batra, Amla

    1977-01-01

    Cell cultures from Arachis hypogaea L. cultivated in a modified medium developed by Murashige and Skoog (1962) showed vigorous qrowth after radiation treatment. Investigations on the effect of various sugars on the chlorophyll formation and growth of the irradiated tissues showed that sucrose was superior to maltose, glucose or fructose as a carbon source. Lactose and mannitol supported growth and development of chlorophyll to a less degree. On prolonging the cultures on a sugar free medium, the tissues failed to regain either growth or chlorophyll content. (author)

  16. Variations on metabolic activities of legume tissues through radiation in tissue culture

    Energy Technology Data Exchange (ETDEWEB)

    Batra, A [Rajasthan Univ., Jaipur (India). Dept. of Botany

    1977-12-01

    Cell cultures from Arachis hypogaea L. cultivated in a modified medium developed by Murashige and Skoog (1962) showed vigorous qrowth after radiation treatment. Investigations on the effect of various sugars on the chlorophyll formation and growth of the irradiated tissues showed that sucrose was superior to maltose, glucose or fructose as a carbon source. Lactose and mannitol supported growth and development of chlorophyll to a less degree. On prolonging the cultures on a sugar free medium, the tissues failed to regain either growth or chlorophyll content.

  17. A simple method for multiday imaging of slice cultures.

    Science.gov (United States)

    Seidl, Armin H; Rubel, Edwin W

    2010-01-01

    The organotypic slice culture (Stoppini et al. A simple method for organotypic cultures of nervous tissue. 1991;37:173-182) has become the method of choice to answer a variety of questions in neuroscience. For many experiments, however, it would be beneficial to image or manipulate a slice culture repeatedly, for example, over the course of many days. We prepared organotypic slice cultures of the auditory brainstem of P3 and P4 mice and kept them in vitro for up to 4 weeks. Single cells in the auditory brainstem were transfected with plasmids expressing fluorescent proteins by way of electroporation (Haas et al. Single-cell electroporation for gene transfer in vivo. 2001;29:583-591). The culture was then placed in a chamber perfused with oxygenated ACSF and the labeled cell imaged with an inverted wide-field microscope repeatedly for multiple days, recording several time-points per day, before returning the slice to the incubator. We describe a simple method to image a slice culture preparation during the course of multiple days and over many continuous hours, without noticeable damage to the tissue or photobleaching. Our method uses a simple, inexpensive custom-built insulator constructed around the microscope to maintain controlled temperature and uses a perfusion chamber as used for in vitro slice recordings. (c) 2009 Wiley-Liss, Inc.

  18. Gastric tissue biopsy and culture

    Science.gov (United States)

    ... symptoms may include: Loss of appetite or weight loss Nausea and vomiting Pain in the upper part of the belly Black stools Vomiting blood or coffee ground-like material A gastric tissue biopsy and culture can help detect: Cancer Infections, most commonly Helicobacter ...

  19. Migration assay on primary culture isolated from patient's primary breast cancer tissue

    Directory of Open Access Journals (Sweden)

    ED Yuliana

    2014-12-01

    Full Text Available Background: Migration is an essential component of breast cancer metastasis, which studyhas been concentrated on culture of established breast cancer cell lines that do not accuratelyrepresent the sophistication and heterogeneity of patient's breast cancer. An attempt toperform migration assay using Boyden Chamber Assay (BCA on primary culture originatingfrom patient's breast cancer tissue was developed to accommodate upcoming study of breastcancer migration in lndonesian patients.Methods: Pathologically proven primary breast cancer tissue samples were obtained fromCiptomangunkusumo Hospital during core (n=4 and incisional (n=3 biopsies of stage llAup to stage lllA breast cancer patients. Following biopsy, the breast cancer tissue samplesunderwent processings to isolate the cancer cells. These cancer cells were -then resuspendedwithin Dulbecco's modified Eagle's medium (DMEM ahd cultured in 12-well plate. The growthof primary culture were observed and compared between the core biopsy and the incisionalbiopsy specimens. Optimization of BCA method was later performed to investigate themigration of the breast cancer primary culture towards different experirnental conditions, whichwere control, Fetal Bovine Serum (FBS, and Stromal Derived Factor-l (SDF-1. Two differentnumber of breast cancer cells were tested for the optimization of the BCA, which were 1 x 105and3x105cells.Results: None of the culture performed on core biopsy specimens grew, while one out ofthree incisional biopsy specimens grew until confluence. The one primary culture that grewwas later assesed using BCA to assess its migration index towards different experimentalconditions. Using 1 x 10s breast cancer cells in the BCA , the result of the absorbance level ofmigrated cells showed that the migration towards SDF-1 (0.529 nearly doubled the migrationtowards controlmedium (0.239 and FBS (0.209. Meanwhile, the absorbance levelwas simiiarbetween the control medium (1.050, FBS (1 .103

  20. Versatile electrochemial sensor for tissue culturing and sample handling

    DEFF Research Database (Denmark)

    Bakmand, Tanya; Kwasny, Dorota; Al Atraktchi, Fatima Al-Zahraa

    2014-01-01

    Culturing of organtypic brain tissues is a routine procedure in neural research. The visual inspection of the medium is the only way of determining the state of the tissue. At the end of culturing, post-processing techniques such as HPLC can be used to measure the concentration of the secreted...

  1. Cell-surface glycoproteins of human sarcomas: differential expression in normal and malignant tissues and cultured cells

    International Nuclear Information System (INIS)

    Rettig, W.F.; Garin-Chesa, P.; Beresford, H.R.; Oettgen, H.F.; Melamed, M.R.; Old, L.J.

    1988-01-01

    Normal differentiation and malignant transformation of human cells are characterized by specific changes in surface antigen phenotype. In the present study, the authors have defined six cell-surface antigens of human sarcomas and normal mesenchymal cells, by using mixed hemadsorption assays and immunochemical methods for the analysis of cultured cells and immunohistochemical staining for the analysis of normal tissues and > 200 tumor specimens. Differential patterns of F19, F24, G171, G253, S5, and Thy-1 antigen expression were found to characterize (i) subsets of cultured sarcoma cell lines, (ii) cultured fibroblasts derived from various organs, (iii) normal resting and activated mesenchymal tissues, and (iv) sarcoma and nonmesenchymal tumor tissues. These results provide a basic surface antigenic map for cultured mesenchymal cells and mesenchymal tissues and permit the classification of human sarcomas according to their antigenic phenotypes

  2. Epigenetics in plant tissue culture

    NARCIS (Netherlands)

    Smulders, M.J.M.; Klerk, de G.J.M.

    2011-01-01

    Plants produced vegetatively in tissue culture may differ from the plants from which they have been derived. Two major classes of off-types occur: genetic ones and epigenetic ones. This review is about epigenetic aberrations. We discuss recent studies that have uncovered epigenetic modifications at

  3. Metabolic labeling of sialic acids in tissue culture cell lines: methods to identify substituted and modified radioactive neuraminic acids

    International Nuclear Information System (INIS)

    Diaz, S.; Varki, A.

    1985-01-01

    The parent sialic acid N-acetylneuraminic acid can be modified or substituted in various ways, giving rise to a family of more than 25 compounds. The definitive identification of these compounds has previously required isolation of nanomole amounts for mass spectrometry or NMR. We have explored the possibility of using the known metabolic precursors of the sialic acids, particularly N-acetyl-[6-3H]mannosamine, to label and identify various forms of sialic acids in tissue culture cells. Firstly, we defined several variables that affect the labeling of sialic acids with N-acetyl-[6-3H]mannosamine. Secondly, we have devised a simple screening method to identify cell lines that synthesize substituted or modified sialic acids. We next demonstrate that it is possible to definitively identify the natures of the various labeled sialic acids without the use of mass spectrometry, even though they are present only in tracer amounts. The methods used include paper chromatography, analytical de-O-acetylation, periodate release of the 9-3H as [3H]formaldehyde (which is subsequently converted to a specific 3H-labeled chromophore), acylneuraminate pyruvate lyase treatment with identification of [3H]acylmannosamines, gas-liquid chromatography with radioactive detection, and two new high-pressure liquid chromatography methods utilizing the amine-adsorption:ion suppression and ion-pair principles. The use of an internal N-acetyl-[4-14C]neuraminic acid standard in each of these methods assures precision and accuracy. The combined use of these methods now allows the identification of radioactive tracer amounts of the various types of sialic acids in well-defined populations of tissue culture cells; it may also allow the identification of hitherto unknown forms of sialic acids

  4. Transcriptomic comparisons between cultured human adipose tissue-derived pericytes and mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Lindolfo da Silva Meirelles

    2016-03-01

    Full Text Available Mesenchymal stromal cells (MSCs, sometimes called mesenchymal stem cells, are cultured cells able to give rise to mature mesenchymal cells such as adipocytes, osteoblasts, and chondrocytes, and to secrete a wide range of trophic and immunomodulatory molecules. Evidence indicates that pericytes, cells that surround and maintain physical connections with endothelial cells in blood vessels, can give rise to MSCs (da Silva Meirelles et al., 2008 [1]; Caplan and Correa, 2011 [2]. We have compared the transcriptomes of highly purified, human adipose tissue pericytes subjected to culture-expansion in pericyte medium or MSC medium, with that of human adipose tissue MSCs isolated with traditional methods to test the hypothesis that their transcriptomes are similar (da Silva Meirelles et al., 2015 [3]. Here, we provide further information and analyses of microarray data from three pericyte populations cultured in pericyte medium, three pericyte populations cultured in MSC medium, and three adipose tissue MSC populations deposited in the Gene Expression Omnibus under accession number GSE67747. Keywords: Mesenchymal stromal cells, Mesenchymal stem cells, Pericytes, Microarrays

  5. Potato transformation and potato cyst nematode infection on potato plantlets in tissue culture

    Science.gov (United States)

    These two protocols describe the methods for generating transgenic potato plants and for evaluating potato cyst nematode (Globodera rostochiensis and G. pallida) infection on potato plantlets in tissue culture. These methods are useful tools that can be used in the study of the interactions between ...

  6. Metabolic Profile of Pancreatic Acinar and Islet Tissue in Culture

    Science.gov (United States)

    Suszynski, Thomas M.; Mueller, Kathryn; Gruessner, Angelika C.; Papas, Klearchos K.

    2016-01-01

    The amount and condition of exocrine impurities may affect the quality of islet preparations especially during culture. In this study, the objective was to determine the oxygen demandand viability of islet and acinar tissue post-isolation and whether they change disproportionately while in culture. We compare the OCR normalized to DNA (OCR/DNA, a measure of fractional viability in units nmol/min/mg DNA), and percent change in OCR and DNA recoveries between adult porcine islet and acinar tissue from the same preparation (paired) over a 6-9 days of standard culture. Paired comparisons were done to quantify differences in OCR/DNA between islet and acinar tissue from the same preparation, at specified time points during culture; the mean (± standard error) OCR/DNA was 74.0 (±11.7) units higher for acinar (vs. islet) tissue on the day of isolation (n=16, p<0.0001), but 25.7 (±9.4) units lower after 1 day (n=8, p=0.03), 56.6 (±11.5) units lower after 2 days (n=12, p=0.0004), and 65.9 (±28.7) units lower after 8 days (n=4, p=0.2) in culture. DNA and OCR recoveries decreased at different rates for acinar versus islet tissue over 6-9 days in culture (n=6). DNA recovery decreased to 24±7% for acinar and 75±8% for islets (p=0.002). Similarly, OCR recovery decreased to 16±3% for acinar and remained virtually constant for islets (p=0.005). Differences in the metabolic profile of acinarand islet tissue should be considered when culturing impure islet preparations. OCR-based measurements may help optimize pre-IT culture protocols. PMID:25131082

  7. Revision washout decreases implant capsule tissue culture positivity: a multicenter study.

    Science.gov (United States)

    Henry, Gerard D; Carson, Culley C; Wilson, Steven K; Wiygul, Jeremy; Tornehl, Chris; Cleves, Mario A; Simmons, Caroline J; Donatucci, Craig F

    2008-01-01

    Positive cultures, visible biofilm and confocal micrography confirm bacterial presence on clinically uninfected inflatable penile prostheses at revision surgery. Salvage irrigation has been proved to rescue patients with clinically infected inflatable penile prostheses. Similar washout at revision for noninfectious reasons significantly lowers subsequent infection rates. We investigated a larger series of patients for positive culture rates and evaluated implant capsule tissue culture rates before and after revision washout. At 4 institutions a total of 148 patients with inflatable penile prostheses underwent revision surgery for noninfectious reasons between June 2001 and September 2005. Swab cultures of the fluid around the pump and visible biofilm were obtained. Also, in 65 patients a wedge of tissue from the capsule that forms around the pump was cultured. After implant removal revision washout of the implant spaces was performed and a second wedge of tissue was cultured. Of the 148 patients 97 (66%) had positive bacterial swab cultures of the fluid around the pump or biofilm. A total of 124 isolates were cultured. Of the 65 implant capsule tissue cultures obtained before washout 28 (43%) were positive for bacteria, while 16 (25%) obtained after revision washout were positive. Positive cultures and visible bacterial biofilm are present on clinically uninfected inflatable penile prostheses at revision surgery in most patients. Revision washout appears to decrease the bacterial load on implant capsule tissue at revision surgery of inflatable penile prostheses for noninfectious reasons.

  8. [Effective productions of plant secondary metabolites having antitumor activity by plant cell and tissue cultures].

    Science.gov (United States)

    Taniguchi, Shoko

    2005-06-01

    Methods for the effective production of plant secondary metabolites with antitumor activity using plant cell and tissue cultures were developed. The factors in tannin productivity were investigated using culture strains producing different types of hydrolyzable tannins, i.e., gallotannins (mixture of galloylglucoses), ellagi-, and dehydroellagitannins. Production of ellagi- and dehydroellagitannins was affected by the concentrations and ratio of nitrogen sources in the medium. The formation of oligomeric ellagitannins in shoots of Oenothera tetraptera was correlated with the differentiation of tissues. Cultured cells of Eriobotrya japonica producing ursane- and oleanane-type triterpenes with antitumor activities were also established.

  9. A method for high purity intestinal epithelial cell culture from adult human and murine tissues for the investigation of innate immune function.

    Science.gov (United States)

    Graves, Christina L; Harden, Scott W; LaPato, Melissa; Nelson, Michael; Amador, Byron; Sorenson, Heather; Frazier, Charles J; Wallet, Shannon M

    2014-12-01

    Intestinal epithelial cells (IECs) serve as an important physiologic barrier between environmental antigens and the host intestinal immune system. Thus, IECs serve as a first line of defense and may act as sentinel cells during inflammatory insults. Despite recent renewed interest in IEC contributions to host immune function, the study of primary IEC has been hindered by lack of a robust culture technique, particularly for small intestinal and adult tissues. Here, a novel adaptation for culture of primary IEC is described for human duodenal organ donor tissue as well as duodenum and colon of adult mice. These epithelial cell cultures display characteristic phenotypes and are of high purity. In addition, the innate immune function of human primary IEC, specifically with regard to Toll-like receptor (TLR) expression and microbial ligand responsiveness, is contrasted with a commonly used intestinal epithelial cell line (HT-29). Specifically, TLR expression at the mRNA level and production of cytokine (IFNγ and TNFα) in response to TLR agonist stimulation is assessed. Differential expression of TLRs as well as innate immune responses to ligand stimulation is observed in human-derived cultures compared to that of HT-29. Thus, use of this adapted method to culture primary epithelial cells from adult human donors and from adult mice will allow for more appropriate studies of IECs as innate immune effectors. Published by Elsevier B.V.

  10. TCUP: A novel hAT transposon active in maize tissue culture

    Directory of Open Access Journals (Sweden)

    Alan eSmith

    2012-01-01

    Full Text Available Transposable elements are capable of inducing heritable de novo genetic variation. The sequences capable of reactivation, and environmental factors that induce mobilization, remain poorly defined even in well-studied genomes such as maize. We treated maize tissue culture with the demethylating agent 5-aza-2-deoxcytidine and examined long-term tissue culture lines to discover silenced transposable elements that have the potential to induce heritable genetic variation. Through these screens we have identified a novel low copy number hAT transposon, Tissue Culture Up-Regulated (TCUP, which is transcribed at high levels in long-term maize Black Mexican Sweet (BMS tissue culture and up-regulated in response to treatment with 5-aza-2-deoxycytidine. Analysis of the TIGR Maize Gene Index revealed that this element is the most frequently represented EST from the BMS cell culture library and is not represented in other tissue libraries, which is the basis for its name. A full-length sequence was assembled in inbred B73 that contains the putative functional motifs required for autonomous movement of a hAT transposon. Transposon display detected movement of TCUP in two long-term tissue cultured cell lines of the genotype Hi-II AxB and BMS. This research implicates TCUP as a transposon that is capable of reactivation and which may also be particularly sensitive to the stress of the tissue culture environment. Our findings are consistent with the hypothesis that epigenetic alterations potentiate genomic responses to stress during clonal propagation of plants.

  11. Micropropagation and maintenance of phytoplasmas in tissue culture.

    Science.gov (United States)

    Bertaccini, Assunta; Paltrinieri, Samanta; Martini, Marta; Tedeschi, Mara; Contaldo, Nicoletta

    2013-01-01

    Maintenance of phytoplasma strains in tissue culture is achievable for all strains transmitted to periwinkle (Catharanthus roseus), and also for other naturally infected plant host species. Shoots of 1-3 cm length are grown in a solid medium containing Murashige and Skoog (MS) micro- and macroelements and 0.12 mg/L benzylaminopurine. The continued presence of phytoplasmas in infected shoots of periwinkle that have been maintained in micropropagation for up to 20 years can be shown by diagnostic methods such as nested PCR tests using the 16S rDNA gene (see Chapters 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,and 26 for phytoplasma diagnostic methods).

  12. Three-dimensional hydrogel cell culture systems for modeling neural tissue

    Science.gov (United States)

    Frampton, John

    Two-dimensional (2-D) neural cell culture systems have served as physiological models for understanding the cellular and molecular events that underlie responses to physical and chemical stimuli, control sensory and motor function, and lead to the development of neurological diseases. However, the development of three-dimensional (3-D) cell culture systems will be essential for the advancement of experimental research in a variety of fields including tissue engineering, chemical transport and delivery, cell growth, and cell-cell communication. In 3-D cell culture, cells are provided with an environment similar to tissue, in which they are surrounded on all sides by other cells, structural molecules and adhesion ligands. Cells grown in 3-D culture systems display morphologies and functions more similar to those observed in vivo, and can be cultured in such a way as to recapitulate the structural organization and biological properties of tissue. This thesis describes a hydrogel-based culture system, capable of supporting the growth and function of several neural cell types in 3-D. Alginate hydrogels were characterized in terms of their biomechanical and biochemical properties and were functionalized by covalent attachment of whole proteins and peptide epitopes. Methods were developed for rapid cross-linking of alginate hydrogels, thus permitting the incorporation of cells into 3-D scaffolds without adversely affecting cell viability or function. A variety of neural cell types were tested including astrocytes, microglia, and neurons. Cells remained viable and functional for longer than two weeks in culture and displayed process outgrowth in 3-D. Cell constructs were created that varied in cell density, type and organization, providing experimental flexibility for studying cell interactions and behavior. In one set of experiments, 3-D glial-endothelial cell co-cultures were used to model blood-brain barrier (BBB) structure and function. This co-culture system was

  13. [Research progress of co-culture system for constructing vascularized tissue engineered bone].

    Science.gov (United States)

    Fu, Weili; Xiang, Zhou

    2014-02-01

    To review the research progress of the co-culture system for constructing vascularized tissue engineered bone. The recent literature concerning the co-culture system for constructing vascularized tissue engineered bone was reviewed, including the selection of osteogenic and endothelial lineages, the design and surface modification of scaffolds, the models and dimensions of the co-culture system, the mechanism, the culture conditions, and their application progress. The construction of vascularized tissue engineered bone is the prerequisite for their survival and further clinical application in vivo. Mesenchymal stem cells (owning the excellent osteogenic potential) and endothelial progenitor cells (capable of directional differentiation into endothelial cell) are considered as attractive cell types for the co-culture system to construct vascularized tissue engineered bone. The culture conditions need to be further optimized. Furthermore, how to achieve the clinical goals of minimal invasion and autologous transplantation also need to be further studied. The strategy of the co-culture system for constructing vascularized tissue engineered bone would have a very broad prospects for clinical application in future.

  14. Improved Diagnosis of Prosthetic Joint Infection by Culturing Periprosthetic Tissue Specimens in Blood Culture Bottles.

    Science.gov (United States)

    Peel, Trisha N; Dylla, Brenda L; Hughes, John G; Lynch, David T; Greenwood-Quaintance, Kerryl E; Cheng, Allen C; Mandrekar, Jayawant N; Patel, Robin

    2016-01-05

    Despite known low sensitivity, culture of periprosthetic tissue specimens on agars and in broths is routine. Culture of periprosthetic tissue samples in blood culture bottles (BCBs) is potentially more convenient, but it has been evaluated in a limited way and has not been widely adopted. The aim of this study was to compare the sensitivity and specificity of inoculation of periprosthetic tissue specimens into blood culture bottles with standard agar and thioglycolate broth culture, applying Bayesian latent class modeling (LCM) in addition to applying the Infectious Diseases Society of America (IDSA) criteria for prosthetic joint infection. This prospective cohort study was conducted over a 9-month period (August 2013 to April 2014) at the Mayo Clinic, Rochester, MN, and included all consecutive patients undergoing revision arthroplasty. Overall, 369 subjects were studied; 117 (32%) met IDSA criteria for prosthetic joint infection, and 82% had late chronic infection. Applying LCM, inoculation of tissues into BCBs was associated with a 47% improvement in sensitivity compared to the sensitivity of conventional agar and broth cultures (92.1 versus 62.6%, respectively); this magnitude of change was similar when IDSA criteria were applied (60.7 versus 44.4%, respectively; P = 0.003). The time to microorganism detection was shorter with BCBs than with standard media (P Prosthetic joint infections are a devastating complication of arthroplasty surgery. Despite this, current microbiological techniques to detect and diagnose infections are imperfect. This study examined a new approach to diagnosing infections, through the inoculation of tissue samples from around the prosthetic joint into blood culture bottles. This study demonstrated that, compared to current laboratory practices, this new technique increased the detection of infection. These findings are important for patient care to allow timely and accurate diagnosis of infection. Copyright © 2016 Peel et al.

  15. Tumor tissue slice cultures as a platform for analyzing tissue-penetration and biological activities of nanoparticles.

    Science.gov (United States)

    Merz, Lea; Höbel, Sabrina; Kallendrusch, Sonja; Ewe, Alexander; Bechmann, Ingo; Franke, Heike; Merz, Felicitas; Aigner, Achim

    2017-03-01

    The success of therapeutic nanoparticles depends, among others, on their ability to penetrate a tissue for actually reaching the target cells, and their efficient cellular uptake in the context of intact tissue and stroma. Various nanoparticle modifications have been implemented for altering physicochemical and biological properties. Their analysis, however, so far mainly relies on cell culture experiments which only poorly reflect the in vivo situation, or is based on in vivo experiments that are often complicated by whole-body pharmacokinetics and are rather tedious especially when analyzing larger nanoparticle sets. For the more precise analysis of nanoparticle properties at their desired site of action, efficient ex vivo systems closely mimicking in vivo tissue properties are needed. In this paper, we describe the setup of organotypic tumor tissue slice cultures for the analysis of tissue-penetrating properties and biological activities of nanoparticles. As a model system, we employ 350μm thick slice cultures from different tumor xenograft tissues, and analyze modified or non-modified polyethylenimine (PEI) complexes as well as their lipopolyplex derivatives for siRNA delivery. The described conditions for tissue slice preparation and culture ensure excellent tissue preservation for at least 14days, thus allowing for prolonged experimentation and analysis. When using fluorescently labeled siRNA for complex visualization, fluorescence microscopy of cryo-sectioned tissue slices reveals different degrees of nanoparticle tissue penetration, dependent on their surface charge. More importantly, the determination of siRNA-mediated knockdown efficacies of an endogenous target gene, the oncogenic survival factor Survivin, reveals the possibility to accurately assess biological nanoparticle activities in situ, i.e. in living cells in their original environment. Taken together, we establish tumor (xenograft) tissue slices for the accurate and facile ex vivo assessment of

  16. Study of the agroindustrial alterations induced by the irradiated tissue culture in sugar cane, variety NA 56-79

    International Nuclear Information System (INIS)

    Figueiredo Junior, O.

    1991-01-01

    The use of plant tissue culture and the application of gamma radiation as mutation inducing agents, in the sugar cane plant, variety NA 5679, are studied. The variation in the contents of brix, pol, fiber, purity, extraction, phosphorus, nitrogen, reducing sugars as well as the morphological characteristics are analysed. The 'callus' obtained by the tissue culture were irradiated with 20, 40, and 60 Gy doses. The statistical analysis indicated that the method of tissue culture may, eventually, increase the contents of the technological parameters and the dosages of gamma radiation were not efficient for such purpose. (M.A.C.)

  17. Plant cell tissue culture: A potential source of chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Scott, C.D.; Dougall, D.K.

    1987-08-01

    Higher plants produce many industrially important products. Among these are drugs and medicinal chemicals, essential oils and flavors, vegetable oils and fats, fine and specialty chemicals, and even some commodity chemicals. Although, currently, whole-plant extraction is the primary means of harvesting these materials, the advent of plant cell tissue culture could be a much more effective method of producing many types of phytochemicals. The use of immobilized plant cells in an advanced bioreactor configuration with excretion of the product into the reactor medium may represent the most straightforward way of commercializing such techniques for lower-value chemicals. Important research and development opportunities in this area include screening for plant cultures for nonmedical, lower-value chemicals; understanding and controlling plant cell physiology and biochemistry; optimizing effective immobilization methods; developing more efficient bioreactor concepts; and perfecting product extraction and purification techniques. 62 refs., 2 figs.

  18. Isolation of Lysosomes from Mammalian Tissues and Cultured Cells.

    Science.gov (United States)

    Aguado, Carmen; Pérez-Jiménez, Eva; Lahuerta, Marcos; Knecht, Erwin

    2016-01-01

    Lysosomes participate within the cells in the degradation of organelles, macromolecules, and a wide variety of substrates. In any study on specific roles of lysosomes, both under physiological and pathological conditions, it is advisable to include methods that allow their reproducible and reliable isolation. However, purification of lysosomes is a difficult task, particularly in the case of cultured cells. This is mainly because of the heterogeneity of these organelles, along with their low number and high fragility. Also, isolation methods, while disrupting plasma membranes, have to preserve the integrity of lysosomes, as the breakdown of their membranes releases enzymes that could damage all cell organelles, including themselves. The protocols described below have been routinely used in our laboratory for the specific isolation of lysosomes from rat liver, NIH/3T3, and other cultured cells, but can be adapted to other mammalian tissues or cell lines.

  19. Mary Jane Hogue (1883-1962): A pioneer in human brain tissue culture.

    Science.gov (United States)

    Zottoli, Steven J; Seyfarth, Ernst-August

    2018-05-16

    The ability to maintain human brain explants in tissue culture was a critical step in the use of these cells for the study of central nervous system disorders. Ross G. Harrison (1870-1959) was the first to successfully maintain frog medullary tissue in culture in 1907, but it took another 38 years before successful culture of human brain tissue was accomplished. One of the pioneers in this achievement was Mary Jane Hogue (1883-1962). Hogue was born into a Quaker family in 1883 in West Chester, Pennsylvania, and received her undergraduate degree from Goucher College in Baltimore, Maryland. Research with the developmental biologist Theodor Boveri (1862-1915) in Würzburg, Germany, resulted in her Ph.D. (1909). Hogue transitioned from studying protozoa to the culture of human brain tissue in the 1940s and 1950s, when she was one of the first to culture cells from human fetal, infant, and adult brain explants. We review Hogue's pioneering contributions to the study of human brain cells in culture, her putative identification of progenitor neuroblast and/or glioblast cells, and her use of the cultures to study the cytopathogenic effects of poliovirus. We also put Hogue's work in perspective by discussing how other women pioneers in tissue culture influenced Hogue and her research.

  20. Media Compositions for Three-Dimensional Mammalian Tissue Growth under Microgravity Culture Conditions

    Science.gov (United States)

    Goodwin, Thomas J. (Inventor)

    1998-01-01

    Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue.The cells are grown in vitro under microgravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

  1. Media Compositions for Three Dimensional Mammalian Tissue Growth Under Microgravity Culture Conditions

    Science.gov (United States)

    Goodwin, Thomas J. (Inventor)

    1998-01-01

    Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue. The cells are grown in vitro under microgravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

  2. Ex vivo culture of patient tissue & examination of gene delivery.

    LENUS (Irish Health Repository)

    Rajendran, Simon

    2012-01-31

    This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial.

  3. Tissue culture and micropropagation for forest biomass production

    Energy Technology Data Exchange (ETDEWEB)

    Mason, E.; Maine, F.W.

    1984-09-01

    An increase in forest production will be necessary in the future when wood becomes a major renewable source of energy and chemicals along with its traditional role of fibre source. This increase could eventually by achieved be proper selection and breeding of trees. Clonal forestry by vegetative propagation of cuttings is becoming a viable alternative to a seedling-based forestry with many advantages, and cutting could be used to quickly propagate large numbers of clones of control-pollinated seedlings. Most forest trees are propagated sexually and seed orchards were started in the US and Canada in the last 40-50 years for breeding purposes. Forests could ultimately be established with improved seedlings instead of from seed with unknown genetic potential, or by natural regeneration. Micropropagation is the term used to refer to the propagation of plants raised by tissue culture methods rather than from seeds or cuttings. Many clonal plantlets could be regenerated asexually in the laboratory and eventually transplanted to permanent sites. In addition the technology could be developed to produce new variants from somatic cells. Tissue culture is a technique which may be useful for plant propagation where conventional methods are inadequate or unsuitable. However, traditional studies of field planting observed over long periods of time would still be necessary. This document has the object of informing those who may wish to know more about these techniques in relation to practical application, and require a general overview rather than experimental details, which are given in an annotated bilbiography. 274 refs., 2 figs., 1 tab.

  4. Characterization of cytoskeletal and junctional proteins expressed by cells cultured from human arachnoid granulation tissue

    Directory of Open Access Journals (Sweden)

    Mehta Bhavya C

    2005-10-01

    Full Text Available Abstract Background The arachnoid granulations (AGs are projections of the arachnoid membrane into the dural venous sinuses. They function, along with the extracranial lymphatics, to circulate the cerebrospinal fluid (CSF to the systemic venous circulation. Disruption of normal CSF dynamics may result in increased intracranial pressures causing many problems including headaches and visual loss, as in idiopathic intracranial hypertension and hydrocephalus. To study the role of AGs in CSF egress, we have grown cells from human AG tissue in vitro and have characterized their expression of those cytoskeletal and junctional proteins that may function in the regulation of CSF outflow. Methods Human AG tissue was obtained at autopsy, and explanted to cell culture dishes coated with fibronectin. Typically, cells migrated from the explanted tissue after 7–10 days in vitro. Second or third passage cells were seeded onto fibronectin-coated coverslips at confluent densities and grown to confluency for 7–10 days. Arachnoidal cells were tested using immunocytochemical methods for the expression of several common cytoskeletal and junctional proteins. Second and third passage cultures were also labeled with the common endothelial markers CD-31 or VE-cadherin (CD144 and their expression was quantified using flow cytometry analysis. Results Confluent cultures of arachnoidal cells expressed the intermediate filament protein vimentin. Cytokeratin intermediate filaments were expressed variably in a subpopulation of cells. The cultures also expressed the junctional proteins connexin43, desmoplakin 1 and 2, E-cadherin, and zonula occludens-1. Flow cytometry analysis indicated that second and third passage cultures failed to express the endothelial cell markers CD31 or VE-cadherin in significant quantities, thereby showing that these cultures did not consist of endothelial cells from the venous sinus wall. Conclusion To our knowledge, this is the first report of

  5. Application of Tissue Culture and Transformation Techniques in Model Species Brachypodium distachyon.

    Science.gov (United States)

    Sogutmaz Ozdemir, Bahar; Budak, Hikmet

    2018-01-01

    Brachypodium distachyon has recently emerged as a model plant species for the grass family (Poaceae) that includes major cereal crops and forage grasses. One of the important traits of a model species is its capacity to be transformed and ease of growing both in tissue culture and in greenhouse conditions. Hence, plant transformation technology is crucial for improvements in agricultural studies, both for the study of new genes and in the production of new transgenic plant species. In this chapter, we review an efficient tissue culture and two different transformation systems for Brachypodium using most commonly preferred gene transfer techniques in plant species, microprojectile bombardment method (biolistics) and Agrobacterium-mediated transformation.In plant transformation studies, frequently used explant materials are immature embryos due to their higher transformation efficiencies and regeneration capacity. However, mature embryos are available throughout the year in contrast to immature embryos. We explain a tissue culture protocol for Brachypodium using mature embryos with the selected inbred lines from our collection. Embryogenic calluses obtained from mature embryos are used to transform Brachypodium with both plant transformation techniques that are revised according to previously studied protocols applied in the grasses, such as applying vacuum infiltration, different wounding effects, modification in inoculation and cocultivation steps or optimization of bombardment parameters.

  6. Tissue culture of ornamental cacti

    Directory of Open Access Journals (Sweden)

    Eugenio Pérez-Molphe-Balch

    2015-12-01

    Full Text Available Cacti species are plants that are well adapted to growing in arid and semiarid regions where the main problem is water availability. Cacti have developed a series of adaptations to cope with water scarcity, such as reduced leaf surface via morphological modifications including spines, cereous cuticles, extended root systems and stem tissue modifications to increase water storage, and crassulacean acid metabolism to reduce transpiration and water loss. Furthermore, seeds of these plants very often exhibit dormancy, a phenomenon that helps to prevent germination when the availability of water is reduced. In general, cactus species exhibit a low growth rate that makes their rapid propagation difficult. Cacti are much appreciated as ornamental plants due to their great variety and diversity of forms and their beautiful short-life flowers; however, due to difficulties in propagating them rapidly to meet market demand, they are very often over-collected in their natural habitats, which leads to numerous species being threatened, endangered or becoming extinct. Therefore, plant tissue culture techniques may facilitate their propagation over a shorter time period than conventional techniques used for commercial purposes; or may help to recover populations of endangered or threatened species for their re-introduction in the wild; or may also be of value to the preservation and conservation of the genetic resources of this important family. Herein we present the state-of-the-art of tissue culture techniques used for ornamental cacti and selected suggestions for solving a number of the problems faced by members of the Cactaceae family.

  7. 21 CFR 864.2240 - Cell and tissue culture supplies and equipment.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cell and tissue culture supplies and equipment. 864.2240 Section 864.2240 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products...

  8. Biomaterials in co-culture systems: towards optimizing tissue integration and cell signaling within scaffolds.

    Science.gov (United States)

    Battiston, Kyle G; Cheung, Jane W C; Jain, Devika; Santerre, J Paul

    2014-05-01

    Most natural tissues consist of multi-cellular systems made up of two or more cell types. However, some of these tissues may not regenerate themselves following tissue injury or disease without some form of intervention, such as from the use of tissue engineered constructs. Recent studies have increasingly used co-cultures in tissue engineering applications as these systems better model the natural tissues, both physically and biologically. This review aims to identify the challenges of using co-culture systems and to highlight different approaches with respect to the use of biomaterials in the use of such systems. The application of co-culture systems to stimulate a desired biological response and examples of studies within particular tissue engineering disciplines are summarized. A description of different analytical co-culture systems is also discussed and the role of biomaterials in the future of co-culture research are elaborated on. Understanding the complex cell-cell and cell-biomaterial interactions involved in co-culture systems will ultimately lead the field towards biomaterial concepts and designs with specific biochemical, electrical, and mechanical characteristics that are tailored towards the needs of distinct co-culture systems. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Organotypic culture of human bone marrow adipose tissue.

    Science.gov (United States)

    Uchihashi, Kazuyoshi; Aoki, Shigehisa; Shigematsu, Masamori; Kamochi, Noriyuki; Sonoda, Emiko; Soejima, Hidenobu; Fukudome, Kenji; Sugihara, Hajime; Hotokebuchi, Takao; Toda, Shuji

    2010-04-01

    The precise role of bone marrow adipose tissue (BMAT) in the marrow remains unknown. The purpose of the present study was therefore to describe a novel method for studying BMAT using 3-D collagen gel culture of BMAT fragments, immunohistochemistry, ELISA and real-time reverse transcription-polymerase chain reaction. Mature adipocytes and CD45+ leukocytes were retained for >3 weeks. Bone marrow stromal cells (BMSC) including a small number of lipid-laden preadipocytes and CD44+/CD105+ mesenchymal stem cell (MSC)-like cells, developed from BMAT. Dexamethasone (10 micromol/L), but not insulin (20 mU/mL), significantly increased the number of preadipocytes. Dexamethasone and insulin also promoted leptin production and gene expression in BMAT. Adiponectin production by BMAT was BMAT, in which adiponectin protein secretion is normally very low, and that BMAT may exhibit a different phenotype from that of the visceral and subcutaneous adipose tissues. BMAT-osteoblast interactions were also examined, and it was found that osteoblasts inhibited the development of BMSC and reduced leptin production, while BMAT inhibited the growth and differentiation of osteoblasts. The present novel method proved to be useful for the study of BMAT biology.

  10. Bridging the gap between cell culture and live tissue

    Directory of Open Access Journals (Sweden)

    Stefan Przyborski

    2017-11-01

    Full Text Available Traditional in vitro two-dimensional (2-D culture systems only partly imitate the physiological and biochemical features of cells in their original tissue. In vivo, in organs and tissues, cells are surrounded by a three-dimensional (3-D organization of supporting matrix and neighbouring cells, and a gradient of chemical and mechanical signals. Furthermore, the presence of blood flow and mechanical movement provides a dynamic environment (Jong et al., 2011. In contrast, traditional in vitro culture, carried out on 2-D plastic or glass substrates, typically provides a static environment, which, however is the base of the present understanding of many biological processes, tissue homeostasis as well as disease. It is clear that this is not an exact representation of what is happening in vivo and the microenvironment provided by in vitro cell culture models are significantly different and can cause deviations in cell response and behaviour from those distinctive of in vivo tissues. In order to translate the present basic knowledge in cell control, cell repair and regeneration from the laboratory bench to the clinical application, we need a better understanding of the cell and tissue interactions. This implies a detailed comprehension of the natural tissue environment, with its organization and local signals, in order to more closely mimic what happens in vivo, developing more physiological models for efficient in vitro systems. In particular, it is imperative to understand the role of the environmental cues which can be mainly divided into those of a chemical and mechanical nature.

  11. The role of activated charcoal in plant tissue culture.

    Science.gov (United States)

    Thomas, T Dennis

    2008-01-01

    Activated charcoal has a very fine network of pores with large inner surface area on which many substances can be adsorbed. Activated charcoal is often used in tissue culture to improve cell growth and development. It plays a critical role in micropropagation, orchid seed germination, somatic embryogenesis, anther culture, synthetic seed production, protoplast culture, rooting, stem elongation, bulb formation etc. The promotary effects of AC on morphogenesis may be mainly due to its irreversible adsorption of inhibitory compounds in the culture medium and substancially decreasing the toxic metabolites, phenolic exudation and brown exudate accumulation. In addition to this activated charcoal is involved in a number of stimulatory and inhibitory activities including the release of substances naturally present in AC which promote growth, alteration and darkening of culture media, and adsorption of vitamins, metal ions and plant growth regulators, including abscisic acid and gaseous ethylene. The effect of AC on growth regulator uptake is still unclear but some workers believe that AC may gradually release certain adsorbed products, such as nutrients and growth regulators which become available to plants. This review focuses on the various roles of activated charcoal in plant tissue culture and the recent developments in this area.

  12. [Effects of endophytic fungi from Dendrobium officinale on host growth and components metabolism of tissue culture seedlings].

    Science.gov (United States)

    Zhu, Bo; Liu, Jing-Jing; Si, Jin-Ping; Qin, Lu-Ping; Han, Ting; Zhao, Li; Wu, Ling-Shang

    2016-05-01

    The paper aims to study the effects of endophytic fungi from D. officinale cultivated on living trees on growth and components metabolism of tissue culture seedlings. Morphological characteristics and agronomic characters of tissue culture seedlings infected and uninfected by endophytic fungus were observed and measured. Polysaccharides and alcohol-soluble extracts contents were determined by phenol-sulfuric acid method and hot-dipmethod, respectively. Monosacchride composition of polysaccharides and alcohol-soluble extracts components were analyzed by pre-column derivatives HPLC and HPLC method, respectively. It showed that effects of turning to purple of stem nodes could be changed by endophytic fungus. Besides, the endophytic fungus could affect the contents and constitutions of polysaccharides and alcohol-soluble extracts. The strains tested, expect DO34, could promote growth and polysaccharides content of tissue culture seedlings. The strains tested, expect DO12, could promote the accumulation of mannose. Furthermore, DO18, DO19 and DO120 could increase alcohol-soluble extracts. On the basis, four superior strains were selected for mechanism research between endophytic fungus and their hosts and microbiology engineering. Copyright© by the Chinese Pharmaceutical Association.

  13. Production of immunoglobulins in gingival tissue explant cultures from juvenile periodontitis patients

    International Nuclear Information System (INIS)

    Hall, E.R.; Falkler, W.A. Jr.; Suzuki, J.B.

    1990-01-01

    B lymphocytes and plasma cells are histologically observed in granulomatous periodontal tissues of juvenile periodontitis (JP) patients. Local immune processes may participate in protective or immunopathologic roles in the pathogenesis of this disease. An in vitro explant culture system was utilized to demonstrate the production of immunoglobulins by diseased JP tissues. Immunodiffusion studies using goat anti-human gamma, alpha, or mu chain serum revealed IgG to be the major immunoglobulin present in 92% of the day 1 supernatant fluids (SF) of the 47 JP gingival tissue explant cultures. IgA was present in 15% of the SF; however, no IgM was detected. Staph Protein A isolated 14C-labeled IgG from the SF, when allowed to react with goat anti-human gamma chain serum, formed lines of precipitation. Positive autoradiographs confirmed the biosynthesis of IgG by the explant cultures. The in vitro gingival tissue explant culture system described provides a useful model for the study of localized immunoglobulins produced by diseased tissues of JP patients

  14. Production of immunoglobulins in gingival tissue explant cultures from juvenile periodontitis patients

    Energy Technology Data Exchange (ETDEWEB)

    Hall, E.R.; Falkler, W.A. Jr.; Suzuki, J.B. (Univ. of Maryland Dental School, Baltimore (USA))

    1990-10-01

    B lymphocytes and plasma cells are histologically observed in granulomatous periodontal tissues of juvenile periodontitis (JP) patients. Local immune processes may participate in protective or immunopathologic roles in the pathogenesis of this disease. An in vitro explant culture system was utilized to demonstrate the production of immunoglobulins by diseased JP tissues. Immunodiffusion studies using goat anti-human gamma, alpha, or mu chain serum revealed IgG to be the major immunoglobulin present in 92% of the day 1 supernatant fluids (SF) of the 47 JP gingival tissue explant cultures. IgA was present in 15% of the SF; however, no IgM was detected. Staph Protein A isolated 14C-labeled IgG from the SF, when allowed to react with goat anti-human gamma chain serum, formed lines of precipitation. Positive autoradiographs confirmed the biosynthesis of IgG by the explant cultures. The in vitro gingival tissue explant culture system described provides a useful model for the study of localized immunoglobulins produced by diseased tissues of JP patients.

  15. Culture of equine fibroblast-like synoviocytes on synthetic tissue scaffolds towards meniscal tissue engineering: a preliminary cell-seeding study

    Directory of Open Access Journals (Sweden)

    Jennifer J. Warnock

    2014-04-01

    Full Text Available Introduction. Tissue engineering is a new methodology for addressing meniscal injury or loss. Synovium may be an ideal source of cells for in vitro meniscal fibrocartilage formation, however, favorable in vitro culture conditions for synovium must be established in order to achieve this goal. The objective of this study was to determine cellularity, cell distribution, and extracellular matrix (ECM formation of equine fibroblast-like synoviocytes (FLS cultured on synthetic scaffolds, for potential application in synovium-based meniscal tissue engineering. Scaffolds included open-cell poly-L-lactic acid (OPLA sponges and polyglycolic acid (PGA scaffolds cultured in static and dynamic culture conditions, and PGA scaffolds coated in poly-L-lactic (PLLA in dynamic culture conditions.Materials and Methods. Equine FLS were seeded on OPLA and PGA scaffolds, and cultured in a static environment or in a rotating bioreactor for 12 days. Equine FLS were also seeded on PGA scaffolds coated in 2% or 4% PLLA and cultured in a rotating bioreactor for 14 and 21 days. Three scaffolds from each group were fixed, sectioned and stained with Masson’s Trichrome, Safranin-O, and Hematoxylin and Eosin, and cell numbers and distribution were analyzed using computer image analysis. Three PGA and OPLA scaffolds from each culture condition were also analyzed for extracellular matrix (ECM production via dimethylmethylene blue (sulfated glycosaminoglycan assay and hydroxyproline (collagen assay. PLLA coated PGA scaffolds were analyzed using double stranded DNA quantification as areflection of cellularity and confocal laser microscopy in a fluorescent cell viability assay.Results. The highest cellularity occurred in PGA constructs cultured in a rotating bioreactor, which also had a mean sulfated glycosaminoglycan content of 22.3 µg per scaffold. PGA constructs cultured in static conditions had the lowest cellularity. Cells had difficulty adhering to OPLA and the PLLA

  16. A hanging drop culture method to study terminal erythroid differentiation.

    Science.gov (United States)

    Gutiérrez, Laura; Lindeboom, Fokke; Ferreira, Rita; Drissen, Roy; Grosveld, Frank; Whyatt, David; Philipsen, Sjaak

    2005-10-01

    To design a culture method allowing the quantitative and qualitative analysis of terminal erythroid differentiation. Primary erythroid progenitors derived either from mouse tissues or from human umbilical cord blood were differentiated using hanging drop cultures and compared to methylcellulose cultures. Cultured cells were analyzed by FACS to assess differentiation. We describe a practical culture method by adapting the previously described hanging drop culture system to conditions allowing terminal differentiation of primary erythroid progenitors. Using minimal volumes of media and small numbers of cells, we obtained quantitative terminal erythroid differentiation within two days of culture in the case of murine cells and 4 days in the case of human cells. The established methods for ex vivo culture of primary erythroid progenitors, such as methylcellulose-based burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) assays, allow the detection of committed erythroid progenitors but are of limited value to study terminal erythroid differentiation. We show that the application of hanging drop cultures is a practical alternative that, in combination with clonogenic assays, enables a comprehensive assessment of the behavior of primary erythroid cells ex vivo in the context of genetic and drug-induced perturbations.

  17. Low technology tissue culture materials for initiation and ...

    African Journals Online (AJOL)

    Low technology tissue culture materials for initiation and multiplication of banana plants. ... African Crop Science Journal ... locally available macronutrients, micronutrients, sugar, equipment and facility reduced the cost of consumable material

  18. Human meniscal proteoglycan metabolism in long-term tissue culture

    NARCIS (Netherlands)

    Verbruggen, G.; Verdonk, R.; Veys, E. M.; van Daele, P.; de Smet, P.; van den Abbeele, K.; Claus, B.; Baeten, D.

    1996-01-01

    For the purpose of human meniscal allografting, menisci have been maintained viable in in vitro culture. The influence of long-term tissue culture on the extracellular matrix metabolism of the meniscus has been studied. Fetal calf serum (FCS) was used as a supplement for the growth factors necessary

  19. Culture Environment-Induced Pluripotency of SACK-Expanded Tissue Stem Cells

    Directory of Open Access Journals (Sweden)

    Jean-François Paré

    2011-01-01

    Full Text Available Previous efforts to improve the efficiency of cellular reprogramming for the generation of induced pluripotent stem cells (iPSCs have focused mainly on transcription factors and small molecule combinations. Here, we report the results of our focus instead on the phenotype of the cells targeted for reprogramming. We find that adult mouse pancreatic tissue stem cells derived by the method of suppression of asymmetric cell kinetics (SACK acquire increased potency simply by culture under conditions for the production and maintenance of pluripotent stem cells. Moreover, supplementation with the SACK agent xanthine, which promotes symmetric self-renewal, significantly increases the efficiency and degree of acquisition of pluripotency properties. In transplantation analyses, clonal reprogrammed pancreatic stem cells produce slow-growing tumors with tissue derivative of all three embryonic germ layers. This acquisition of pluripotency, without transduction with exogenous transcription factors, supports the concept that tissue stem cells are predisposed to cellular reprogramming, particularly when symmetrically self-renewing.

  20. Renal cell carcinoma primary cultures maintain genomic and phenotypic profile of parental tumor tissues

    International Nuclear Information System (INIS)

    Cifola, Ingrid; Magni, Fulvio; Signorini, Stefano; Battaglia, Cristina; Perego, Roberto A; Bianchi, Cristina; Mangano, Eleonora; Bombelli, Silvia; Frascati, Fabio; Fasoli, Ester; Ferrero, Stefano; Di Stefano, Vitalba; Zipeto, Maria A

    2011-01-01

    Clear cell renal cell carcinoma (ccRCC) is characterized by recurrent copy number alterations (CNAs) and loss of heterozygosity (LOH), which may have potential diagnostic and prognostic applications. Here, we explored whether ccRCC primary cultures, established from surgical tumor specimens, maintain the DNA profile of parental tumor tissues allowing a more confident CNAs and LOH discrimination with respect to the original tissues. We established a collection of 9 phenotypically well-characterized ccRCC primary cell cultures. Using the Affymetrix SNP array technology, we performed the genome-wide copy number (CN) profiling of both cultures and corresponding tumor tissues. Global concordance for each culture/tissue pair was assayed evaluating the correlations between whole-genome CN profiles and SNP allelic calls. CN analysis was performed using the two CNAG v3.0 and Partek software, and comparing results returned by two different algorithms (Hidden Markov Model and Genomic Segmentation). A very good overlap between the CNAs of each culture and corresponding tissue was observed. The finding, reinforced by high whole-genome CN correlations and SNP call concordances, provided evidence that each culture was derived from its corresponding tissue and maintained the genomic alterations of parental tumor. In addition, primary culture DNA profile remained stable for at least 3 weeks, till to third passage. These cultures showed a greater cell homogeneity and enrichment in tumor component than original tissues, thus enabling a better discrimination of CNAs and LOH. Especially for hemizygous deletions, primary cultures presented more evident CN losses, typically accompanied by LOH; differently, in original tissues the intensity of these deletions was weaken by normal cell contamination and LOH calls were missed. ccRCC primary cultures are a reliable in vitro model, well-reproducing original tumor genetics and phenotype, potentially useful for future functional approaches

  1. Substituted Indoleacetic Acids Tested in Tissue Cultures

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1978-01-01

    Monochloro substituted IAA inhibited shoot induction in tobacco tissue cultures about as much as IAA. Dichloro substituted IAA inhibited shoot formation less. Other substituted IAA except 5-fluoro- and 5-bromoindole-3-acetic acid were less active than IAA. Callus growth was quite variable...

  2. Functional enhancement of chitosan and nanoparticles in cell culture, tissue engineering, and pharmaceutical applications

    Directory of Open Access Journals (Sweden)

    Wenjuan eGao

    2012-08-01

    Full Text Available Abstract: As a biomaterial, chitosan has been widely used in tissue engineering, wound healing, drug delivery, and other biomedical applications. It can be formulated in a variety of forms, such as powder, film, sphere, gel and fiber. These features make chitosan an almost ideal biomaterial in cell culture applications, and cell cultures arguably constitute the most practical way to evaluate biocompatibility and biotoxicity. The advantages of cell cultures are that they can be performed under totally controlled environments, allow high throughput functional screening, and are less costly, as compared to other assessment methods. Chitosan can also be modified into multilayer composite by combining with other polymers and moieties to alter the properties of chitosan for particular biomedical applications. This review briefly depicts and discusses applications of chitosan and nanoparticles in cell culture, in particular, the effects of chitosan and nanoparticles on cell adhesion, cell survival, and the underlying molecular mechanisms: both stimulatory and inhibitory influences are discussed. Our aim is to update the current status of how nanoparticles can be utilized to modify the properties of chitosan to advance the art of tissue engineering by using cell cultures.

  3. 21 CFR 864.2220 - Synthetic cell and tissue culture media and components.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Synthetic cell and tissue culture media and components. 864.2220 Section 864.2220 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture...

  4. Diagnostic utility of melanin production by fungi: Study on tissue sections and culture smears with Masson-Fontana stain

    Directory of Open Access Journals (Sweden)

    Challa Sundaram

    2014-01-01

    Full Text Available Background: Dematiaceous fungi appear brown in tissue section due to melanin in their cell walls. When the brown color is not seen on routine H and E and culture is not available, differentiation of dematiaceous fungi from other fungi is difficult on morphology alone. Aims and Objective: To study if melanin production by dematiaceous fungi can help differentiate them from other types of fungi. Materials and Methods: Fifty tissue sections of various fungal infections and 13 smears from cultures of different species of fungi were stained with Masson Fontana stain to assess melanin production. The tissue sections included biopsies from 26 culture-proven fungi and 24 biopsies of filamentous fungi diagnosed on morphology alone with no culture confirmation. Results: All culture-proven dematiaceous fungi and Zygomycetes showed strong positivity in sections and culture smears. Aspergillus sp showed variable positivity and intensity. Cryptococcus neoformans showed strong positivity in tissue sections and culture smears. Tissue sections of septate filamentous fungi (9/15, Zygomycetes (4/5, and fungi with both hyphal and yeast morphology (4/4 showed positivity for melanin. The septate filamentous fungi negative for melanin were from biopsy samples of fungal sinusitis including both allergic and invasive fungal sinusitis and colonizing fungal balls. Conclusion: Melanin is produced by both dematiaceous and non-dematiaceous fungi. Masson-Fontana stain cannot reliably differentiate dematiaceous fungi from other filamentous fungi like Aspergillus sp; however, absence of melanin in the hyphae may be used to rule out dematiaceous fungi from other filamentous fungi. In the differential diagnosis of yeast fungi, Cryptococcus sp can be differentiated from Candida sp by Masson-Fontana stain in tissue sections.

  5. Growing tissues in real and simulated microgravity: new methods for tissue engineering.

    Science.gov (United States)

    Grimm, Daniela; Wehland, Markus; Pietsch, Jessica; Aleshcheva, Ganna; Wise, Petra; van Loon, Jack; Ulbrich, Claudia; Magnusson, Nils E; Infanger, Manfred; Bauer, Johann

    2014-12-01

    Tissue engineering in simulated (s-) and real microgravity (r-μg) is currently a topic in Space medicine contributing to biomedical sciences and their applications on Earth. The principal aim of this review is to highlight the advances and accomplishments in the field of tissue engineering that could be achieved by culturing cells in Space or by devices created to simulate microgravity on Earth. Understanding the biology of three-dimensional (3D) multicellular structures is very important for a more complete appreciation of in vivo tissue function and advancing in vitro tissue engineering efforts. Various cells exposed to r-μg in Space or to s-μg created by a random positioning machine, a 2D-clinostat, or a rotating wall vessel bioreactor grew in the form of 3D tissues. Hence, these methods represent a new strategy for tissue engineering of a variety of tissues, such as regenerated cartilage, artificial vessel constructs, and other organ tissues as well as multicellular cancer spheroids. These aggregates are used to study molecular mechanisms involved in angiogenesis, cancer development, and biology and for pharmacological testing of, for example, chemotherapeutic drugs or inhibitors of neoangiogenesis. Moreover, they are useful for studying multicellular responses in toxicology and radiation biology, or for performing coculture experiments. The future will show whether these tissue-engineered constructs can be used for medical transplantations. Unveiling the mechanisms of microgravity-dependent molecular and cellular changes is an up-to-date requirement for improving Space medicine and developing new treatment strategies that can be translated to in vivo models while reducing the use of laboratory animals.

  6. Dynamic culture induces a cell type-dependent response impacting on the thickness of engineered connective tissues.

    Science.gov (United States)

    Fortier, Guillaume Marceau; Gauvin, Robert; Proulx, Maryse; Vallée, Maud; Fradette, Julie

    2013-04-01

    Mesenchymal cells are central to connective tissue homeostasis and are widely used for tissue-engineering applications. Dermal fibroblasts and adipose-derived stromal cells (ASCs) allow successful tissue reconstruction by the self-assembly approach of tissue engineering. This method leads to the production of multilayered tissues, devoid of exogenous biomaterials, that can be used as stromal compartments for skin or vesical reconstruction. These tissues are formed by combining cell sheets, generated through cell stimulation with ascorbic acid, which favours the cell-derived production/organization of matrix components. Since media motion can impact on cell behaviour, we investigated the effect of dynamic culture on mesenchymal cells during tissue reconstruction, using the self-assembly method. Tissues produced using ASCs in the presence of a wave-like movement were nearly twice thicker than under standard conditions, while no difference was observed for tissues produced from dermal fibroblasts. The increased matrix deposition was not correlated with an increased proliferation of ASCs, or by higher transcript levels of fibronectin or collagens I and III. A 30% increase of type V collagen mRNA was observed. Interestingly, tissues engineered from dermal fibroblasts featured a four-fold higher level of MMP-1 transcripts under dynamic conditions. Mechanical properties were similar for tissues reconstructed using dynamic or static conditions. Finally, cell sheets produced using ASCs under dynamic conditions could readily be manipulated, resulting in a 2 week reduction of the production time (from 5 to 3 weeks). Our results describe a distinctive property of ASCs' response to media motion, indicating that their culture under dynamic conditions leads to optimized tissue engineering. Copyright © 2011 John Wiley & Sons, Ltd.

  7. Solid tissue culture for cytogenetic analysis: a collaborative survey for the Association of Clinical Cytogeneticists.

    Science.gov (United States)

    Rodgers, C S; Creasy, M R; Fitchett, M; Maliszewska, C T; Pratt, N R; Waters, J J

    1996-01-01

    AIMS: To survey the diagnostic service provided by UK laboratories for the culture of solid tissue samples (excluding tumours) and in particular to examine the variation in culture success rates and the problems of maternal cell overgrowth. METHODS: Twenty seven laboratories took part in a collaborative survey during 1992. Each laboratory submitted data on up to a maximum of 60 consecutive specimens (n = 1361) over a six month period. RESULTS: Skin specimens, the largest category received (n = 520), were the most problematic (51% success rate). Culture success rates were significantly lower (43%) when skin specimens (n = 140) were transported dry to the laboratory. Success rates for skin specimens also varied, depending on the origin of the specimen, from 18% for intra-uterine deaths (IUD) (n = 94) to 85% for neonatal deaths (n = 33) and 83% for live patients (n = 54). Culture of selected extra-fetal tissues from IUD, stillbirths and following elective termination of pregnancy (TOP) gave comparable success rates to those achieved for skin samples from neonatal deaths and live births. Skewed sex ratios, female > male, were identified for products of conception (POC) (n = 298) and placental biopsy specimens (n = 97). CONCLUSIONS: By appropriate selection, transport and processing of tissues, and in particular by avoiding relying solely on skin samples from IUD, stillbirths and TOP, an increase in culture success rates for solid tissue samples submitted for cytogenetic analysis could be achieved. The high risk of maternal cell contamination from POC and placental biopsy specimens was also identified in this survey. PMID:8881913

  8. Advanced cell culture technology for generation of in vivo-like tissue models

    Directory of Open Access Journals (Sweden)

    Stefan Przyborski

    2017-06-01

    Full Text Available Human tissues are mostly composed of different cell types, that are often highly organised in relation to each other. Often cells are arranged in distinct layers that enable signalling and cell-to-cell interactions. Here we describe the application of scaffold-based technology, that can be used to create advanced organotypic 3D models of various tissue types that more closely resemble in vivo-like conditions (Knight et al., 2011. The scaffold comprises a highly porous polystyrene material, engineered into a 200 micron thick membrane that is presented in various ways including multi-welled plates and well inserts, for use with conventional culture plasticware and medium perfusion systems. This technology has been applied to generate numerous unique types of co-culture model. For example: 1 a full thickness human skin construct comprising dermal fibroblasts and keratinocytes, raised to the air-liquid interface to induce cornification of the upper layers (Fig.1 (Hill et al., 2015; 2 a neuron-glial co-culture to enable the study of neurite outgrowth interacting with astroglial cells to model and investigate the glial scar found in spinal cord injury (Clarke et al., 2016; 3 formation of a sub-mucosa consisting of a polarised simple epithelium, layer of ECM proteins simulating the basement membrane, and underlying stromal tissues (e.g. intestinal mucosa. These organotypic models demonstrate the versatility of scaffold membranes and the creation of advanced in vivo-like tissue models. Creating a layered arrangement more closely simulates the true anatomy and organisation of cells within many tissue types. The addition of different cell types in a temporal and spatial fashion can be used to study inter-cellular relationships and create more physiologically relevant in vivo-like cell-based assays. Methods that are relatively straightforward to use and that recreate the organised structure of real tissues will become valuable research tools for use in

  9. Beauveria bassiana (Balsamo) Vuillemin as an endophyte in tissue culture banana (Musa spp.).

    Science.gov (United States)

    Akello, Juliet; Dubois, Thomas; Gold, Clifford S; Coyne, Daniel; Nakavuma, Jessica; Paparu, Pamela

    2007-09-01

    Beauveria bassiana is considered a virulent pathogen against the banana weevil Cosmopolites sordidus. However, current field application techniques for effective control against this pest remain a limitation and an alternative method for effective field application needs to be investigated. Three screenhouse experiments were conducted to determine the ability of B. bassiana to form an endophytic relationship with tissue culture banana (Musa spp.) plants and to evaluate the plants for possible harmful effects resulting from this relationship. Three Ugandan strains of B. bassiana (G41, S204 and WA) were applied by dipping the roots and rhizome in a conidial suspension, by injecting a conidial suspension into the plant rhizome and by growing the plants in sterile soil mixed with B. bassiana-colonized rice substrate. Four weeks after inoculation, plant growth parameters were determined and plant tissue colonization assessed through re-isolation of B. bassiana. All B. bassiana strains were able to colonize banana plant roots, rhizomes and pseudostem bases. Dipping plants in a conidial suspension achieved the highest colonization with no negative effect on plant growth or survival. Beauveria bassiana strain G41 was the best colonizer (up to 68%, 79% and 41% in roots, rhizome and pseudostem base, respectively) when plants were dipped. This study demonstrated that, depending on strain and inoculation method, B. bassiana can form an endophytic relationship with tissue culture banana plants, causing no harmful effects and might provide an alternative method for biological control of C. sordidus.

  10. Rotating three-dimensional dynamic culture of adult human bone marrow-derived cells for tissue engineering of hyaline cartilage.

    Science.gov (United States)

    Sakai, Shinsuke; Mishima, Hajime; Ishii, Tomoo; Akaogi, Hiroshi; Yoshioka, Tomokazu; Ohyabu, Yoshimi; Chang, Fei; Ochiai, Naoyuki; Uemura, Toshimasa

    2009-04-01

    The method of constructing cartilage tissue from bone marrow-derived cells in vitro is considered a valuable technique for hyaline cartilage regenerative medicine. Using a rotating wall vessel (RWV) bioreactor developed in a NASA space experiment, we attempted to efficiently construct hyaline cartilage tissue from human bone marrow-derived cells without using a scaffold. Bone marrow aspirates were obtained from the iliac crest of nine patients during orthopedic operation. After their proliferation in monolayer culture, the adherent cells were cultured in the RWV bioreactor with chondrogenic medium for 2 weeks. Cells from the same source were cultured in pellet culture as controls. Histological and immunohistological evaluations (collagen type I and II) and quantification of glycosaminoglycan were performed on formed tissues and compared. The engineered constructs obtained using the RWV bioreactor showed strong features of hyaline cartilage in terms of their morphology as determined by histological and immunohistological evaluations. The glycosaminoglycan contents per microg DNA of the tissues were 10.01 +/- 3.49 microg/microg DNA in the case of the RWV bioreactor and 6.27 +/- 3.41 microg/microg DNA in the case of the pellet culture, and their difference was significant. The RWV bioreactor could provide an excellent environment for three-dimensional cartilage tissue architecture that can promote the chondrogenic differentiation of adult human bone marrow-derived cells.

  11. Development of human nervous tissue upon differentiation of embryonic stem cells in three-dimensional culture.

    Science.gov (United States)

    Preynat-Seauve, Olivier; Suter, David M; Tirefort, Diderik; Turchi, Laurent; Virolle, Thierry; Chneiweiss, Herve; Foti, Michelangelo; Lobrinus, Johannes-Alexander; Stoppini, Luc; Feki, Anis; Dubois-Dauphin, Michel; Krause, Karl Heinz

    2009-03-01

    Researches on neural differentiation using embryonic stem cells (ESC) require analysis of neurogenesis in conditions mimicking physiological cellular interactions as closely as possible. In this study, we report an air-liquid interface-based culture of human ESC. This culture system allows three-dimensional cell expansion and neural differentiation in the absence of added growth factors. Over a 3-month period, a macroscopically visible, compact tissue developed. Histological coloration revealed a dense neural-like neural tissue including immature tubular structures. Electron microscopy, immunochemistry, and electrophysiological recordings demonstrated a dense network of neurons, astrocytes, and oligodendrocytes able to propagate signals. Within this tissue, tubular structures were niches of cells resembling germinal layers of human fetal brain. Indeed, the tissue contained abundant proliferating cells expressing markers of neural progenitors. Finally, the capacity to generate neural tissues on air-liquid interface differed for different ESC lines, confirming variations of their neurogenic potential. In conclusion, this study demonstrates in vitro engineering of a human neural-like tissue with an organization that bears resemblance to early developing brain. As opposed to previously described methods, this differentiation (a) allows three-dimensional organization, (b) yields dense interconnected neural tissue with structurally and functionally distinct areas, and (c) is spontaneously guided by endogenous developmental cues.

  12. Whole genome characterization of non-tissue culture adapted HRSV strains in severely infected children

    Directory of Open Access Journals (Sweden)

    Kumaria Rajni

    2011-07-01

    Full Text Available Abstract Background Human respiratory syncytial virus (HRSV is the most important virus causing lower respiratory infection in young children. The complete genetic characterization of RSV clinical strains is a prerequisite for understanding HRSV infection in the clinical context. Current information about the genetic structure of the HRSV genome has largely been obtained using tissue culture adapted viruses. During tissue culture adaptation genetic changes can be introduced into the virus genome, which may obscure subtle variations in the genetic structure of different RSV strains. Methods In this study we describe a novel Sanger sequencing strategy which allowed the complete genetic characterisation of 14 clinical HRSV strains. The viruses were sequenced directly in the nasal washes of severely hospitalized children, and without prior passage of the viruses in tissue culture. Results The analysis of nucleotide sequences suggested that vRNA length is a variable factor among primary strains, while the phylogenetic analysis suggests selective pressure for change. The G gene showed the greatest sequence variation (2-6.4%, while small hydrophobic protein and matrix genes were completely conserved across all clinical strains studied. A number of sequence changes in the F, L, M2-1 and M2-2 genes were observed that have not been described in laboratory isolates. The gene junction regions showed more sequence variability, and in particular the intergenic regions showed a highest level of sequence variation. Although the clinical strains grew slower than the HRSVA2 virus isolate in tissue culture, the HRSVA2 isolate and clinical strains formed similar virus structures such as virus filaments and inclusion bodies in infected cells; supporting the clinical relevance of these virus structures. Conclusion This is the first report to describe the complete genetic characterization of HRSV clinical strains that have been sequenced directly from clinical

  13. Lipid-mediated glial cell line-derived neurotrophic factor gene transfer to cultured porcine ventral mesencephalic tissue

    DEFF Research Database (Denmark)

    Bauer, Matthias; Meyer, Morten; Brevig, Thomas

    2002-01-01

    Transplantation of dopaminergic ventral mesencephalic (VM) tissue into the basal ganglia of patients with Parkinson's disease (PD) shows at best moderate symptomatic relief in some of the treated cases. Experimental animal studies and clinical trials with allogenic and xenogenic pig-derived VM...... tissue grafts to PD patients indicate that one reason for the poor outcome of neural transplantation is the low survival and differentiation of grafted dopaminergic neurons. To improve dopaminergic cell survival through a gene-therapeutic approach we have established and report here results of lipid-mediated...... numbers of tyrosine hydroxylase-positive neurons in the cultured VM tissue. We conclude that lipid-mediated gene transfer employed on embryonic pig VM explant cultures is a safe and effective method to improve survival of dopaminergic neurons and may become a valuable tool to improve allo...

  14. Renal cell carcinoma primary cultures maintain genomic and phenotypic profile of parental tumor tissues.

    Science.gov (United States)

    Cifola, Ingrid; Bianchi, Cristina; Mangano, Eleonora; Bombelli, Silvia; Frascati, Fabio; Fasoli, Ester; Ferrero, Stefano; Di Stefano, Vitalba; Zipeto, Maria A; Magni, Fulvio; Signorini, Stefano; Battaglia, Cristina; Perego, Roberto A

    2011-06-13

    Clear cell renal cell carcinoma (ccRCC) is characterized by recurrent copy number alterations (CNAs) and loss of heterozygosity (LOH), which may have potential diagnostic and prognostic applications. Here, we explored whether ccRCC primary cultures, established from surgical tumor specimens, maintain the DNA profile of parental tumor tissues allowing a more confident CNAs and LOH discrimination with respect to the original tissues. We established a collection of 9 phenotypically well-characterized ccRCC primary cell cultures. Using the Affymetrix SNP array technology, we performed the genome-wide copy number (CN) profiling of both cultures and corresponding tumor tissues. Global concordance for each culture/tissue pair was assayed evaluating the correlations between whole-genome CN profiles and SNP allelic calls. CN analysis was performed using the two CNAG v3.0 and Partek software, and comparing results returned by two different algorithms (Hidden Markov Model and Genomic Segmentation). A very good overlap between the CNAs of each culture and corresponding tissue was observed. The finding, reinforced by high whole-genome CN correlations and SNP call concordances, provided evidence that each culture was derived from its corresponding tissue and maintained the genomic alterations of parental tumor. In addition, primary culture DNA profile remained stable for at least 3 weeks, till to third passage. These cultures showed a greater cell homogeneity and enrichment in tumor component than original tissues, thus enabling a better discrimination of CNAs and LOH. Especially for hemizygous deletions, primary cultures presented more evident CN losses, typically accompanied by LOH; differently, in original tissues the intensity of these deletions was weaken by normal cell contamination and LOH calls were missed. ccRCC primary cultures are a reliable in vitro model, well-reproducing original tumor genetics and phenotype, potentially useful for future functional approaches

  15. Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues.

    Science.gov (United States)

    Eiraku, Mototsugu; Sasai, Yoshiki

    2011-12-15

    Generation of compound tissues with complex structures is a major challenge in cell biology. In this article, we describe a protocol for mouse embryonic stem cell (ESC) culture for in vitro generation of three-dimensional retinal tissue, comparing it with the culture protocol for cortical tissue generation. Dissociated ESCs are reaggregated in a 96-well plate with reduced cell-plate adhesion and cultured as floating aggregates. Retinal epithelium is efficiently generated when ESC aggregates are cultured in serum-free medium containing extracellular matrix proteins, spontaneously forming hemispherical vesicles and then progressively transforming into a shape reminiscent of the embryonic optic cup in 9-10 d. In long-term culture, the ESC-derived optic cup generates a fully stratified retinal tissue consisting of all major neural retinal components. In contrast, the cortical differentiation culture can be started without exogenous extracellular matrix proteins, and it generates stratified cortical epithelia consisting of four distinct layers in 13 d.

  16. Discrimination and similarity evaluation of tissue-cultured and wild Dendrobium species using Fourier transform infrared spectroscopy

    Science.gov (United States)

    Chen, Nai-dong; Chen, Han; Li, Jun; Sang, Mang-mang; Ding, Shen; Yu, Hao

    2015-04-01

    The FTIR method was applied to evaluate the similarity of tissue-cultured and wild Dendrobium huoshanense C.Z. Tang et S.J. Cheng, Dendrobium officinale Kimura et Migo and Dendrobium moniliforme (Linn.) Sw and discriminate different Dendrobium species, especially D. huoshanense and its main goldbrick Dendrobium henanense J.L. Lu et L.X. Gao. Despite the general pattern of the IR spectra, different intensities, shapes and peak positions were found in the IR spectra of these samples, especially in the range of 1800-600 cm-1, which could be used to discriminate them. The methanol, aqueous extracting procedure and the second derivative transformation obviously enlarged the tiny spectral differences among these samples. The similarity evaluation based on the IR spectra and the second derivative IR spectrum revealed that the similarity of the methanol extracts between tissue-cultured and wild Dendrobiums might be lower than that between different Dendrobium species. The similarities of the powders and aqueous extracts between tissue-cultured and wild Dendrobiums were higher than those between different Dendrobium species. The further principal component analysis showed that the first three components explained 99.7%, 87.7% and 85.1% of data variance for powder, methanol extract and aqueous extract, respectively, demonstrating a good discrimination between samples. Our research suggested that the variations of secondary metabolites between different origins of the investigated Dendrobiums might be higher than what we had supposed. Tissue culture techniques were widely used in the conversation of rare and endangered medicinal amedica, however, our study suggested that the chemical constituents of tissue-cultured plants might be quite different from their wild correspondences.

  17. Microfluidic 3D cell culture: potential application for tissue-based bioassays

    Science.gov (United States)

    Li, XiuJun (James); Valadez, Alejandra V.; Zuo, Peng; Nie, Zhihong

    2014-01-01

    Current fundamental investigations of human biology and the development of therapeutic drugs, commonly rely on two-dimensional (2D) monolayer cell culture systems. However, 2D cell culture systems do not accurately recapitulate the structure, function, physiology of living tissues, as well as highly complex and dynamic three-dimensional (3D) environments in vivo. The microfluidic technology can provide micro-scale complex structures and well-controlled parameters to mimic the in vivo environment of cells. The combination of microfluidic technology with 3D cell culture offers great potential for in vivo-like tissue-based applications, such as the emerging organ-on-a-chip system. This article will review recent advances in microfluidic technology for 3D cell culture and their biological applications. PMID:22793034

  18. A Simplified Method for Three-Dimensional (3-D Ovarian Tissue Culture Yielding Oocytes Competent to Produce Full-Term Offspring in Mice.

    Directory of Open Access Journals (Sweden)

    Carolyn M Higuchi

    Full Text Available In vitro growth of follicles is a promising technology to generate large quantities of competent oocytes from immature follicles and could expand the potential of assisted reproductive technologies (ART. Isolated follicle culture is currently the primary method used to develop and mature follicles in vitro. However, this procedure typically requires complicated, time-consuming procedures, as well as destruction of the normal ovarian microenvironment. Here we describe a simplified 3-D ovarian culture system that can be used to mature multilayered secondary follicles into antral follicles, generating developmentally competent oocytes in vitro. Ovaries recovered from mice at 14 days of age were cut into 8 pieces and placed onto a thick Matrigel drop (3-D culture for 10 days of culture. As a control, ovarian pieces were cultured on a membrane filter without any Matrigel drop (Membrane culture. We also evaluated the effect of activin A treatment on follicle growth within the ovarian pieces with or without Matrigel support. Thus we tested four different culture conditions: C (Membrane/activin-, A (Membrane/activin+, M (Matrigel/activin-, and M+A (Matrigel/activin+. We found that the cultured follicles and oocytes steadily increased in size regardless of the culture condition used. However, antral cavity formation occurred only in the follicles grown in the 3-D culture system (M, M+A. Following ovarian tissue culture, full-grown GV oocytes were isolated from the larger follicles to evaluate their developmental competence by subjecting them to in vitro maturation (IVM and in vitro fertilization (IVF. Maturation and fertilization rates were higher using oocytes grown in 3-D culture (M, M+A than with those grown in membrane culture (C, A. In particular, activin A treatment further improved 3-D culture (M+A success. Following IVF, two-cell embryos were transferred to recipients to generate full-term offspring. In summary, this simple and easy 3-D ovarian

  19. Smallholder adoption and economic impacts of tissue culture ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-12-01

    Dec 1, 2009 ... ISSN 1684–5315 © 2009 Academic Journals. Full Length ... Key words: Biotechnology, adoption, tissue culture bananas, Kenya. INTRODUCTION ... Recent studies about the agronomic and economic impacts of biotech- ..... accused scientist for 'playing God', others have supported biotechnologies.

  20. Tissue culture of black pepper (piper nigrum l.) in Pakistan

    International Nuclear Information System (INIS)

    Hussain, A.; Naz, S.; Nazir, H.; Shinwari, Z.K.

    2011-01-01

    Black pepper (Piper nigrum L.) the 'King of Spices' is a universal table condiment. It is extensively used in Pakistani cuisines and herbal medicines and imported in bulk from neighboring countries. The black pepper vine is generally cultivated by seed because other vegetative propagation methods are slow and time consuming. Therefore the tissue culture technique is considered more efficient and reliable method for rapid and mass propagation of this economically important plant. The present study was initiated to develop protocol for micro-propagation of black pepper vine. The stem, leaf and shoot tip explants from mature vine were cultured on MS medium supplemented with different concentrations of plant growth regulators (2,4-D, BA, IBA). Best callus was produced on MS medium with 1.5 mg/l BA by shoot tip explant. Shoot regeneration was excellent on MS medium with 0.5 mg/l BA. The plantlets formed were rooted best on 1.5 mg/l IBA. The rooted plants were transplanted in soil medium and acclimatized in growth room. The plants raised were test planted under the local conditions of Hattar. (author)

  1. EXPLANTATION OF MESANGIAL CELL HILLOCKS - A METHOD FOR OBTAINING HUMAN MESANGIAL CELLS IN CULTURE

    NARCIS (Netherlands)

    MULLER, EW; KIM, Y; MICHAEL, AF; VERNIER, RL; VANDERHEM, GK; VANDERWOUDE, FJ

    A simple method is presented for selective cell culture of human mesangial cells using explanatation of mesangial cell hillocks. Glomeruli which had been incubated with collagenase were explanted on plastic tissue culture flasks. Three to 6 weeks after explantation, a rapidly growing multilayer of

  2. Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ.

    Science.gov (United States)

    Marciniak, Anja; Selck, Claudia; Friedrich, Betty; Speier, Stephan

    2013-01-01

    Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.

  3. Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ.

    Directory of Open Access Journals (Sweden)

    Anja Marciniak

    Full Text Available Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.

  4. Demonstration of the economic feasibility of plant tissue culture for jojoba (Simmondsia chinensis) and Euphorbia spp

    Energy Technology Data Exchange (ETDEWEB)

    Sluis, C.

    1980-09-01

    The economic feasibility of plant tissue culture was demonstrated as applied to two plants: jojoba (Simmondsia chinensis) and Euphorbia spp. The gopher weed (Euphorbia lathyris) was selected as the species of Euphorbia to research due to the interest in this plant as a potential source of hydrocarbon-like compounds. High yield female selections of jojoba were chosen from native stands and were researched to determine the economic feasibility of mass producing these plants via a tissue culture micropropagation program. The female jojoba selection was successfully mass produced through tissue culture. Modifications in initiation techniques, as well as in multiplication media and rooting parameters, were necessary to apply the tissue culture system, which had been developed for juvenile seedling tissue, to mature jojobas. Since prior attempts at transfer of tissue cultured plantlets were unsuccessful, transfer research was a major part of the project and has resulted in a system for transfer of rooted jojoba plantlets to soil. Euphorbia lathyris was successfully cultured using shoot tip cultures. Media and procedures were established for culture initiation, multiplication of shoots, callus induction and growth, and root initiation. Well-developed root systems were not attained and root initiation percentages should be increased if the system is to become commercially feasible.

  5. Mass spectrometric characterization of elements and molecules in cell cultures and tissues

    International Nuclear Information System (INIS)

    Arlinghaus, H.F.; Kriegeskotte, C.; Fartmann, M.; Wittig, A.; Sauerwein, W.; Lipinsky, D.

    2006-01-01

    Time-of-flight secondary ion mass spectrometry (ToF-SIMS) and laser post-ionization secondary neutral mass spectrometry (laser-SNMS) have been used to image and quantify targeted compounds, intrinsic elements and molecules with subcellular resolution in single cells of both cell cultures and tissues. Special preparation procedures for analyzing cell cultures and tissue materials were developed. Cancer cells type MeWo, incubated with boronated compounds, were sandwiched between two substrates, cryofixed, freeze-fractured and freeze-dried. Also, after injection with boronated compounds, different types of mouse tissues were extracted, prepared on a special specimen carrier and plunged with high velocity into LN 2 -cooled propane for cryofixation. After trimming, these tissue blocks were freeze-dried. The measurements of the K/Na ratio demonstrated that for both cell cultures and tissue materials the special preparation techniques used were appropriate for preserving the chemical and structural integrity of the living cell. The boron images show inter- and intracellular boron signals with different intensities. Molecular images show distinct features partly correlated with the cell structure. A comparison between laser-SNMS and ToF-SIMS showed that especially laser-SNMS is particularly well-suited for identifying specific cell structures and imaging ultratrace element concentrations in tissues

  6. Soft tissue tumors - imaging methods

    International Nuclear Information System (INIS)

    Arlart, I.P.

    1985-01-01

    Soft Tissue Tumors - Imaging Methods: Imaging methods play an important diagnostic role in soft tissue tumors concerning a preoperative evaluation of localization, size, topographic relationship, dignity, and metastatic disease. The present paper gives an overview about diagnostic methods available today such as ultrasound, thermography, roentgenographic plain films and xeroradiography, radionuclide methods, computed tomography, lymphography, angiography, and magnetic resonance imaging. Besides sonography particularly computed tomography has the most important diagnostic value in soft tissue tumors. The application of a recently developed method, the magnetic resonance imaging, cannot yet be assessed in its significance. (orig.) [de

  7. A method for quantifying mechanical properties of tissue following viral infection.

    Directory of Open Access Journals (Sweden)

    Vy Lam

    Full Text Available Viral infection and replication involves the reorganization of the actin network within the host cell. Actin plays a central role in the mechanical properties of cells. We have demonstrated a method to quantify changes in mechanical properties of fabricated model three-dimensional (3D connective tissue following viral infection. Using this method, we have characterized the impact of infection by the human herpesvirus, cytomegalovirus (HCMV. HCMV is a member of the herpesvirus family and infects a variety of cell types including fibroblasts. In the body, fibroblasts are necessary for maintaining connective tissue and function by creating mechanical force. Using this 3D connective tissue model, we observed that infection disrupted the cell's ability to generate force and reduced the cumulative contractile force of the tissue. The addition of HCMV viral particles in the absence of both viral gene expression and DNA replication was sufficient to disrupt tissue function. We observed that alterations of the mechanical properties are, in part, due to a disruption of the underlying complex actin microfilament network established by the embedded fibroblasts. Finally, we were able to prevent HCMV-mediated disruption of tissue function by the addition of human immune globulin against HCMV. This study demonstrates a method to quantify the impact of viral infection on mechanical properties which are not evident using conventional cell culture systems.

  8. [Extraction and analysis of chemical components of essential oil in Thymus vulgaris of tissue culture].

    Science.gov (United States)

    Li, Xiao-Dong; Yang, Li; Xu, Shi-Qian; Li, Jian-Guo; Cheng, Zhi-Hui; Dang, Jian-Zhang

    2011-10-01

    To extract the essential oils from the Seedlings, the Aseptic Seedlings and the Tissue Culture Seedlings of Thymus vulgaris and analyze their chemical components and the relative contents. The essential oils were extracted by steam distillation, the chemical components and the relative contents were identified and analyzed by gas chromatography-mass spectrometry (GC/MS) and peak area normalization method. The main chemical components of essential oil in these three samples had no significant difference, they all contained the main components of essential oil in Thymus vulgaris: Thymol, Carvacrol, o-Cymene, gamma-Terpinene, Caryophyllene et al. and only had a slight difference in the relative content. This study provides important theoretical foundation and data reference for further study on production of essential oil in thyme by tissue culture technology.

  9. GROWTH AND ROOTING SYSTEM OF ACACIA MANGIUM OBTAINED BY TISSUE CULTURE

    Directory of Open Access Journals (Sweden)

    SUPRIYANTO

    1991-01-01

    Full Text Available Since 1980/1981, the government of Indonesia through the Ministry of Forestry has started to reforest logged-over, alang-alang, unproductive areas and to convert them to Forest Industry Plantation. The target is 300 000 ha per year. It means, 750 million seedlings should be provided per year (planting distance 2 m x 2 m. The tree species to be planted in forest industry plantation should have shorter life cycle (8 - 10 years, good stem-form, good rooting system, and should be fast growing. Acacia mangium has been selected as one of the important tree species for forest industry plantation due to its growth, quality of fiber wood (pulp and paper industry and rooting system (produce a lot of secondary root and nitrogen fixater (Soebardjo 1986. The reforestation of logged-over Dipterocarp forests in Malaysia with A. mangium has also been considered (Appanah and Weinland 1989. Generally, reforestation with A. mangium is done with seedlings obtained by seed germination. A. mangium produce a lot of seeds but its production is still limited by the season, while the conventional method of vegetative propagation through cuttings gave very low percentage of rooted-cuttings (1% (Umboh and Syamsul Yani 1989. The micropropagation of A. mangium through tissue culture is a promising method. The production of A. mangium plantlets through that method has been done at the Forest Genetic Laboratory, Tropical Forest Biology, SEAMEO BIOTROP (Situmorang 1988, Umboh 1988, Umboh et al. 1989, 1990. These rooted-plantlets (plantlings were first put in the green house (acclimatization before planting in the field. Field tests of some agricultural plants have been done but information on forest trees species is still lacking because the production of plantlings through tissue culture is still limited as there are still problems of their rooting. In fact, the progress of reproducing woody plants by tissue culture has been much slower than with herbaceous plants. The major

  10. A novel culture method reveals unique neural stem/progenitors in mature porcine iris tissues that differentiate into neuronal and rod photoreceptor-like cells.

    Science.gov (United States)

    Royall, Lars N; Lea, Daniel; Matsushita, Tamami; Takeda, Taka-Aki; Taketani, Shigeru; Araki, Masasuke

    2017-11-15

    Iris neural stem/progenitor cells from mature porcine eyes were investigated using a new protocol for tissue culture, which consists of dispase treatment and Matrigel embedding. We used a number of culture conditions and found an intense differentiation of neuronal cells from both the iris pigmented epithelial (IPE) cells and the stroma tissue cells. Rod photoreceptor-like cells were also observed but mostly in a later stage of culture. Neuronal differentiation does not require any additives such as fetal bovine serum or FGF2, although FGF2 and IGF2 appeared to promote neural differentiation in the IPE cultures. Furthermore, the stroma-derived cells were able to be maintained in vitro indefinitely. The evolutionary similarity between humans and domestic pigs highlight the potential for this methodology in the modeling of human diseases and characterizing human ocular stem cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Banana Musa tissue culture plants enhanced by endophytic fungi

    African Journals Online (AJOL)

    Mo

    Merging biotechnology with biological control: Banana Musa tissue culture plants enhanced by endophytic .... While working in the laminar flow cabinet, sterile filter papers were placed in ..... University of Bonn, Bonn, Germany. Niere, B., 2001.

  12. Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture.

    Directory of Open Access Journals (Sweden)

    Pek-Lan Chan

    Full Text Available BACKGROUND: The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR. With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. RESULTS: In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569 outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN. PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. CONCLUSIONS: Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection

  13. Evaluation of Reference Genes for Quantitative Real-Time PCR in Oil Palm Elite Planting Materials Propagated by Tissue Culture

    Science.gov (United States)

    Chan, Pek-Lan; Rose, Ray J.; Abdul Murad, Abdul Munir; Zainal, Zamri; Leslie Low, Eng-Ti; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

    2014-01-01

    Background The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. Results In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Conclusions Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate

  14. Skin equivalent tissue-engineered construct: co-cultured fibroblasts/ keratinocytes on 3D matrices of sericin hope cocoons.

    Directory of Open Access Journals (Sweden)

    Sunita Nayak

    Full Text Available The development of effective and alternative tissue-engineered skin replacements to autografts, allografts and xenografts has became a clinical requirement due to the problems related to source of donor tissue and the perceived risk of disease transmission. In the present study 3D tissue engineered construct of sericin is developed using co-culture of keratinocytes on the upper surface of the fabricated matrices and with fibroblasts on lower surface. Sericin is obtained from "Sericin Hope" silkworm of Bombyx mori mutant and is extracted from cocoons by autoclave. Porous sericin matrices are prepared by freeze dried method using genipin as crosslinker. The matrices are characterized biochemically and biophysically. The cell proliferation and viability of co-cultured fibroblasts and keratinocytes on matrices for at least 28 days are observed by live/dead assay, Alamar blue assay, and by dual fluorescent staining. The growth of the fibroblasts and keratinocytes in co-culture is correlated with the expression level of TGF-β, b-FGF and IL-8 in the cultured supernatants by enzyme-linked immunosorbent assay. The histological analysis further demonstrates a multi-layered stratified epidermal layer of uninhibited keratinocytes in co-cultured constructs. Presence of involucrin, collagen IV and the fibroblast surface protein in immuno-histochemical stained sections of co-cultured matrices indicates the significance of paracrine signaling between keratinocytes and fibroblasts in the expression of extracellular matrix protein for dermal repair. No significant amount of pro inflammatory cytokines (TNF-α, IL-1β and nitric oxide production are evidenced when macrophages grown on the sericin matrices. The results all together depict the potentiality of sericin 3D matrices as skin equivalent tissue engineered construct in wound repair.

  15. Human colon tissue in organ culture: calcium and multi-mineral-induced mucosal differentiation.

    Science.gov (United States)

    Dame, Michael K; Veerapaneni, Indiradevi; Bhagavathula, Narasimharao; Naik, Madhav; Varani, James

    2011-01-01

    We have recently shown that a multi-mineral extract from the marine red algae, Lithothamnion calcareum, suppresses colon polyp formation and inflammation in mice. In the present study, we used intact human colon tissue in organ culture to compare responses initiated by Ca(2+) supplementation versus the multi-mineral extract. Normal human colon tissue was treated for 2 d in culture with various concentrations of calcium or the mineral-rich extract. The tissue was then prepared for histology/immunohistochemistry, and the culture supernatants were assayed for levels of type I procollagen and type I collagen. At higher Ca(2+) concentrations or with the mineral-rich extract, proliferation of epithelial cells at the base and walls of the mucosal crypts was suppressed, as visualized by reduced Ki67 staining. E-cadherin, a marker of differentiation, was more strongly expressed at the upper third of the crypt and at the luminal surface. Treatment with Ca(2+) or with the multi-mineral extract influenced collagen turnover, with decreased procollagen and increased type I collagen. These data suggest that calcium or mineral-rich extract has the capacity to (1) promote differentiation in human colon tissue in organ culture and (2) modulate stromal function as assessed by increased levels of type I collagen. Taken together, these data suggest that human colon tissue in organ culture (supporting in vivo finding in mice) will provide a valuable model for the preclinical assessment of agents that regulate growth and differentiation in the colonic mucosa.

  16. Skeletal muscle tissue engineering: methods to form skeletal myotubes and their applications.

    Science.gov (United States)

    Ostrovidov, Serge; Hosseini, Vahid; Ahadian, Samad; Fujie, Toshinori; Parthiban, Selvakumar Prakash; Ramalingam, Murugan; Bae, Hojae; Kaji, Hirokazu; Khademhosseini, Ali

    2014-10-01

    Skeletal muscle tissue engineering (SMTE) aims to repair or regenerate defective skeletal muscle tissue lost by traumatic injury, tumor ablation, or muscular disease. However, two decades after the introduction of SMTE, the engineering of functional skeletal muscle in the laboratory still remains a great challenge, and numerous techniques for growing functional muscle tissues are constantly being developed. This article reviews the recent findings regarding the methodology and various technical aspects of SMTE, including cell alignment and differentiation. We describe the structure and organization of muscle and discuss the methods for myoblast alignment cultured in vitro. To better understand muscle formation and to enhance the engineering of skeletal muscle, we also address the molecular basics of myogenesis and discuss different methods to induce myoblast differentiation into myotubes. We then provide an overview of different coculture systems involving skeletal muscle cells, and highlight major applications of engineered skeletal muscle tissues. Finally, potential challenges and future research directions for SMTE are outlined.

  17. COMPARISON OF CULTURE OF SYNOVIAL FLUID, PERIPROSTHETIC TISSUE AND PROSTHESIS SONICATE FOR THE DIAGNOSIS OF KNEE PROSTHESIS INFECTION

    Directory of Open Access Journals (Sweden)

    Andrej Trampuž

    2003-03-01

    Full Text Available Background. Synovial fluid and periprosthetic tissue specimens are the standard specimens cultured for the diagnosis of prosthetic joint infection (PJI. We hypothesize that ultrasonication of the explanted prosthesis may improve diagnosis of PJI by dislodging biofilm bacteria from the prosthesis surface and improve the sensitivity and specificity of diagnosis of PJI.Methods. Included were patients undergoing knee prosthesis exchange for septic or biomechanical failure and have not received antimicrobial therapy in the last 2 weeks prior specimen collection. Cultures of synovial fluid and periprosthetic tissue specimens were performed per the usual clinical practice. Additionally, explanted joint components were sonicated for 5 minutes at frequency 40 kHz in sterile Ringer’s solution; aliquots of 0.5 ml sonicate were plated onto five aerobic and five anaerobic blood agar plates, and incubated at 37 °C and examined for the next seven days. The number and identity of each colony morphology was recorded.Results. 35 patients undergoing knee replacement have been studied (24 for aseptic biomechanical failure and 11 for suspected PJI. In patients with PJI, coagulase-negative staphylococci (7 cases, Corynebacterium spp. (2 cases, Staphylococcus aureus (1 case, and viridans group streptococcus (1 case were recovered. Culture sensitivity and specificity were for synovial fluid 88% and 100%, for periprosthetic tissue 83% and 81%, and for explant sonicate 91% and 100%, respectively. In sonicate cultures higher numbers of microorganisms than in periprosthetic tissue cultures were consistently detected.Conclusions. Using synovial fluid, periprosthetic tissue, and explant sonicate cultures, 12%, 17% and 9% of PJI were missed, respectively. Explant sonicate cultures were the most sensitive with respect to the diagnosis of PJI, indicating that explant ultrasonication may improve bacterial recovery. In sonicate cultures, infecting organisms were detected in

  18. Advanced tissue culture used by Twyfords to build up jojoba clones

    Energy Technology Data Exchange (ETDEWEB)

    1983-01-01

    Twyford Plant Laboratories Ltd. in the UK, using their own advanced methods of plant tissue culture, have built up a bank of 30 different male and female clones of jojoba, the arid land crop whose seeds produced a liquid wax which - amongst other uses - can be substituted for sperm whale oil. The technique involves growing microscopic parts of a parent plant on a medium containing all the necessary growth hormones, salts, vitamins and other nutrients. Growth takes place under artificial light in an all-electric controlled, air-conditioned environment. No other method is so successful for rapidly multiplying plants, particularly those that do not breed true from seed. These include most fruits and some flowers and vegetables.

  19. Plasmid-dependent attachment of Agrobacterium tumefaciens to plant tissue culture cells.

    Science.gov (United States)

    Matthysse, A G; Wyman, P M; Holmes, K V

    1978-11-01

    Kinetic, microscopic, and biochemical studies show that virulent Ti (tumor inducing)-plasmid-containing strains of Agrobacterium attach to normal tobacco and carrot tissue culture cells. Kinetic studies showed that virulent strains of A. tumefaciens attach to the plant tissue culture cells in increasing numbers during the first 1 to 2 h of incubation of the bacteria with the plant cells. Five Ti-plasmid-containing virulent Agrobacterium strains showed greater attachment to tobacco cells than did five avirulent strains. Light and scanning electron microscopic observations confirmed that virulent strains showed little attachment. Bacterial attachment was blocked by prior incubation of the plant cells with lipopolysaccharide extracted from A. tumefaciens, but not from A. radiobacter, suggesting that bacterial lipopolysaccharide is one of the components involved in the attachment process. At least one other bacterial product may be required for attachment in tissue culture because the virulent A. tumefaciens NT1, which lacks the Ti plasmid, does not itself attach to tobacco cells, but its lipopolysaccharide does inhibit the attachment of virulent strains.

  20. Influence of postmortem time on the outcome of blood cultures among cadaveric tissue donors.

    Science.gov (United States)

    Saegeman, V; Verhaegen, J; Lismont, D; Verduyckt, B; De Rijdt, T; Ectors, N

    2009-02-01

    Tissue banks provide tissues of human cadaver donors for transplantation. The maximal time limit for tissue retrieval has been set at 24 h postmortem. This study aimed at evaluating the evidence for this limit from a microbiological point of view. The delay of growth in postmortem blood cultures, the identification of the species isolated and clinical/environmental factors were investigated among 100 potential tissue donors. No significant difference was found in the rate of donors with grown blood cultures within (25/65=38%) compared with after (24/65=37%) 24 h of death. Coagulase-negative staphylococci and gastro-intestinal microorganisms were isolated within and after 24 h of death. Two factors--antimicrobial therapy and "delay before body cooling"--were significantly inversely related with donors' blood culture results. From a microbiological point of view, there is no evidence for avoiding tissue retrieval among donors after 24 h of death.

  1. Improved method for HPLC analysis of polyamines, agmatine and aromatic monoamines in plant tissue

    Science.gov (United States)

    Slocum, R. D.; Flores, H. E.; Galston, A. W.; Weinstein, L. H.

    1989-01-01

    The high performance liquid chromatographic (HPLC) method of Flores and Galston (1982 Plant Physiol 69: 701) for the separation and quantitation of benzoylated polyamines in plant tissues has been widely adopted by other workers. However, due to previously unrecognized problems associated with the derivatization of agmatine, this important intermediate in plant polyamine metabolism cannot be quantitated using this method. Also, two polyamines, putrescine and diaminopropane, also are not well resolved using this method. A simple modification of the original HPLC procedure greatly improves the separation and quantitation of these amines, and further allows the simulation analysis of phenethylamine and tyramine, which are major monoamine constituents of tobacco and other plant tissues. We have used this modified HPLC method to characterize amine titers in suspension cultured carrot (Daucas carota L.) cells and tobacco (Nicotiana tabacum L.) leaf tissues.

  2. Usefulness of fibroblast culture for testing of cattle tissues polluted with heavy metals

    International Nuclear Information System (INIS)

    Weglarz, L.; Drozdz, M.Wa.; Wardas, M.; Kula, B.; Pawlaczyk-Szpilowa, M.

    1990-01-01

    Cattle tissues (liver, kidney, brain, and lung) that had been polluted with heavy metals were tested for their ability to alter fibroblast culture growth, cellular protein and DNA content, and fibroblast DNA synthesis. At 72 hr of incubation a significant increase in cellular DNA and [14C]thymidine incorporation was noted in the primary cultures as well as in the subcultures compared to controls. Fibroblast cultures also displayed growth inhibition and reduction in protein content. The measurement of basic biochemical parameters of the fibroblast culture may represent a sensitive means of assessing rapidly the activity of heavy metals deposited in the tissues of cattle as a result of their grazing on polluted soil

  3. Project on production of mutants by irradiation of in vitro cultured tissues of coconut and banana and their mass propagation by the tissue culture technique

    International Nuclear Information System (INIS)

    Guzman, E.V. de

    1975-01-01

    Fruit pulp tissue, ovary segments with or without ovules and sections from shoot tips of banana were used for studies on growth stimulating or morphogenetic effects of irradiation. Irradiation at 0.1-1.0 kR tended to induce faster callus growth in the otherwise slow-growing cultures. The physical condition and composition of the culture media especially with respect to growth regulators were studied, as were techniques to overcome discoloration of explants, the best choice of plant tissue for explant, and radiation effects on growth and morphogenesis. Due to the difficulty of callus induction with coconut, only the effects of irradiation on embryos cultured in vitro were studied. They were irradiated at various stages of development, i.e. during the early and final stage of liquid culture, and several days after transfer to a solid medium. Adverse effects of irradiation became evident only during the subsequent growth in solid, during the latter stage of which morphological changes were observed. Whereas irradiation of the liquid as well as solid media up to 50 kR had no adverse effect; survival and development became adversely affected at a dose of 1 kR

  4. Substrate specific hydrolysis of aromatic and aromatic-aliphatic esters in orchid tissue cultures

    Directory of Open Access Journals (Sweden)

    Agnieszka Mironowicz

    2014-01-01

    Full Text Available We found that tissue cultures of higher plants were able, similarly as microorganisms, to transform low-molecular-weight chemical compounds. In tissue cultures of orchids (Cymbidium 'Saint Pierre' and Dendrobium phalaenopsis acetates of phenols and aromatic-aliphatic alcohols were hydrolyzed, whereas methyl esters of aromatic and aromatic-aliphatic acids did not undergo this reaction. Acetates of racemic aromatic-aliphatic alcohols were hydrolyzed with distinct enantiospecificity.

  5. Picroside I and Picroside II from Tissue Cultures of Picrorhiza kurroa

    Science.gov (United States)

    Ganeshkumar, Yamjala; Ramarao, Ajmera; Veeresham, Ciddi

    2017-01-01

    Background: Picrorhiza kurroa (PK) belongs to Scrophulariaceae family and is a representative endemic, medicinal herb, widely distributed throughout the higher altitudes of alpine Himalayas from west to east, between 3000 and 4500 m above mean sea level. Objective: The objective of the present study is to assess the production of picroside I and picroside II from tissue cultures of PK. Materials and Methods: Auxiliary shoot tips of PK were incubated in Murashige and Skoog medium supplemented with indole-3-butyric acid and kinetin phytohormones. The callus produced was collected at different time intervals and was processed for extraction of picroside I and picroside II followed by thin layer chromatography and high-performance liquid chromatography HPLC analysis. Results: The maximum growth index was found to be 5.109 ± 0.159 at 16-week-old callus culture. The estimation of picroside-I and picroside-II was carried out by (HPLC) analysis; quantity of secondary metabolite found to be 16.37 ± 0.0007 mg/g for PK-I and 6.34 ± 0.0012 mg/g for PK-II. Conclusion: This is the first attempt to produce the Picroside-I and II in large amount by the tissue culture technique. It can be observed that the method of callus culture can be used in production of secondary metabolites Picroside-I and II from PK SUMMARY Picrorhiza kurroa is a high value medicinal herb due to rich source of hepatoprotective metabolites, Picroside-I and Picroside-II. The medicinal importance of P. kurroa is due to its pharmacological properties like hepatoprotective, antioxidant (particularly in liver), antiallergic and antiasthamatic, anticancer activity particularly in liver and immunomodulatory. Shoot apices which were produced a good response was inoculated on selected medium i.e., on MS medium containing 2, 4 D (mg/l) + KN (1mg/l) for induction of callus. The initiation of callus was observed after 4weeks and it was light green and fragile Maximum growth was observed with 3% w/v of sucrose

  6. Monitoring Dynamic Interactions between Breast Cancer Cells and Human Bone Tissue in a Co-Culture Model

    Science.gov (United States)

    Contag, Christopher H.; Lie, Wen-Rong; Bammer, Marie C.; Hardy, Jonathan W.; Schmidt, Tobi L.; Maloney, William J.; King, Bonnie L.

    2015-01-01

    Purpose Bone is a preferential site of breast cancer metastasis and models are needed to study this process at the level of the microenvironment. We have used bioluminescence imaging (BLI) and multiplex biomarker immunoassays to monitor dynamic breast cancer cell behaviors in co-culture with human bone tissue. Procedures Femur tissue fragments harvested from hip replacement surgeries were co-cultured with luciferase-positive MDA-MB-231-fLuc cells. BLI was performed to quantify breast cell division and track migration relative to bone tissue. Breast cell colonization of bone tissues was assessed with immunohistochemistry. Biomarkers in co-culture supernatants were profiled with MILLIPLEX® immunoassays. Results BLI demonstrated increased MDA-MB-231-fLuc proliferation (pbones, and revealed breast cell migration toward bone. Immunohistochemistry illustrated MDA-MB-231-fLuc colonization of bone, and MILLIPLEX® profiles of culture supernatants suggested breast/bone crosstalk. Conclusions Breast cell behaviors that facilitate metastasis occur reproducibly in human bone tissue co-cultures and can be monitored and quantified using BLI and multiplex immunoassays. PMID:24008275

  7. The use of tissue culture techniques to detect irradiated vegetables

    International Nuclear Information System (INIS)

    Al-Safadi, B.; Sharabi, N.E.; Nabulsi, I

    2001-01-01

    the ability of two tissue culture methods, callus and vegetable growth induction, to detect irradiated vegetables was evaluated. Potato tubers, carrot roots, garlic cloves and onion bulbs were subjected to various gamma radiation doses (0, 25, 100, 150, 250, 500, 750, and 1000 Gy). Irradiated vegetables were cultured in vitro and in vivo (pots). Gamma irradiation significantly reduced callus-forming ability especially in carrot and potato where no callus was observed in doses higher than 50 Gy. Length of shoots and roots growing from irradiated garlic and onion explants was considerably reduced starting from the 25 Gy dose. No roots were formed on garlic explants at any irradiation dose. Garlic leaves growing from irradiated explants were spotted with purple to brown spots. The intensity of these spots increased as gamma ray dosage increased. In the pot experiment, potato plant appeared in the control only. On the contrary, a complete sprouting of garlic and onion was seen in all irradiation treatments. It was not possible to distinguish between the various irradiation treatments and the control 3 days after planting in pots. The two in vitro techniques, tested in our study, may effectively be used to detect irradiated vegetables and estimate the range of doses used. The callus formation method is more useful for potato and carrot, since regeneration of shoots in vitro from these two plants takes along time, making this method unpractical. The other technique is very useful in the case of onion and garlic since it is rapid. The two techniques can be used with most of the vegetables that can be cultured in vitro. (Author)

  8. Air exposure induced characteristics of dry eye in conjunctival tissue culture.

    Directory of Open Access Journals (Sweden)

    Hui Lin

    Full Text Available There are several animal models illustrating dry eye pathophysiology. Current study would like to establish an ex vivo tissue culture model for characterizing dry eye. Human conjunctival explants were cultured under airlift or submerged conditions for up to 2 weeks, and only airlifted conjunctival cultures underwent increased epithelial stratification. Starting on day 4, the suprabasal cells displayed decreased K19 expression whereas K10 keratin became evident in airlift group. Pax6 nuclear expression attenuated already at 2 days, while its perinuclear and cytoplasmic expression gradually increased. MUC5AC and MUC19 expression dramatically decreased whereas the full thickness MUC4 and MUC16 expression pattern disappeared soon after initiating the airlift condition. Real time PCR showed K16, K10 and MUC16 gene up-regulated while K19, MUC5AC, MUC19 and MUC4 down-regulated on day 8 and day 14. On day 2 was the appearance of apoptotic epithelial and stromal cells appeared. The Wnt signaling pathway was transiently activated from day 2 to day 10. The inflammatory mediators IL-1β, TNF-α, and MMP-9 were detected in the conditioned media after 6 to 8 days. In conclusion, airlifted conjunctival tissue cultures demonstrated Wnt signaling pathway activation, coupled with squamous metaplasia, mucin pattern alteration, apoptosis and upregulation of proinflammatory cytokine expression. These changes mimic the pathohistological alterations described in dry eye. This correspondence suggests that insight into the pathophysiology of dry eye may be aided through the use of airlifted conjunctival tissue cultures.

  9. Primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue.

    Science.gov (United States)

    Tamashiro, Tami T; Dalgard, Clifton Lee; Byrnes, Kimberly R

    2012-08-15

    Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted. Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study. The principle and protocol of this methodology have been described in the literature. Additionally, alternate methodologies to isolate primary microglia are well described. Homogenized brain tissue may be separated

  10. Pathogen and biological contamination management in plant tissue culture: phytopathogens, vitro pathogens, and vitro pests.

    Science.gov (United States)

    Cassells, Alan C

    2012-01-01

    The ability to establish and grow plant cell, organ, and tissue cultures has been widely exploited for basic and applied research, and for the commercial production of plants (micro-propagation). Regardless of whether the application is for research or commerce, it is essential that the cultures be established in vitro free of biological contamination and be maintained as aseptic cultures during manipulation, growth, and storage. The risks from microbial contamination are spurious experimental results due to the effects of latent contaminants or losses of valuable experimental or commercial cultures. Much of the emphasis in culture contamination management historically focussed on the elimination of phytopathogens and the maintenance of cultures free from laboratory contamination by environmental bacteria, fungi (collectively referred to as "vitro pathogens", i.e. pathogens or environmental micro-organisms which cause culture losses), and micro-arthropods ("vitro pests"). Microbial contamination of plant tissue cultures is due to the high nutrient availability in the almost universally used Murashige and Skoog (Physiol Plant 15:473-497, 1962) basal medium or variants of it. In recent years, it has been shown that many plants, especially perennials, are at least locally endophytically colonized intercellularly by bacteria. The latter, and intracellular pathogenic bacteria and viruses/viroids, may pass latently into culture and be spread horizontally and vertically in cultures. Growth of some potentially cultivable endophytes may be suppressed by the high salt and sugar content of the Murashige and Skoog basal medium and suboptimal temperatures for their growth in plant tissue growth rooms. The management of contamination in tissue culture involves three stages: disease screening (syn. disease indexing) of the stock plants with disease and endophyte elimination where detected; establishment and pathogen and contaminant screening of established initial cultures

  11. Methods for suspension culture, protoplast extraction, and transformation of high-biomass yielding perennial grass Arundo donax.

    Science.gov (United States)

    Pigna, Gaia; Dhillon, Taniya; Dlugosz, Elizabeth M; Yuan, Joshua S; Gorman, Connor; Morandini, Piero; Lenaghan, Scott C; Stewart, C Neal

    2016-12-01

    Arundo donax L. is a promising biofuel feedstock in the Mediterranean region. Despite considerable interest in its genetic improvement, Arundo tissue culture and transformation remains arduous. The authors developed methodologies for cell- and tissue culture and genetic engineering in Arundo. A media screen was conducted, and a suspension culture was established using callus induced from stem axillary bud explants. DBAP medium, containing 9 µM 2,4-D and 4.4 µM BAP, was found to be the most effective medium among those tested for inducing cell suspension cultures, which resulted in a five-fold increase in tissue mass over 14 days. In contrast, CIM medium containing 13 µM 2,4-D, resulted in just a 1.4-fold increase in mass over the same period. Optimized suspension cultures were superior to previously-described solidified medium-based callus culture methods for tissue mass increase. Suspension cultures proved to be very effective for subsequent protoplast isolation. Protoplast electroporation resulted in a 3.3 ± 1.5% transformation efficiency. A dual fluorescent reporter gene vector enabled the direct comparison of the CAMV 35S promoter with the switchgrass ubi2 promoter in single cells of Arundo. The switchgrass ubi2 promoter resulted in noticeably higher reporter gene expression compared with that conferred by the 35S promoter in Arundo. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Discarded human fetal tissue and cell cultures for transplantation research

    International Nuclear Information System (INIS)

    Hay, R.J.; Phillips, T.; Thompson, A.; Vilner, L.; Cleland, M.; Tchaw-ren Chen; Zabrenetzky, V.

    1999-01-01

    A feasibility study has been performed to explore the utility of various tissues from discarded human abortuses for transplantation and related research. Specifically, aborted fetuses plus parental blood samples and all relevant clinical data were obtained through a local hospital complex. Whenever possible, pancreas, skin and skeletal muscle, heart, liver, kidney, cartilage and lung tissues were removed, dissociated and subfractionated for cryopreservation, characterization and cultivation trials in vitro. Existing protocols for these manipulations were compared and improved upon as required. Clonal culture, cell aggregate maintenance techniques and use of feeder cell populations have been utilized where appropriate to develop quantitative comparative data. Histological and biochemical assays were applied both to evaluate separation/cultivation methods and to identify optimal culture conditions for maintaining functional cells. Immunochemical and molecular biological procedures were applied to study expression of Major Histocompatibility Vomplex (MHC) class 1 and 11 molecules on cell lines derived. Tissue and cell culture populations were examined for infections with bacteria, ftingi, mycoplasma, HIV, CMV, hepatitis B and other viruses. Only 1% of the abortuses tested were virally infected. Cytogenetic analyses confin-ned the normal diploid status in the vast majority (>98%) of lines tested. A total of over 250 abortuses have been obtained and processed. Only 25 were found to be contaminated with bacteria or fungi and unsuitable for further cultivation trials. A total of over 200 cell populations were isolated, characterized and cryopreserved for further study. Included were kidney, lung, liver and epidermal epithelia: cartilage-derived cells from the spine and epiphyses plus myogenic myoblasts. Selected lines have been immortalized using HPV I 6E6/E7 sequences. Epithelia from the liver and pancreas and cardiac myocytes were the most problematic in that initial

  13. Selection of seed lots of Pinus taeda L. for tissue culture

    Directory of Open Access Journals (Sweden)

    Diego Pascoal Golle

    2014-06-01

    Full Text Available The aim of this work was to identify the fungi genera associated with three Pinus taeda L. seed lots and to assess the sanitary and physiological quality of these lots for use as selection criteria for tissue culture and evaluate the in vitro establishment of explants from seminal origin in different nutritive media. It was possible to discriminate the lots on the sanitary and physiological quality, as well as to establish in vitro plants of Pinus taeda from cotyledonary nodes obtained from aseptic seed germination of a selected lot by the sanitary and physiological quality higher. The nutritive media MS, ½ MS and WPM were equally suitable for this purpose. For the sanitary analysis the fungal genera Fusarium, Penicillium and Trichoderma were those of the highest sensitivity. For the physiological evaluation were important the variables: abnormal seedlings, strong normal seedlings; length, fresh and dry weight of strong normal seedlings. The analyzes were favorable to choose lots of seeds for in vitro culture and all culture media were adequate for the establishment of this species in tissue culture.

  14. New method for the study of Amaryllidaceae alkaloid biosynthesis using biotransformation of deuterium-labeled precursor in tissue cultures

    International Nuclear Information System (INIS)

    Tahchy, A. E.; Boisbrun, M.; Chretien, F.; Henry, M.; Chapleur, Y.; Laurain-Mattar, D.; Ptak, A.; Dupire, F.

    2010-01-01

    Biotransformation of deuterated-4'-O-methylnorbelladine into alkaloids galanthamine and lycorine in tissue cultures of Leucojum aestivum was demonstrated using HPLC coupled to mass spectrometry. GC-MS screening was also carried to investigate other native and deuterated alkaloids. A total of six labeled alkaloids were identified indicating that 4'-O-methyl-d3-norbelladine is incorporated into three different groups of Amaryllidaceae alkaloids that are biosynthesized by three modes of intramolecular oxidative phenol coupling. (authors)

  15. Frozen and fresh ovarian tissue require different culture media to promote in vitro development of bovine preantral follicles.

    Science.gov (United States)

    Castro, Simone Vieira; Carvalho, Adeline Andrade; Silva, Cleidson Manoel Gomes; Santos, Francielli Weber; Campello, Cláudio Cabral; de Figueiredo, José Ricardo; Rodrigues, Ana Paula Ribeiro

    2014-10-01

    The aim of this study was to evaluate the efficiency of different media in the in vitro culture of bovine preantral follicles that were used either fresh or following slow freezing treatment. Frozen and fresh noncultured or cultured ovarian fragments were processed for histological, viability, and cell proliferation analyses. For cryopreservation, a solution containing 1.5 M ethylene glycol was frozen in a programmable biological freezer. After thawing, a portion of the samples was destined for frozen controls. The remainder were cultured in vitro for 5 days in three media: α-MEM, McCoy, or M199. Samples from these culture media were collected on days 1 and 5 for quantification of reactive oxygen species (ROS) and for hormonal assays. In fresh-cultured tissues, the percentage of morphologically normal follicles was significantly higher when cultured in M199 compared to that in the other media. In frozen-cultured tissues, McCoy medium was significantly superior to the other media, and was the only treatment that helped in maintaining the viability similar to fresh and frozen controls. Upon quantification of the nucleolus organizer region, we observed greater proliferation of granulosa cells in the frozen-cultured tissues with McCoy medium, and lesser proliferation in fresh-cultured tissues only with α-MEM. In frozen-cultured tissues, ROS levels were highest at day 1 and progressively reduced during culture, independent of the media used. In conclusion, under the conditions used in this study, the M199 and McCoy media are recommended for the culture of follicles derived from fresh and frozen ovarian tissues, respectively.

  16. [18S-25S rDNA variation in tissue culture of some Gentiana L. species].

    Science.gov (United States)

    Mel'nyk, V M; Andrieiev, I O; Spiridonova, K V; Strashniuk, N M; Kunakh, V A

    2007-01-01

    18S-25S rDNA of intact plants and tissue cultures of G. acaulis, G. punctata and G. lutea have been investigated by using blot-hybridization. The decrease of rDNA amount was found in the callus cultures as compared with the plants. In contrast to other species, G. lutea showed intragenome heterogeneity of rRNA genes as well as qualitative rDNA changes in tissue culture, in particular appearance of altered repeats. The relationship between the peculiarities of rRNA gene structure and their rearrangements in in vitro culture was suggested.

  17. Macroporous Hydrogel Scaffolds for Three-Dimensional Cell Culture and Tissue Engineering.

    Science.gov (United States)

    Fan, Changjiang; Wang, Dong-An

    2017-10-01

    Hydrogels have been promising candidate scaffolds for cell delivery and tissue engineering due to their tissue-like physical properties and capability for homogeneous cell loading. However, the encapsulated cells are generally entrapped and constrained in the submicron- or nanosized gel networks, seriously limiting cell growth and tissue formation. Meanwhile, the spatially confined settlement inhibits attachment and spreading of anchorage-dependent cells, leading to their apoptosis. In recent years, macroporous hydrogels have attracted increasing attention in use as cell delivery vehicles and tissue engineering scaffolds. The introduction of macropores within gel scaffolds not only improves their permeability for better nutrient transport but also creates space/interface for cell adhesion, proliferation, and extracellular matrix deposition. Herein, we will first review the development of macroporous gel scaffolds and outline the impact of macropores on cell behaviors. In the first part, the advantages and challenges of hydrogels as three-dimensional (3D) cell culture scaffolds will be described. In the second part, the fabrication of various macroporous hydrogels will be presented. Third, the enhancement of cell activities within macroporous gel scaffolds will be discussed. Finally, several crucial factors that are envisaged to propel the improvement of macroporous gel scaffolds are proposed for 3D cell culture and tissue engineering.

  18. An electrochemical approach to monitor pH change in agar media during plant tissue culture.

    Science.gov (United States)

    Wang, Min; Ha, Yang

    2007-05-15

    In this work, metal oxide microelectrodes were developed to monitor pH change in agar media during plant tissue culture. An antimony wire was produced by a new approach "capillary melt method". The surface of the obtained antimony wire was oxidized in a potassium nitrate melt to fabricate an antimony oxide film for pH sensing. Characterization results show that the oxide layer grown on the wire surface consists of Sb(2)O(3) crystal phase. The sensing response, open-circuit potential, of the electrode has a good linear relationship (R(2)=1.00) with pH value of the test solution. Adding organic compounds into the test media would not affect the linear relationship, although the slope of the lines varied with different ingredients added. The antimony oxide electrodes were employed to continuously monitor pH change of agar culture media during a 2-week plant tissue culture of Dendrobium candidum. The antimony oxide electrode fabricated this way has the advantages of low cost, easy fabrication, fast response, and almost no contamination introduced into the system. It would be suitable for in situ and continuous pH measurement in many bio applications.

  19. Pre-irradiation of tissue culture flasks leads to diminished stem and progenitor cell production in long-term bone marrow cultures

    International Nuclear Information System (INIS)

    Rooney, P.; Wright, E.G.

    1993-01-01

    Empty plastic tissue culture flasks were exposed to X-irradiation doses of 0.3-10.0 Gy, prior to the establishment of long-term bone marrow cultures. During the course of a 10 week culture period, all irradiated plastic flasks exhibited a dramatic decrease in the number of both haemopoietic stem cells and myeloid progenitor cells, in the non-adherent layer, when compared with controls. This decrease was not due to a decrease in the number of non-adherent cells produced. Histological examination of non-adherent cells showed an increase in mature granulocytic cells with few blast cells. Morphologically, the adherent layers of irradiated flasks demonstrated a delay in appearance or absence of fat cell production. X-irradiation of glass tissue culture flasks had no deleterious effect. (author)

  20. Using organotypic (raft) epithelial tissue cultures for the biosynthesis and isolation of infectious human papillomaviruses.

    Science.gov (United States)

    Ozbun, Michelle A; Patterson, Nicole A

    2014-08-01

    Papillomaviruses have a strict tropism for epithelial cells, and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitro-wherein virion morphogenesis occurs under cooperative viral and cellular cues-requires the cultivation of epithelium. Presented in the first section of this unit is a protocol to grow differentiating epithelial tissues that mimic many important morphological and biochemical aspects of normal skin. The technique involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname "raft" cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, or keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single-step virus growth cycle is achieved in this process, as it is unlikely that progeny virions are released to initiate subsequent rounds of infection. Copyright © 2014 John Wiley

  1. In Vivo-Like Culture Conditions in a Bioreactor Facilitate Improved Tissue Quality in Corneal Storage.

    Science.gov (United States)

    Schmid, Richard; Tarau, Ioana-Sandra; Rossi, Angela; Leonhardt, Stefan; Schwarz, Thomas; Schuerlein, Sebastian; Lotz, Christian; Hansmann, Jan

    2018-01-01

    The cornea is the most-transplanted tissue worldwide. However, the availability and quality of grafts are limited due to the current methods of corneal storage. In this study, a dynamic bioreactor system is employed to enable the control of intraocular pressure and the culture at the air-liquid interface. Thereby, in vivo-like storage conditions are achieved. Different media combinations for endothelium and epithelium are tested in standard and dynamic conditions to enhance the viability of the tissue. In contrast to culture conditions used in eye banks, the combination of the bioreactor and biochrom medium 1 allows to preserve the corneal endothelium and the epithelium. Assessment of transparency, swelling, and the trans-epithelial-electrical-resistance (TEER) strengthens the impact of the in vivo-like tissue culture. For example, compared to corneas stored under static conditions, significantly lower optical densities and significantly higher TEER values were measured (p-value <0.05). Furthermore, healing of epithelial defects is enabled in the bioreactor, characterized by re-epithelialization and initiated stromal regeneration. Based on the obtained results, an easy-to-use 3D-printed bioreactor composed of only two parts was derived to translate the technology from the laboratory to the eye banks. This optimized bioreactor facilitates noninvasive microscopic monitoring. The improved storage conditions ameliorate the quality of corneal grafts and the storage time in the eye banks to increase availability and reduce re-grafting. © 2017 The Authors. Biotechnology Journal Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  2. Effects of Apollo 12 lunar material on lipid levels of tobacco tissue and slash pine cultures

    Science.gov (United States)

    Weete, J. D.

    1972-01-01

    Investigations of the lipid components of pine tissues (Pinus elloitii) are discussed, emphasizing fatty acids and steroids. The response by slash pine tissue cultures to growth in contact with Apollo lunar soil, earth basalt, and Iowa soil is studied. Tissue cultures of tobacco grown for 12 weeks in contact with lunar material from Apollo 12 flight contained 21 to 35 percent more total pigment than control tissues. No differences were noted in the fresh or dry weight of the experimental and control samples.

  3. Chemical evaluation of strawberry plants produced by tissue culturing of gamma irradiated seedlings

    International Nuclear Information System (INIS)

    Maraei, R.W.

    2007-01-01

    studies were conducted to evaluate the influence of gamma irradiation as a supplementary factor precedes tissue culture application on strawberry seedlings (c.v.Rosa Linda). the strawberry seedling were irradiated using 8 doses of co 60 gamma rays 50.75.100.125 ,150,250, 350 and 500 gray. tissue culture technique was applied on irradiated and unirradiated strawberry seedling. different characteristics of plantlets, plant and fruit of strawberry produced from the double treatment (irradiation followed by tissue culture) were studied as well as the early, total and exportable fruit yields. data indicated that, low radiation doses 50,75 and 100 gray increased all morphological and chemical characteristics of the plantlets, plant and fruit of strawberry, whereas radiation doses higher than 100 gray decreased them significantly. moreover 350 and gray were lethal doses. radiation dose 50 gray increased the survival percentage and the length of plantlets by 1.5% and 50% respectively more than the unirradiated treatment in all multiplication stages

  4. Studies on the reaction in tissue culture of tomato genotypes under biotic stress

    Directory of Open Access Journals (Sweden)

    Ewa Hanus-Fajerska

    2014-01-01

    Full Text Available Plant regeneration in vitro from virus-infected somatic tomato (Lycopersicon sp. tissue was performed. Regeneration experiments were started after the determination of virus presence, using enzyme-linked immunosorbent assay, in leaves used as a source of explants. Leaf explants infected with selected strains of tomato mosaic Tobamovirus or cucumber mosaic Cucumovirus respectively, were cultured on a standarised MS agar medium to induce adventitious shoots, which were afterwards excised, rooted in vitro and cultured to plants. Explants were also screened for their ability to produce callus. Diverse effects of viral infection, ranging from stimulation to inhibition of callus formation and of morphogenesis rate, were observed. The health condition of the tissue proved to affect regeneration potential of Lycopersicon esculentum, whereas wild accesions did not react in that case so distinctly. In cultivated tomato was encountered the decline in competence to reproduce shoots adventitiously in infected tissue. There was also relationship between donor plant health condition and adventitious root formation in regenerated shoots. Experiments with short-term cultures of L. esculenum reveled also that a certain number of shoots regenerated from diseased tissue can be virus-free.

  5. Apollo 12 lunar material - Effects on lipid levels of tobacco tissue cultures.

    Science.gov (United States)

    Weete, J. D.; Walkinshaw, C. H.; Laseter, J. L.

    1972-01-01

    Tobacco tissue cultures grown in contact with lunar material from Apollo 12, for a 12-week period, resulted in fluctuations of both the relative and absolute concentrations of endogenous sterols and fatty acids. The experimental tissues contained higher concentrations of sterols than the controls did. The ratio of campesterol to stigmasterol was greater than 1 in control tissues, but less than 1 in the experimental tissues after 3 weeks. High relative concentrations (17.1 to 22.2 per cent) of an unidentified compound or compounds were found only in control tissues that were 3 to 9 weeks of age.

  6. Co-culture with infrapatellar fat pad differentially stimulates proteoglycan synthesis and accumulation in cartilage and meniscus tissues.

    Science.gov (United States)

    Nishimuta, James F; Bendernagel, Monica F; Levenston, Marc E

    2017-09-01

    Although osteoarthritis is widely viewed as a disease of the whole joint, relatively few studies have focused on interactions among joint tissues in joint homeostasis and degeneration. In particular, few studies have examined the effects of the infrapatellar fat pad (IFP) on cartilaginous tissues. The aim of this study was to test the hypothesis that co-culture with healthy IFP would induce degradation of cartilage and meniscus tissues. Bovine articular cartilage, meniscus, and IFP were cultured isolated or as cartilage-fat or meniscus-fat co-cultures for up to 14 days. Conditioned media were assayed for sulfated glycosaminoglycan (sGAG) content, nitrite content, and matrix metalloproteinase (MMP) activity, and explants were assayed for sGAG and DNA contents. Co-cultures exhibited increased cumulative sGAG release and sGAG release rates for both cartilage and meniscus, and the cartilage (but not meniscus) exhibited a substantial synergistic effect of co-culture (sGAG release in co-culture was significantly greater than the summed release from isolated cartilage and fat). Fat co-culture did not significantly alter the sGAG content of either cartilage or meniscus explants, indicating that IFP co-culture stimulated net sGAG production by cartilage. Nitrite release was increased relative to isolated tissue controls in co-cultured meniscus, but not the cartilage, with no synergistic effect of co-culture. Interestingly, MMP-2 production was decreased by co-culture for both cartilage and meniscus. This study demonstrates that healthy IFP may modulate joint homeostasis by stimulating sGAG production in cartilage. Counter to our hypothesis, healthy IFP did not promote degradation of either cartilage or meniscus tissues.

  7. Distribution of phospholipase C isozymes in various rat tissues and cultured cells

    International Nuclear Information System (INIS)

    Suh, P.G.; Ryu, S.H.; Choi, W.C.; Lee, K.Y.; Rhee, S.G.

    1987-01-01

    Monoclonal antibodies prepared against PLC-I or PLC-II enzyme did not cross-react with the other. Using a pair of antibodies which recognizes 2 different antigenic sites on the same molecule, radioimmunoassays were developed for the quantitation of PLC-I and PLC-II in homogenates of various tissues and cultured cells, prepared by homogenization in a 2 M KCl buffer. The contents of PLC enzymes were measured in 19 rat tissues, in human platelets and in 17 cultured cells. Results indicate that the concentration of PLC-I and PLC-II is very high in brain, PLC-I is localized mainly in brain and partly in seminal vesicles, PLC-II is found in most tissues and cells. PLC-I is highly localized even in brain: 5 different neuroblastoma did not contain PLC-I while 2 glioma and 1 astrocytoma contained significant amounts

  8. Glioma tissue obtained by modern ultrasonic aspiration with a simple sterile suction trap for primary cell culture and pathological evaluation.

    Science.gov (United States)

    Schroeteler, Juliane; Reeker, Ralf; Suero Molina, Eric; Brokinkel, Benjamin; Holling, Markus; Grauer, Oliver M; Senner, Volker; Stummer, Walter; Ewelt, Christian

    2014-01-01

    Ultrasonic aspiration is widely used in the resection of brain tumors. Nevertheless, tumor tissue fragments obtained by ultrasonic aspiration are usually discarded. In this study, we demonstrate that these fragments are possible sources of material for histopathological study and tissue culture and compare their microscopic features and viability in tissue culture of cavitron ultrasonic surgical aspirator tissue fragments. Brain tumor tissue collected by ultrasonic aspiration (CUSA EXcel®; Integra Radionics Inc.) in a simple sterile suction trap during resection was processed for primary cell culture. Cell viability and immunohistological markers were measured by the WST-1 test, microscopy and immunofluorescent evaluation. Six gliomas are presented to demonstrate that these tissue fragments show good preservation of histological detail and tissue viability in culture. Utilization of this material may facilitate pathological interpretation by providing a more representative sample of tumor histology as well as an adequate and sterile biosource of material for tissue culture studies.

  9. Comparison of Fluorescence Microscopy and Different Growth Media Culture Methods for Acanthamoeba Keratitis Diagnosis.

    Science.gov (United States)

    Peretz, Avi; Geffen, Yuval; Socea, Soergiu D; Pastukh, Nina; Graffi, Shmuel

    2015-08-01

    Acanthamoeba keratitis (AK), a potentially blinding infection of the cornea, is caused by a free-living protozoan. Culture and microscopic examination of corneal scraping tissue material is the conventional method for identifying Acanthamoeba. In this article, we compared several methods for AK diagnosis of 32 patients: microscopic examination using fluorescent dye, specific culture on growth media-non-nutrient agar (NNA), culture on liquid growth media-peptone yeast glucose (PYG), and TYI-S-33. AK was found in 14 patients. Thirteen of the specimens were found AK positive by fluorescence microscopic examination, 11 specimens were found AK positive on PYG growth media, and 9 specimens were found AK positive on TYI-S-33 growth media. Only five specimens were found AK positive on NNA growth media. Therefore, we recommend using fluorescence microscopy technique and culture method, especially PYG liquid media. © The American Society of Tropical Medicine and Hygiene.

  10. Estimation of the in vitro eye irritating and inflammatory potential of lipopolysaccharide (LPS) and dust by using reconstituted human corneal epithelium tissue cultures

    DEFF Research Database (Denmark)

    Cao, Yi; Arenholt-Bindslev, Dorthe; Kjærgaard, Søren K

    2015-01-01

    CONTEXT: Eye irritation is a common complaint in indoor environment, but the causes have still not been identified among the multiple exposures in house environments. To identify the potential environmental factors responsible for eye irritation and study the possible mechanisms, an in vitro model...... AND CONCLUSION: LPS and dust showed in vitro eye irritating and inflammatory potential, and cytokines/chemokines like IL-1β and IL-8 may be involved in the mechanisms of eye irritation. The HCE tissue culture may be used as an in vitro model to study environmental exposure induced eye irritation and inflammation....... for eye irritation is suggested. MATERIALS AND METHODS: In this study, reconstituted human corneal epithelium (HCE) tissue cultures were used to study the eye irritating and inflammatory potential of lipopolysaccharide (LPS) and dust. HCE tissue cultures were exposed to a range of concentrations of LPS...

  11. Prevention of pink-pigmented methylotrophic bacteria (Methylohacterium mesophilicum) contamination of plant tissue cultures.

    Science.gov (United States)

    Chanprame, S; Todd, J J; Widholm, J M

    1996-12-01

    Pink-pigmented facultative methylotrophic bacteria (PPFMs) have been found on the surfaces of leaves of most plants tested. We found PPFMs on the leaf surfaces of all 40 plants (38 species) tested and on soybean pods by pressing onto AMS medium with methanol as the sole carbon source. The abundance ranged from 0.5 colony forming unit (cfu) /cm(2) to 69.4 cfu/cm(2) on the leaf surfaces. PPFMs were found in homogenized leaf tissues of only 4 of the species after surface disinfestation with 1.05% sodium hypochlorite and were rarely found in cultures initiated from surface disinfested Datura innoxia leaves or inside surface disinfested soybean pods. Of 20 antibiotics tested for PPFM growth inhibition, rifampicin was the most effective and of seven others which also inhibited PPFM growth, cefotaxime should be the most useful due to the expected low plant cell toxicity. These antibiotics could be used in concert with common surface sterilization procedures to prevent the introduction or to eliminate PPFM bacteria in tissue cultures. Thus, while PPFMs are present on the surfaces of most plant tissues, surface disinfestation alone can effectively remove them so that uncontaminated tissue cultures can be initiated in most cases.

  12. Plant regeneration from petiole segments of some species in tissue culture

    Directory of Open Access Journals (Sweden)

    Krystyna Klimaszewska

    2013-12-01

    Full Text Available The regeneration ability of 21 plant species belonging to 14 families was tested. The method of tissue culture in vitro was applied, on basic MS medium with an addition of growth regulators from the auxin and cytokinin groups. From among the investigated plant groups Peperomia scandens and Caladium × hortulanum were capable of plant regeneration, Passiilora coerulea regenerated shoots, Hedera helix, Begonia glabra, Coleus blumei, Fuchsia hybrida, Passiflora suberosa and Peperomia eburnea formed callus and roots, Kalanchoe blossfeldiana, Pelargonium grandiflorum, P. peltatum, P. radula, Coleus shirensis and Magnolia soulangeana produced callus, Philodendron scandens, Rhododendron smirnovii, Hibiscus rosa-sinensis, Coprosma baueri, Cestrum purpureum and Solanum rantonnetii did not exhibit any regeneration reactions.

  13. Long-term culture of human liver tissue with advanced hepatic functions.

    Science.gov (United States)

    Ng, Soon Seng; Xiong, Anming; Nguyen, Khanh; Masek, Marilyn; No, Da Yoon; Elazar, Menashe; Shteyer, Eyal; Winters, Mark A; Voedisch, Amy; Shaw, Kate; Rashid, Sheikh Tamir; Frank, Curtis W; Cho, Nam Joon; Glenn, Jeffrey S

    2017-06-02

    A major challenge for studying authentic liver cell function and cell replacement therapies is that primary human hepatocytes rapidly lose their advanced function in conventional, 2-dimensional culture platforms. Here, we describe the fabrication of 3-dimensional hexagonally arrayed lobular human liver tissues inspired by the liver's natural architecture. The engineered liver tissues exhibit key features of advanced differentiation, such as human-specific cytochrome P450-mediated drug metabolism and the ability to support efficient infection with patient-derived inoculums of hepatitis C virus. The tissues permit the assessment of antiviral agents and maintain their advanced functions for over 5 months in culture. This extended functionality enabled the prediction of a fatal human-specific hepatotoxicity caused by fialuridine (FIAU), which had escaped detection by preclinical models and short-term clinical studies. The results obtained with the engineered human liver tissue in this study provide proof-of-concept determination of human-specific drug metabolism, demonstrate the ability to support infection with human hepatitis virus derived from an infected patient and subsequent antiviral drug testing against said infection, and facilitate detection of human-specific drug hepatotoxicity associated with late-onset liver failure. Looking forward, the scalability and biocompatibility of the scaffold are also ideal for future cell replacement therapeutic strategies.

  14. Mass Spectrometry-Based Proteomics in Molecular Diagnostics: Discovery of Cancer Biomarkers Using Tissue Culture

    Directory of Open Access Journals (Sweden)

    Debasish Paul

    2013-01-01

    Full Text Available Accurate diagnosis and proper monitoring of cancer patients remain a key obstacle for successful cancer treatment and prevention. Therein comes the need for biomarker discovery, which is crucial to the current oncological and other clinical practices having the potential to impact the diagnosis and prognosis. In fact, most of the biomarkers have been discovered utilizing the proteomics-based approaches. Although high-throughput mass spectrometry-based proteomic approaches like SILAC, 2D-DIGE, and iTRAQ are filling up the pitfalls of the conventional techniques, still serum proteomics importunately poses hurdle in overcoming a wide range of protein concentrations, and also the availability of patient tissue samples is a limitation for the biomarker discovery. Thus, researchers have looked for alternatives, and profiling of candidate biomarkers through tissue culture of tumor cell lines comes up as a promising option. It is a rich source of tumor cell-derived proteins, thereby, representing a wide array of potential biomarkers. Interestingly, most of the clinical biomarkers in use today (CA 125, CA 15.3, CA 19.9, and PSA were discovered through tissue culture-based system and tissue extracts. This paper tries to emphasize the tissue culture-based discovery of candidate biomarkers through various mass spectrometry-based proteomic approaches.

  15. Mass Spectrometry-Based Proteomics in Molecular Diagnostics: Discovery of Cancer Biomarkers Using Tissue Culture

    Science.gov (United States)

    Paul, Debasish; Kumar, Avinash; Gajbhiye, Akshada; Santra, Manas K.; Srikanth, Rapole

    2013-01-01

    Accurate diagnosis and proper monitoring of cancer patients remain a key obstacle for successful cancer treatment and prevention. Therein comes the need for biomarker discovery, which is crucial to the current oncological and other clinical practices having the potential to impact the diagnosis and prognosis. In fact, most of the biomarkers have been discovered utilizing the proteomics-based approaches. Although high-throughput mass spectrometry-based proteomic approaches like SILAC, 2D-DIGE, and iTRAQ are filling up the pitfalls of the conventional techniques, still serum proteomics importunately poses hurdle in overcoming a wide range of protein concentrations, and also the availability of patient tissue samples is a limitation for the biomarker discovery. Thus, researchers have looked for alternatives, and profiling of candidate biomarkers through tissue culture of tumor cell lines comes up as a promising option. It is a rich source of tumor cell-derived proteins, thereby, representing a wide array of potential biomarkers. Interestingly, most of the clinical biomarkers in use today (CA 125, CA 15.3, CA 19.9, and PSA) were discovered through tissue culture-based system and tissue extracts. This paper tries to emphasize the tissue culture-based discovery of candidate biomarkers through various mass spectrometry-based proteomic approaches. PMID:23586059

  16. Enlargement of induced variations by combined method of chronic irradiations with callus culture in sugarcane

    International Nuclear Information System (INIS)

    Nagatomi, Shigeki

    1993-01-01

    The present study was conducted to elucidate the effects of gamma ray irradiation and callus culture upon induced variation of the regeneratives. The populations regenerated from young leaf tissue of chronic irradiated plnats grown under a gamma field receiving a total dose of 300 and 100 Gy, showed rather wider variation on quantitative characters than plants from populations of the non-irradiated. This variation extended in both negative and positive directions. Analysis of variance also revealed that variation and heritability in broad sense of most agronomic characters increased significantly among the subclones as the irradiation done rose. Principal component analysis also indicated that the subclones from the irradiated population were more variable than the non-irradiated. Such variation with higher heritability could be transmitted to the following generations by clonal propagation and utilized as genetic sources in mutation breeding. The combined method with chronic irradiation followed by tissue culture is evaluated as an effective method of widening mutation spectrum and increasing mutation frequency in regenerated plants. In addition, this method is valid to improve any crop species which can regenerate plants through callus culture. (author)

  17. Three-dimensional spheroid culture targeting versatile tissue bioassays using a PDMS-based hanging drop array.

    Science.gov (United States)

    Kuo, Ching-Te; Wang, Jong-Yueh; Lin, Yu-Fen; Wo, Andrew M; Chen, Benjamin P C; Lee, Hsinyu

    2017-06-29

    Biomaterial-based tissue culture platforms have emerged as useful tools to mimic in vivo physiological microenvironments in experimental cell biology and clinical studies. We describe herein a three-dimensional (3D) tissue culture platform using a polydimethylsiloxane (PDMS)-based hanging drop array (PDMS-HDA) methodology. Multicellular spheroids can be achieved within 24 h and further boosted by incorporating collagen fibrils in PDMS-HDA. In addition, the spheroids generated from different human tumor cells exhibited distinct sensitivities toward drug chemotherapeutic agents and radiation as compared with two-dimensional (2D) cultures that often lack in vivo-like biological insights. We also demonstrated that multicellular spheroids may enable key hallmarks of tissue-based bioassays, including drug screening, tumor dissemination, cell co-culture, and tumor invasion. Taken together, these results offer new opportunities not only to achieve the active control of 3D multicellular spheroids on demand, but also to establish a rapid and cost-effective platform to study anti-cancer therapeutics and tumor microenvironments.

  18. Cost-effective nutrient sources for tissue culture of cassava ( Manihot ...

    African Journals Online (AJOL)

    Application of tissue culture technology is constrained by high costs making seedlings unaffordable. The objective of this study was to evaluate the possibility of using locally available fertilizers as alternative nutrient sources for cassava micropropagation. A Low Cost Medium (LCM) whereby the conventional sources of four ...

  19. The effect of plant growth regulators on optimization of tissue culture ...

    African Journals Online (AJOL)

    USER

    2010-04-05

    Apr 5, 2010 ... ISSN 1684–5315 © 2010 Academic Journals ... tissue culture system in Malaysian upland rice ... Scientists believe that using new cultivars which have potential ..... providing the financial support and to Firouzeh Ashjazadeh ...

  20. Anaerobic Cultures from Preserved Tissues of Baby Mammoth

    Science.gov (United States)

    Pikuta, Elena V.; Hoover, Richard B.; Fisher, Daniel

    2011-01-01

    Microbiological analysis of several cold-preserved tissue samples from the Siberian baby mammoth known as Lyuba revealed a number of culturable bacterial strains that were grown on anaerobic media at 4 C. Lactic acid produced by LAB (lactic acid bacteria) group, usually by members of the genera Carnobacterium and Lactosphera, appears to be a wonderful preservative that prevents other bacteria from over-dominating a system. Permafrost and lactic acid preserved the body of this one-month old baby mammoth and kept it in exceptionally good condition, resulting in this mammoth being the most complete such specimen ever recovered. The diversity of novel anaerobic isolates was expressed on morphological, physiological and phylogenetic levels. Here we discuss the specifics of the isolation of new strains, differentiation from trivial contamination, and preliminary results for the characterization of cultures.

  1. The gene expression profile of non-cultured, highly purified human adipose tissue pericytes: Transcriptomic evidence that pericytes are stem cells in human adipose tissue

    Energy Technology Data Exchange (ETDEWEB)

    Silva Meirelles, Lindolfo da, E-mail: lindolfomeirelles@gmail.com [Center for Cell-Based Therapy (CEPID/FAPESP), Regional Center for Hemotherapy of Ribeirão Preto, University of São Paulo, Rua Tenente Catão Roxo 2501, 14051-140 Ribeirão Preto, SP (Brazil); Laboratory for Stem Cells and Tissue Engineering, PPGBioSaúde, Lutheran University of Brazil, Av. Farroupilha 8001, 92425-900 Canoas, RS (Brazil); Deus Wagatsuma, Virgínia Mara de; Malta, Tathiane Maistro; Bonini Palma, Patrícia Viana [Center for Cell-Based Therapy (CEPID/FAPESP), Regional Center for Hemotherapy of Ribeirão Preto, University of São Paulo, Rua Tenente Catão Roxo 2501, 14051-140 Ribeirão Preto, SP (Brazil); Araújo, Amélia Goes; Panepucci, Rodrigo Alexandre [Laboratory of Large-Scale Functional Biology (LLSFBio), Regional Center for Hemotherapy of Ribeirão Preto, University of São Paulo, Rua Tenente Catão Roxo 2501, 14051-140 Ribeirão Preto, SP (Brazil); and others

    2016-12-10

    Pericytes (PCs) are a subset of perivascular cells that can give rise to mesenchymal stromal cells (MSCs) when culture-expanded, and are postulated to give rise to MSC-like cells during tissue repair in vivo. PCs have been suggested to behave as stem cells (SCs) in situ in animal models, although evidence for this role in humans is lacking. Here, we analyzed the transcriptomes of highly purified, non-cultured adipose tissue (AT)-derived PCs (ATPCs) to detect gene expression changes that occur as they acquire MSC characteristics in vitro, and evaluated the hypothesis that human ATPCs exhibit a gene expression profile compatible with an AT SC phenotype. The results showed ATPCs are non-proliferative and express genes characteristic not only of PCs, but also of AT stem/progenitor cells. Additional analyses defined a gene expression signature for ATPCs, and revealed putative novel ATPC markers. Almost all AT stem/progenitor cell genes differentially expressed by ATPCs were not expressed by ATMSCs or culture-expanded ATPCs. Genes expressed by ATMSCs but not by ATPCs were also identified. These findings strengthen the hypothesis that PCs are SCs in vascularized tissues, highlight gene expression changes they undergo as they assume an MSC phenotype, and provide new insights into PC biology. - Highlights: • Non-cultured adipose tissue-derived human pericytes (ncATPCs) exhibit a distinctive gene expression signature. • ncATPCs express key adipose tissue stem cell genes previously described in vivo in mice. • ncATPCs express message for anti-proliferative and antiangiogenic molecules. • Most ncATPC-specific transcripts are absent in culture-expanded pericytes or ATMSCs • Gene expression changes ncATPCs undergo as they acquire a cultured ATMSC phenotype are pointed out.

  2. The gene expression profile of non-cultured, highly purified human adipose tissue pericytes: Transcriptomic evidence that pericytes are stem cells in human adipose tissue

    International Nuclear Information System (INIS)

    Silva Meirelles, Lindolfo da; Deus Wagatsuma, Virgínia Mara de; Malta, Tathiane Maistro; Bonini Palma, Patrícia Viana; Araújo, Amélia Goes; Panepucci, Rodrigo Alexandre

    2016-01-01

    Pericytes (PCs) are a subset of perivascular cells that can give rise to mesenchymal stromal cells (MSCs) when culture-expanded, and are postulated to give rise to MSC-like cells during tissue repair in vivo. PCs have been suggested to behave as stem cells (SCs) in situ in animal models, although evidence for this role in humans is lacking. Here, we analyzed the transcriptomes of highly purified, non-cultured adipose tissue (AT)-derived PCs (ATPCs) to detect gene expression changes that occur as they acquire MSC characteristics in vitro, and evaluated the hypothesis that human ATPCs exhibit a gene expression profile compatible with an AT SC phenotype. The results showed ATPCs are non-proliferative and express genes characteristic not only of PCs, but also of AT stem/progenitor cells. Additional analyses defined a gene expression signature for ATPCs, and revealed putative novel ATPC markers. Almost all AT stem/progenitor cell genes differentially expressed by ATPCs were not expressed by ATMSCs or culture-expanded ATPCs. Genes expressed by ATMSCs but not by ATPCs were also identified. These findings strengthen the hypothesis that PCs are SCs in vascularized tissues, highlight gene expression changes they undergo as they assume an MSC phenotype, and provide new insights into PC biology. - Highlights: • Non-cultured adipose tissue-derived human pericytes (ncATPCs) exhibit a distinctive gene expression signature. • ncATPCs express key adipose tissue stem cell genes previously described in vivo in mice. • ncATPCs express message for anti-proliferative and antiangiogenic molecules. • Most ncATPC-specific transcripts are absent in culture-expanded pericytes or ATMSCs • Gene expression changes ncATPCs undergo as they acquire a cultured ATMSC phenotype are pointed out.

  3. Clonal multiplication of Cymbidiums through tissue culture of the shoot meristem

    Energy Technology Data Exchange (ETDEWEB)

    Wimber, Donald E.

    1963-09-01

    The propagation of clonal varieties of some orchids is at times exasperatingly slow and occasionally an almost futile effort. Clonal multiplication is generally confined to dlvidlng mature plants and to starting plants from pseudobulbs. There is, of course, the specialized technique for obtaining Phalaenopsis plantlets from the aseptic culture of inflorescence nodes, but this is basically the same thing as propagating plants from pseudobulbs. In certain cases it is highly desirable to rapidly multiply certain clones of orchids. Awarded varieties could thereby be dispersed with great rapidity where now it may take decades for some clones to became fairly common. Commercial flower production would be very much enhanced if certain desirable clones could be multiplied ad infinitum within a short time. Orchid flower production could then be placed more on a par with many of the other cut flowers and the clonal peculiarities of some fo the current hybrids could be pampered instead of ignored. This paper describes a tissue culture method for the rapid propagation of Cymbidium clones.

  4. INVESTIGATION OF HYPOLIPIDEMIC EFFECT OF SESQUITERPENE Γ-LACTONE AHILLIN IN HEPATOMA TISSUE CULTURE (HTC CELLS

    Directory of Open Access Journals (Sweden)

    V. V. Ivanov

    2014-01-01

    Full Text Available Objective. Investigation of hypolipidemic effect of sesquiterpene γ-lactone ahillin in hepatoma tissue culture (HTC cells.Material and methods. In this study we’ve evaluated the effect of γ-lactone sesquiterpene aсhillin and gemfibrozil (comparator drug on the lipid content in the hepatoma tissue culture (HTC cell which were incubated with a fat emulsion lipofundin by fluorescent method with vital dye Nile Redand staining the cells with the dye Oil Red O. The cell viability was investigated using the MTT-test and staining with Trypan blue.Results. Cultivation cells HTC with aсhillin and gemfibrozilat concentrations ranging from 0.5 to1.5 mM and from0.25 mM to0.5 mM, respectively, resulted in dose-dependent decrease of the fluorescence’s intensity Nile Red. It reflects a decrease in lipid content in the cells. At these concentrations the drugs didn’t have cytotoxic effect and the cell viability didn’t change compared to the control culture.An experimental hyperlipidemia in the hepatoma culture cells was induced by adding to the incubation medium a fat emulsion lipofundin at a final concentration 0.05%. The intensity of fluorescence Nile Red in the cells was increased 4 fold (p < 0.05. This result suggests the significant accumulation of lipids in the cell’s cytosol and confirmed by microscopy after staining neutral lipids with the dye Oil Red O. Under these conditions aсhillin and gemfibrozil reduced lipid content in cells and hadthe effect at concentrations of0.5 mM and0.25 mM respectively.Conclusion. In the lipofundin-mediated model of hyperlipidemia the sesquiterpene lactone aсhillin prevents the lipid accumulation in cells. It confirms by decrease of fluorescence Nile Red and reduction lipid drops which were stained with Oil Red O in cytosol. To establish the molecular targets of aсhillin’saction on lipid metabolism in cell culture HTC we need to investigate a gene expression of key enzymes of lipid metabolism.

  5. Rapid detection of Mannheimia haemolytica in lung tissues of sheep and from bacterial culture

    Directory of Open Access Journals (Sweden)

    Jyoti Kumar

    2015-09-01

    Full Text Available Aim: This study was aimed to detect Mannheimia haemolytica in lung tissues of sheep and from a bacterial culture. Introduction: M. haemolytica is one of the most important and well-established etiological agents of pneumonia in sheep and other ruminants throughout the world. Accurate diagnosis of M. haemolytica primarily relies on bacteriological examination, biochemical characteristics and, biotyping and serotyping of the isolates. In an effort to facilitate rapid M. haemolytica detection, polymerase chain reaction assay targeting Pasteurella haemolytica serotype-1 specific antigens (PHSSA, Rpt2 and 12S ribosomal RNA (rRNA genes were used to detect M. haemolytica directly from lung tissues and from bacterial culture. Materials and Methods: A total of 12 archived lung tissues from sheep that died of pneumonia on an organized farm were used. A multiplex polymerase chain reaction (mPCR based on two-amplicons targeted PHSSA and Rpt2 genes of M. haemolytica were used for identification of M. haemolytica isolates in culture from the lung samples. All the 12 lung tissue samples were tested for the presence M. haemolytica by PHSSA and Rpt2 genes based PCR and its confirmation by sequencing of the amplicons. Results: All the 12 lung tissue samples tested for the presence of PHSSA and Rpt2 genes of M. haemolytica by mPCR were found to be positive. Amplification of 12S rRNA gene fragment as internal amplification control was obtained with each mPCR reaction performed from DNA extracted directly from lung tissue samples. All the M. haemolytica were also positive for mPCR. No amplified DNA bands were observed for negative control reactions. All the three nucleotide sequences were deposited in NCBI GenBank (Accession No. KJ534629, KJ534630 and KJ534631. Sequencing of the amplified products revealed the identity of 99-100%, with published sequence of PHSSA and Rpt2 genes of M. haemolytica available in the NCBI database. Sheep specific mitochondrial 12S r

  6. In vitro differentiation of rat spermatogonia into round spermatids in tissue culture.

    Science.gov (United States)

    Reda, A; Hou, M; Winton, T R; Chapin, R E; Söder, O; Stukenborg, J-B

    2016-09-01

    Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in

  7. Effect of radiation and other cytotoxic agents on the growth of cells cultured from normal and tumor tissues from the female genital tract

    International Nuclear Information System (INIS)

    Mothersill, C.; Seymour, C.B.; Bonnar, J.

    1990-01-01

    A technique is presented which allows the response of human gynecological tissue to radiation and cytotoxic drugs to be assessed using a tissue culture explant system. The technique is simple to use and gives results in line with those obtained for human tissues by more complex culture methods. Data are presented showing how the explant technique developed by the group for other tissues can be adapted to yield acceptable results for normal tissue response to radiation. The potential of the technique for use in predictive testing of individual tumor response is then assessed in five cases of gynecological malignancy. It is clear that variations in sensitivity to different radio- and chemotherapy agents and combinations can be detected. The results obtained require clinical validation and it is hoped that this will come over the next few years from evaluation of patient response to treatment using individually optimized, rather than empirical therapy

  8. Biocompatible, biodegradable polymer-based, lighter than or light as water scaffolds for tissue engineering and methods for preparation and use thereof

    Science.gov (United States)

    Khan, Mohammed Yusuf (Inventor); Laurencin, Cato T. (Inventor); Lu, Helen H. (Inventor); Botchwey, Edward (Inventor); Pollack, Solomon R. (Inventor); Levine, Elliot (Inventor)

    2012-01-01

    Scaffolds for tissue engineering prepared from biocompatible, biodegradable polymer-based, lighter than or light as water microcarriers and designed for cell culturing in vitro in a rotating bioreactor are provided. Methods for preparation and use of these scaffolds as tissue engineering devices are also provided.

  9. Tissue culture-induced alteration in cytosine methylation in new rice ...

    African Journals Online (AJOL)

    Zizania DNA introgression could induce a large number of genetic and epigenetic changes of the new rice recombinant inbred lines genome. In this present study, we employed inter-simple sequence repeat (ISSR) to further study the genetic and epigenetic changes that are induced by tissue culture. Changes induced by ...

  10. Method and Apparatus for a Miniature Bioreactor System for Long-Term Cell Culture

    Science.gov (United States)

    Kleis, Stanley J. (Inventor); Geffert, Sandra K. (Inventor); Gonda, Steve R. (Inventor)

    2015-01-01

    A bioreactor and method that permits continuous and simultaneous short, moderate, or long term cell culturing of one or more cell types or tissue in a laminar flow configuration is disclosed, where the bioreactor supports at least two laminar flow zones, which are isolated by laminar flow without the need for physical barriers between the zones. The bioreactors of this invention are ideally suited for studying short, moderate and long term studies of cell cultures and the response of cell cultures to one or more stressors such as pharmaceuticals, hypoxia, pathogens, or any other stressor. The bioreactors of this invention are also ideally suited for short, moderate or long term cell culturing with periodic cell harvesting and/or medium processing for secreted cellular components.

  11. Cells in human postmortem brain tissue slices remain alive for several weeks in culture

    NARCIS (Netherlands)

    Verwer, Ronald W. H.; Hermens, Wim T. J. M. C.; Dijkhuizen, PaulaA; ter Brake, Olivier; Baker, Robert E.; Salehi, Ahmad; Sluiter, Arja A.; Kok, Marloes J. M.; Muller, Linda J.; Verhaagen, Joost; Swaab, Dick F.

    2002-01-01

    Animal models for human neurological and psychiatric diseases only partially mimic the underlying pathogenic processes. Therefore, we investigated the potential use of cultured postmortem brain tissue from adult neurological patients and controls. The present study shows that human brain tissue

  12. Citrus tissue culture employing vegetative explants.

    Science.gov (United States)

    Chaturvedi, H C; Singh, S K; Sharma, A K; Agnihotri, S

    2001-11-01

    Citrus being a number one fruit of the world due to its high nutritional value, huge production of fruits and fruit products, the citrus industry may be considered a major fruit industry. Though citrus orchard area in India is comparable to USA, the produce is far less, while its export is nil. Biotechnology has played an outstanding role in boosting the citrus industry, e.g., in Spain, which is now the biggest exporter of citrus fruit with the application of micrografting. Amongst the fruit trees, perhaps the maximum tissue culture research has been done in citrus during the past four decades, however, the results of practical value are meagre. The shortfalls in citrus tissue culture research and some advancements made in this direction along with bright prospects are highlighted, restricting the review to vegetative explants only. Whilst utilization of nucellar embryogenesis is limited to rootstocks, the other aspects, like, regeneration and proliferation of shoot meristems measuring 200 microm in length--a global breakthrough--of two commercially important scion species, Citrus aurantifolia and C. sinensis and an important rootstock, C. limonia, improvement of micrografting technique, cloning of the same two scion species as well as some Indian rootstock species, employing nodal stem segments of mature trees, of immense practical value have been elaborated. A rare phenomenon of shift in the morphogenetic pattern of differentiation from shoot bud differentiation to embryoid formation occurred during the long-term culture of stem callus of C. grandis. Stem callus-regenerated plants of C. aurantifolia, C. sinensis and C. grandis showed variation in their ploidy levels and a somaclonal variant of C. sinensis, which produced seedless fruits was isolated. Tailoring of rooting in microshoots to a tap root-like system by changing the inorganic salt composition of the rooting medium, resulting in 100% transplant success, and germplasm preservation through normal growth

  13. An efficient method for the establishment of cell suspension cultures in potato (Solanum tuberosum L.)

    International Nuclear Information System (INIS)

    Sajid, Z.A.

    2016-01-01

    Cell suspension cultures offers an In vitro system that can be used as a tool for various studies involving mutant selection, mass propagation, protoplast isolation, gene transfer and selection of cell-lines which are resistant to various biotic or abiotic stresses. Research work on the development of cell suspension cultures was carried out to establish the most efficient method in Potato (cv. Desiree). Healthy, well-proliferating tissues from different types of callus cultures (compact, friable, embryogenic or non-embryogenic) were inoculated on various media combinations, i.e., MS, MS2 or AA liquid medium containing 18.09 micro M 2, 4-D. A fixed quantity (0.5-1.0 g) of callus tissue from 60-day-old callus cultures was transferred to 10-25 ml of liquid medium in 100 ml Erlenmeyer flask. Cultures were placed on an orbital shaker and agitated at different speeds (75, 100 or 125 rpm) under 16-h photoperiod at 25 ± 2 degree C. Medium was changed after every 3 days and fractionated tissue was filtered after every 6 days through sterile mesh (100-800 micro m) to develop a cell-line by transferring resulting suspension to fresh medium under the same conditions. Results indicated that eight-week-old translucent, friable, off-white callus cultures were an excellent starting material for the initiation of homogeneous cell suspension cultures as compared to other tested sources. Of the three tested media (MS, MS2 or AA medium containing 18.09 micro M 2, 4-D), MS2 was found to be a better medium for the initiation of cell suspension cultures. Cell suspension cultures, placed in 16-h photoperiod at 25 ± 2 degree C and agitated at 120 rpm using a gyratory shaker showed excellent results. Several other factors influencing quick establishment of cell suspension cultures in this cultivar are also discussed in this communication. (author)

  14. Canine osteosarcoma karyotypes from an original tumor, its metastasis, and tumor cells in tissue culture

    International Nuclear Information System (INIS)

    Taylor, N.; Shifrine, M.; Wolf, H.G.; Trommershausen-Smith, A.

    1975-01-01

    Radiation-induced osteosarcoma, its metastasis, and cells grown in tissue culture were karyotyped. Both hypodiploid and hyperdiploid stem lines were observed. The hypodiploid line contained 45-55 chromosomes with 10 to 15 abnormal metacentric and submetacentric chromosomes and one subtelocentric marker. The hyperdiploid line contained 90 to 105 chromosomes with 20 to 30 abnormal metacentric and submetacentric chromosomes with two subtelocentric markers. Karyotypic analysis can be used to monitor osteosarcomas maintained in tissue culture

  15. [Variability of nuclear 18S-25S rDNA of Gentiana lutea L. in nature and in tissue culture in vitro].

    Science.gov (United States)

    Mel'nyk, V M; Spiridonova, K V; Andrieiev, I O; Strashniuk, N M; Kunakh, V A

    2004-01-01

    18S-25S rDNA sequence in genomes of G. lutea plants from different natural populations and from tissue culture has been studied with blot-hybridization method. It was shown that ribosomal repeats are represented by the variants which differ for their size and for the presence of additional HindIII restriction site. Genome of individual plant usually possesses several variants of DNA repeats. Interpopulation variability according to their quantitative ratio and to the presence of some of them has been shown. Modifications of the range of rDNA repeats not exceeding intraspecific variability were observed in callus tissues in comparison with the plants of initial population. Non-randomness of genome modifications in the course of cell adaptation to in vitro conditions makes it possible to some extent to forecast these modifications in tissue culture.

  16. Tissue culture of three species of Laurencia complex

    Science.gov (United States)

    Shen, Songdong; Wu, Xunjian; Yan, Binlun; He, Lihong

    2010-05-01

    To establish a micropropagation system of three Laurencia complex species ( Laurencia okamurai, Laurencia tristicha, and Chondrophycus undulatus) by tissue culture techniques, we studied the regeneration characteristics and optimal culture conditions of axenic algal fragments cultured on solid medium and in liquid medium. Regeneration structures were observed and counted regularly under a reverse microscope to investigate the regeneration process, polarity and optimal illumination, and temperature and salinity levels. The results show that in most cultures of the three species, we obtained bud regeneration on solidified medium with 0.5% agar and in liquid medium. Rhizoid-like regeneration was filamentous and developed from the lower cut surface of fragments in L. okamurai, but was discoid and developed from the apical back side of bud regeneration in L. tristicha and C. undulatus. Regeneration polarity was localized to the apical part of algal fronds in all three species, and on fragments cut from the basal part of algae buds could develop from both the upper and the lower cut surfaces. Buds could develop from both the medullary and the cortical portions in L. okamurai and C. undulatus, while in L. tristicha, buds only emerged from the cortex. The optimal culture conditions for L. okamurai were 4 500 lx, 20°C and 35 (salinity); for C. undulatus, 4 500 lx, 20°C and 30; and for L. tristicha, 4 500 lx, 25°C and 30.

  17. Low cost options for tissue culture technology in developing countries. Proceedings of a technical meeting

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2004-02-01

    Tissue culture technology is used for the production of doubled haploids, cryopreservation, propagating new plant varieties, conserving rare and endangered plants, difficult-to-propagate plants, and to produce secondary metabolites and transgenic plants. The production of high quality planting material of crop plants and fruit trees, propagated from vegetative parts, has created new opportunities in global trading, benefited growers, farmers, and nursery owners, and improved rural employment. However, there are still major opportunities to produce and distribute high quality planting material, e.g. crops like banana, date palm, cassava, pineapple, plantain, potato, sugarcane, sweet potato, yams, ornamentals, fruit and forest trees. The main advantage of tissue culture technology lies in the production of high quality and uniform planting material that can be multiplied on a year-round basis under disease-free conditions anywhere irrespective of the season and weather. However, the technology is capital, labor and energy intensive. Although, labor is cheap in many developing countries, the resources of trained personnel and equipment are often not readily available. In addition, energy, particularly electricity, and clean water are costly. The energy requirements for tissue culture technology depend on day temperature, day-length and relative humidity, and they have to be controlled during the process of propagation. Individual plant species also differ in their growth requirements. Hence, it is necessary to have low cost options for weaning, hardening of micropropagated plants and finally growing them in the field. This publication describes options for reducing costs to establish and operate tissue culture facilities and primarily focus on plant micropropagation. It includes papers on the basics of tissue culture technology, low cost options for the design of laboratories, use of culture media and containers, energy and labor saving, integration and adoption of

  18. Low cost options for tissue culture technology in developing countries. Proceedings of a technical meeting

    International Nuclear Information System (INIS)

    2004-02-01

    Tissue culture technology is used for the production of doubled haploids, cryopreservation, propagating new plant varieties, conserving rare and endangered plants, difficult-to-propagate plants, and to produce secondary metabolites and transgenic plants. The production of high quality planting material of crop plants and fruit trees, propagated from vegetative parts, has created new opportunities in global trading, benefited growers, farmers, and nursery owners, and improved rural employment. However, there are still major opportunities to produce and distribute high quality planting material, e.g. crops like banana, date palm, cassava, pineapple, plantain, potato, sugarcane, sweet potato, yams, ornamentals, fruit and forest trees. The main advantage of tissue culture technology lies in the production of high quality and uniform planting material that can be multiplied on a year-round basis under disease-free conditions anywhere irrespective of the season and weather. However, the technology is capital, labor and energy intensive. Although, labor is cheap in many developing countries, the resources of trained personnel and equipment are often not readily available. In addition, energy, particularly electricity, and clean water are costly. The energy requirements for tissue culture technology depend on day temperature, day-length and relative humidity, and they have to be controlled during the process of propagation. Individual plant species also differ in their growth requirements. Hence, it is necessary to have low cost options for weaning, hardening of micropropagated plants and finally growing them in the field. This publication describes options for reducing costs to establish and operate tissue culture facilities and primarily focus on plant micropropagation. It includes papers on the basics of tissue culture technology, low cost options for the design of laboratories, use of culture media and containers, energy and labor saving, integration and adoption of

  19. Tissue culture of osteogenic sarcoma in rats, induced by radioactive phosphorus P-32 and the effect of the anti-cancerous agents on these tumor cells under tissue culture

    International Nuclear Information System (INIS)

    Osaka, Shunzo

    1976-01-01

    Small pieces of osteogenic sarcoma, induced into albino rats of the C.F. Wistar strain by injection of radioactive phosphorus 32 P, were cultured in mixtures of Eagle's minimum essential medium and 20% calf serum. The tumor cells cultured in this way were transplanted into the subcutaneous tissue or the intraabdominal cavity to healthy albino rats. The effect of the anticancerous agents was evaluated by the decrease of nucleic acid composition in these cultured tumor cells. As anti-cancerous agents, cyclophosphamide (CPA), mitomycin C(MMC), and 5-fluorouracil(5-FU) were put into contact with the tumor cells in cultures for two hours under the following dilutions: CPA; 10 -6 , 10 -5 , 10 -4 g/ml. MMC; 2 x 10 -8 , 2 x 10 -7 , 2 x 10 -6 g/ml. 5-FU; 2 x 10 -6 , 2 x 10 -5 , 2 x 10 -4 g/ml. The results are as follows: Three of the seven osteogenic sarcomas in rats were successfully cultured, one of them through more than eighteen generations. After about five hundred thousand cultured cells had been transplanted into the subcutaneous tissues or abdominal cavities of rats, tumors grew in all of them. The histological findings of the tumors in the second generation were quite similar to those of the original tumor. The same process was repeated three times and the tumor showed histogical findings similar to those of the original ones. The capability of nucleic acid synthesis in these cells was decreased at twenty fours after CPA contact and at forty eight hours after MMC. (J.P.N.)

  20. Constructing Failure: Leonard Hayflick, Biomedicine, and the Problems with Tissue Culture.

    Science.gov (United States)

    Park, Hyung Wook

    2016-07-01

    By examining the use of tissue culture in post-war American biomedicine, this paper investigates how scientists experience and manage failure. I study how Leonard Hayflick forged his new definition of failure and ways of managing it by refuting Alexis Carrel's definition of failure alongside his theory of the immortality of cultured cells. Unlike Carrel, Hayflick claimed that every vertebrate somatic cell should eventually die, unless it transformed into a tumour cell. This claim defined cell death, which had been a problem leading to a laboratory failure, as a normal phenomenon. On the other hand, permanent life, which had been considered a normal cellular characteristic, became a major factor causing scientific failure, since it implied malignant transformation that scientists hoped to control. Hayflick then asserted that his cell strains and method would partly enable scientists to manage this factor-especially that occurred through viral infection-alongside other causes of failure in routine tasks, including bacterial contamination. I argue that the growing biomedical enterprise fostered this work of Hayflick's, which had repercussions in both his career and the uses of cells in diverse investigations. His redefinition of failure in the age of biomedicine resulted in the broad dissemination of his cells, medium, and method as well as his long struggle with the National Institutes of Health (NIH), which caused his temporarily failed career.

  1. Tobacco clones derived from tissue culture with supersensitivity to ozone

    International Nuclear Information System (INIS)

    Sun, E.J.; Kang, H.W.

    2003-01-01

    New tobacco clones supersensitive to ozone were obtained from tissue culture. - At least two supersensitive tobacco somaclones were obtained from tissue culture (TC) , when this approach was used to asexually propagate Bel-W3 tobacco indicator plants. These somaclones can detect as low as 30 ppb ozone for a 4-h exposure duration both within CSTR exposure chambers and in ambient air. Comparison of the injury index and their coefficient of variance showed that the TC plantlets usually have more uniform performance in response to ozone in addition to their higher sensitivity. A quick regeneration procedure was established to preserve the supersensitive germplasm immediately when it was found. The TC plantlets will flower and produce seed similar to seed-grown tobacco. The TC approach proved to be a better propagation system for valuable indicator plant species. The mechanism that causes the variation and the possible difference in their genome from seed-grown tobacco is still unknown. Further studies are needed in the future to determine if factors in the TC system may be responsible for the sensitivity difference

  2. Organ and plantlet regeneration of Menyanthes trifoliata through tissue culture

    Directory of Open Access Journals (Sweden)

    Urszula Adamczyk-Rogozińska

    2014-01-01

    Full Text Available The conditions for the regeneration of plants through organogenesis from callus tissues of Menyanthes trifoliata are described. The shoot multiplication rate was affected by basal culture media, the type and concentration of cytokinin and subculture number. The best response was obtained when caulogenic calli were cultured on the modified Schenk and Hildebrandt medium (SH-M containing indole-3-acetic acid (IAA 0,5 mg/l and 6-benzyladenine (BA 1 mg/l or zeatin (2 mg/l. Under these conditions ca 7 shoots (mostly 1 cm or more in length per culture in the 5th and 6th passages could be developed. In older cultures (after 11-12 passages there was a trend for more numerous but shorter shoot formation. All regenerated shoots could be rooted on the SH-M medium supplemented with 0.5 mg/l IAA within 6 weeks; 80% of in vitro rooted plantlets survived their transfer to soil.

  3. Treatment of chronic desquamative gingivitis using tissue-engineered human cultured gingival epithelial sheets: a case report.

    Science.gov (United States)

    Okuda, Kazuhiro; Momose, Manabu; Murata, Masashi; Saito, Yoshinori; lnoie, Masukazu; Shinohara, Chikara; Wolff, Larry F; Yoshie, Hiromasa

    2004-04-01

    Human cultured gingival epithelial sheets were used as an autologous grafting material for regenerating gingival tissue in the maxillary left and mandibular right quadrants of a patient with chronic desquamative gingivitis. Six months post-surgery in both treated areas, there were gains in keratinized gingiva and no signs of gingival inflammation compared to presurgery. In the maxillary left quadrant, preoperative histopathologic findings revealed the epithelium was separated from the connective tissue and inflammatory cells were extensive. After grafting with the gingival epithelial sheets, inflammatory cells were decreased and separation between epithelium and connective tissue was not observed. The human cultured gingival epithelial sheets fabricated using tissue engineering technology showed significant promise for gingival augmentation in periodontal therapy.

  4. Heritability of regeneration in tissue cultures of sweet potato (Ipomoea batatas L.).

    Science.gov (United States)

    Templeton-Somers, K M; Collins, W W

    1986-03-01

    A population of open-pollinated progeny from 12 parents, and the 12 parents, was surveyed for in vitro growth and regeneration characteristics. Four different tissue culture procedures involving different media and the use of different explants to initiate the cultures were used. Petiole explants from young leaves were used as explants for initiation of callus cultures. These were evaluated for callus growth rate, friability, and callus color and texture, before transferring to each of three different regeneration media for evaluation of morphogenetic potential. Small shoot tips also were used to initiate callus cultures, which were evaluated for the same growth characteristics and transferred to growth-regulator free regeneration media. Regeneration occurred through root or shoot regeneration or through embryogenesis. Tissue culture treatment effects, as well as genotypic effects, were highly significant in determining: the types of callus produced, callus growth rates, color and texture on the two types of media used for the second and third subcultures. The family x treatment interaction was generally not statistically significant, affecting only callus color. Estimates of narrow sense heritability for callus growth rate in both the second and third subcultures were high enough (0.35 and 0.63, respectively) for the evaluation of parental lines for selection procedures. These characteristics were also the only early culture callus traits that were consistently correlated with later morphogenesis of the cultures. They were negatively correlated with root or shoot regeneration. The occurence of somatic embryogenesis was not correlated with early callus growth characteristics. Genetic and treatment effects were highly significant in the evaluation of morphogenetic potential, through root or shoot regeneration, or through embryogenesis. Regeneration of all types was of low frequency for all procedures, expressed in ≦ 11% of the cultures of the total population.

  5. Culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in different media

    Directory of Open Access Journals (Sweden)

    Gesiane Ribeiro

    2013-12-01

    Full Text Available The objective of this study was to evaluate the culture of equine bone marrow mononuclear fraction and adipose tissue - derived stromal vascular fraction cells in two different cell culture media. Five adult horses were submitted to bone marrow aspiration from the sternum, and then from the adipose tissue of the gluteal region near the base of the tail. Mononuclear fraction and stromal vascular fraction were isolated from the samples and cultivated in DMEM medium supplemented with 10% fetal bovine serum or in AIM-V medium. The cultures were observed once a week with an inverted microscope, to perform a qualitative analysis of the morphology of the cells as well as the general appearance of the cell culture. Colony-forming units (CFU were counted on days 5, 15 and 25 of cell culture. During the first week of culture, differences were observed between the samples from the same source maintained in different culture media. The number of colonies was significantly higher in samples of bone marrow in relation to samples of adipose tissue.

  6. Autoradiographic demonstration of unscheduled DNA synthesis in oral tissues treated with chemical carcinogens in short-term organ culture

    International Nuclear Information System (INIS)

    Ide, F.; Umemura, S.; Ishikawa, T.; Takayama, S.

    1981-01-01

    A system in which oral tissues of inbred F344 adult rats and Syrian golden hamster embryos were used in combination with autoradiography was developed for measurement of unscheduled DNA synthesis (UDS). For this, oral mucosa, submandibular gland, tooth germ and mandible in short-term organ cultures were treated with 4-nitroquinoline l-oxide or N-methyl-N-nitrosourea plus (methyl- 3 H)thymidine. Significant numbers of silver grains, indicating UDS, were detected over the nuclei of cells of all these tissues except rat salivary gland after treatment with carcinogens. This autoradiographic method is suitable for detection of UDS in oral tissues in conditions mimicking those in vivo. Results obtained in this study indicated a potential use of this system for studies on the mechanism of carcinogenesis at a cellular level comparable to in vivo carcinogenesis studies on oral tissues. (author)

  7. Co-cultures and cell sheet engineering as relevant tools to improve the outcome of bone tissue engineering strategies

    OpenAIRE

    Pirraco, Rogério

    2011-01-01

    Taking into consideration the complex biology of bone tissue it is quite clear that the understanding of the cellular interactions that regulate the homeostasis and regeneration of this remarkable tissue is essential for a successful Tissue Engineering strategy. The in vitro study of these cellular interactions relies on co-culture systems, a tremendously useful methodology where two or more cell types are cultured at the same time. Such strategy increases the complexity of typ...

  8. Joint use of developed collagen-containing complexes and cell cultures in creating new tissue equivalents

    Directory of Open Access Journals (Sweden)

    K. V. Kulakova

    2016-01-01

    Full Text Available The purpose of the study is to assess the possibility of applying the integrated module as the basis of a celltissue equivalent for treatment of wounds of skin and soft tissues. In the frame of the set task the following problems were being solved: research of the spatial structure and architectonics of the surface of the developed base collagen-containing materials and their biocompatibility with cell cultures.Materials and methods. The study of a material which is a two-layer complex film, consisting of collagen and polysaccharide components was carried out. The collagen was separated from the dermis and was then impregnated with particulate demineralized bone matrix (DCM according to the original methodology. For the purposes of the study the dehydrated material was created in the form of a film. Electron microscopic examination of surfaces was performed on scanning electron microscope JEOL JSM-IT300LV in high vacuum and at low values of probe current (< 0,1 nА. Studies to assess the viability of the cells cultivated on films of collagen material (tested for cytotoxicity and the adhesive capacity were performed in vitro using strains of diploid human fibroblasts 4–6 passage. The culture condition was visually assessed using an inverted Leica microscope DM IL (Carl Zeiss, Austria, equipped with a computerizes program of control of culture growth (Leica IM 1000.Results. The data obtained in the study of the surface structure of the developed complex module showed that it seems to be promising as a basic component of the cellular-tissue system with its large number of structural formations for fixation of the cells and a well-organized barrier layer capable of vapor - permeability. Experiments in vitro confirmed the absence of toxicity of the material being studied in relation to the culture of dermal human fibroblasts, suggesting the possibility of creation on its basis of cell-tissue complex and further experimental studies in vivo

  9. Protocols for the in vitro design of animal articular cartilage based on tissue engineering methods

    Directory of Open Access Journals (Sweden)

    Diego Correa

    2002-01-01

    Full Text Available The articular cartilage is the structure that covers the joint ends. It has some specific tasks crucial to the correct joint physiology. It may experience a large amount of injuries that could generate considerable disabilities. Unfortunately its selfrepair capacity is too limited; therefore, many treatments have been developed with partial success, given the suboptimal biomechanical behavior of the resultant tissue. Given that, Tissue Engineering offers an alternative, based on the design of a new tissue with biological and biomechanical features which resembles the native tissue. In this work, the authors describe the methodologies followed to accomplish that goal, studying the chondrocytes harvesting, the cellular cultures, the scaffold seeding processes, the mechanical stimulation and the structural and biomechanical evaluation. Finally, exposed some of the preliminary results, as a experimental validation of the methods proposed are.

  10. Online grading method for tissue culture seedlings ofSpathiphyllum floribundum based on machine vision%基于机器视觉的白掌组培苗在线分级方法

    Institute of Scientific and Technical Information of China (English)

    杨意; 初麒; 杨艳丽; 张祥接; 徐祥朋; 辜松

    2016-01-01

    白掌在观叶类花卉中占有很大比例,其育苗多采用组织栽培法,且组培苗生产具有规模化。为提高成苗出苗品质,需要在组培苗炼苗前对其分级,而目前常用分级法不能有效解决自然状态下水平放置的白掌组培苗存在的叶片扭曲和重叠问题,因此该文提出一种基于机器视觉实现白掌组培苗在线分级的方法,通过对自然状态下水平放置的白掌组培苗的叶片面积、苗高、地径以及投影面积的分析,得到其投影面积与叶片面积呈线性关系,相关度为0.9344;投影面积与地径呈多项式函数关系,相关性为0.9067,故确定组培苗投影面积和苗高为实际生产中的分级指标。该文采用基于颜色模板匹配算法测量组培苗投影面积,得到的叶片面积和地径与实际叶片面积和地径的变异系数相对误差分别为0.35%和7.95%;利用最小外接矩形法(MBR,minimum bounding rectangle)测量苗高,得到的苗高和实际苗高变异系数相对误差为1.44%。通过整机分级试验发现在输送间距为0.25 m,输送速度为0.5 m/s,分级级别为3级的条件下,该分级装置的分级成功率可达96%,对应生产率为7200株/h。%At present, most of young plants ofSpathiphyllum floribundum are breeding by the technique of tissue culture. Due to absence of grading machine specially designed for primary-growth plants that is small, irregular and young, the grading of tissue culture seedlings are normally handled manually. In this paper, we proposed an automated online grading method for Spathiphyllum floribundum tissue culture seedlings based on the technique of machine vision. SinceSpathiphyllum floribundum is a foliage flower, the leaf area is one of the most important parameters in grading, along with seedling height and diameter. Direct measurement not only would do damage to young plant because of its tenderness, but also the manpower productivity

  11. Bioprinting for Neural Tissue Engineering.

    Science.gov (United States)

    Knowlton, Stephanie; Anand, Shivesh; Shah, Twisha; Tasoglu, Savas

    2018-01-01

    Bioprinting is a method by which a cell-encapsulating bioink is patterned to create complex tissue architectures. Given the potential impact of this technology on neural research, we review the current state-of-the-art approaches for bioprinting neural tissues. While 2D neural cultures are ubiquitous for studying neural cells, 3D cultures can more accurately replicate the microenvironment of neural tissues. By bioprinting neuronal constructs, one can precisely control the microenvironment by specifically formulating the bioink for neural tissues, and by spatially patterning cell types and scaffold properties in three dimensions. We review a range of bioprinted neural tissue models and discuss how they can be used to observe how neurons behave, understand disease processes, develop new therapies and, ultimately, design replacement tissues. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. A Protocol for Rapid, Measurable Plant Tissue Culture Using Stem Disc Meristem Micropropagation of Garlic ("Allium Sativum L.")

    Science.gov (United States)

    Peat, Gerry; Jones, Meriel

    2012-01-01

    Plant tissue culture is becoming an important technique for the mass propagation of plants. Problems with existing techniques, such as slow growth and contamination, have restricted the practical work in plant tissue culture carried out in schools. The new protocol using garlic meristematic stem discs explained in this article addresses many of…

  13. Influence of hydroxyurea on nucleic acids content and 3H-uridine incorporation in callus and tumorous tobacco tissues cultured in vitro

    Directory of Open Access Journals (Sweden)

    A. Bielecka

    2015-01-01

    Full Text Available In callus and tumor tissues of Nicotiana tabacum cultured for 39 days in media supplemented with various concentrations of hydroxyurea (1.3 x 10-4 M - 1.3 x 10-3 M a decrease of DNA content (ca. 24 per cent in callus tissue and ca. 23 per cent in tumour tissue and a decrease of RNA content (over 10 per cent and ca. 9 per cent in callus and tumour tissue, respectively was observed. The autoradiographic method showed that a long-lasting action of this com-pound inhibits RNA synthesis. A stronger inhibitory influence of hydroxyurea upon incorporation of 3H-uridine from the incubation medium was revealed.

  14. Aplicações da cultura de tecidos em plantas medicinais Applications of tissue culture in medicinal plants

    Directory of Open Access Journals (Sweden)

    T.P. Morais

    2012-01-01

    Full Text Available Esta revisão tem por objetivo levantar dados de literatura sobre o histórico e a situação atual das técnicas de cultura de tecidos em plantas medicinais. Para tanto, foi realizada uma revisão de publicações do período de 1976 a 2009. A cultura de tecidos é muito utilizada em pesquisas envolvendo plantas medicinais, com destaque para a técnica de micropropagação. A aplicação das técnicas de cultura de tecidos em plantas medicinais tem como perspectivas a obtenção de germoplasma competitivo e adaptado a diversos métodos de cultivo, escolha de novas espécies que servirão como fonte de compostos biologicamente ativos e aprimoramento da produção de fitofármacos, a fim de assegurar exploração sustentável destas espécies.The aim of this literature review is to conduct a survey concerning the history and current situation of tissue culture techniques in medicinal plants. Therefore, a review was done considering the period from 1976 to 2009. Tissue culture is widely applied in medicinal plants researches, especially micropropagation. The perspectives of tissue culture techniques in medicinal plants are related to the development of competitive germoplasm adapted to diverse methods of cultivation, the election of new species that will serve as source of biological active composts, and the improvement of phytochemicals production, in order to assure sustainable exploration of these species.

  15. Tissue culture-induced genetic and epigenetic alterations in rice pure-lines, F1 hybrids and polyploids.

    Science.gov (United States)

    Wang, Xiaoran; Wu, Rui; Lin, Xiuyun; Bai, Yan; Song, Congdi; Yu, Xiaoming; Xu, Chunming; Zhao, Na; Dong, Yuzhu; Liu, Bao

    2013-05-05

    Genetic and epigenetic alterations can be invoked by plant tissue culture, which may result in heritable changes in phenotypes, a phenomenon collectively termed somaclonal variation. Although extensive studies have been conducted on the molecular nature and spectrum of tissue culture-induced genomic alterations, the issue of whether and to what extent distinct plant genotypes, e.g., pure-lines, hybrids and polyploids, may respond differentially to the tissue culture condition remains poorly understood. We investigated tissue culture-induced genetic and epigenetic alterations in a set of rice genotypes including two pure-lines (different subspecies), a pair of reciprocal F1 hybrids parented by the two pure-lines, and a pair of reciprocal tetraploids resulted from the hybrids. Using two molecular markers, amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP), both genetic and DNA methylation alterations were detected in calli and regenerants from all six genotypes, but genetic alteration is more prominent than epigenetic alteration. While significant genotypic difference was observed in frequencies of both types of alterations, only genetic alteration showed distinctive features among the three types of genomes, with one hybrid (N/9) being exceptionally labile. Surprisingly, difference in genetic alteration frequencies between the pair of reciprocal F1 hybrids is much greater than that between the two pure-line subspecies. Difference also exists in the pair of reciprocal tetraploids, but is to a less extent than that between the hybrids. The steady-state transcript abundance of genes involved in DNA repair and DNA methylation was significantly altered in both calli and regenerants, and some of which were correlated with the genetic and/or epigenetic alterations. Our results, based on molecular marker analysis of ca. 1,000 genomic loci, document that genetic alteration is the major cause of somaclonal variation in rice

  16. Characterization of microbes in prosthetic joint specimens by culture-independent molecular methods

    DEFF Research Database (Denmark)

    Xu, Yijuan; Rudkjøbing, Vibeke Børsholt; Simonsen, Ole

    Prosthetic joint infection (PJI) is one of the most challenging complications of joint alloplasty. Formation of biofilm is a prominent feature of PJIs and constitutes a challenge to current sampling procedures and culture practices to obtain a reliable diagnosis. The aim of the study was to inves......Prosthetic joint infection (PJI) is one of the most challenging complications of joint alloplasty. Formation of biofilm is a prominent feature of PJIs and constitutes a challenge to current sampling procedures and culture practices to obtain a reliable diagnosis. The aim of the study...... was to investigate the microbial diversity in surgical samples (eg. synovial fluid, periprosthetic tissue, removed prosthesis) from 22 prosthetic patients using a range of culture-independent molecular methods including broad range 16S rRNA gene PCR, cloning, phylogeny, quantitative PCR (qPCR), and fluorescence...

  17. Dynamic Culturing of Cartilage Tissue: The Significance of Hydrostatic Pressure

    Science.gov (United States)

    Pereira, Ana L.; Duarte, Ana R.C.; Frias, Ana M.; Pedro, Adriano J.; Oliveira, João T.; Sousa, Rui A.; Reis, Rui L.

    2012-01-01

    Human articular cartilage functions under a wide range of mechanical loads in synovial joints, where hydrostatic pressure (HP) is the prevalent actuating force. We hypothesized that the formation of engineered cartilage can be augmented by applying such physiologic stimuli to chondrogenic cells or stem cells, cultured in hydrogels, using custom-designed HP bioreactors. To test this hypothesis, we investigated the effects of distinct HP regimens on cartilage formation in vitro by either human nasal chondrocytes (HNCs) or human adipose stem cells (hASCs) encapsulated in gellan gum (GG) hydrogels. To this end, we varied the frequency of low HP, by applying pulsatile hydrostatic pressure or a steady hydrostatic pressure load to HNC-GG constructs over a period of 3 weeks, and evaluated their effects on cartilage tissue-engineering outcomes. HNCs (10×106 cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 3 weeks: (1) 0.4 MPa Pulsatile HP; (2) 0.4 MPa Steady HP; and (3) Static. Subsequently, we applied the pulsatile regimen to hASC-GG constructs and varied the amplitude of loading, by generating both low (0.4 MPa) and physiologic (5 MPa) HP levels. hASCs (10×106 cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 4 weeks: (1) 0.4 MPa Pulsatile HP; (2) 5 MPa Pulsatile HP; and (3) Static. In the HNC study, the best tissue development was achieved by the pulsatile HP regimen, whereas in the hASC study, greater chondrogenic differentiation and matrix deposition were obtained for physiologic loading, as evidenced by gene expression of aggrecan, collagen type II, and sox-9; metachromatic staining of cartilage extracellular matrix; and immunolocalization of collagens. We thus propose that both HNCs and hASCs detect and respond to physical forces, thus resembling joint loading, by enhancing cartilage tissue development in a frequency- and

  18. Tissue culture of adult larch as a tool for breeding purposes

    Energy Technology Data Exchange (ETDEWEB)

    Ewald, D.; Kretzschmar, U. [Federal Research Centre of Forestry and Forest Products, Waldsieversdorf (Germany). Inst. for Forest Tree Biology

    1995-12-31

    Aimed at the identical reproduction of genotypes which are considered superior different methods were tested to establish and to propagate tissue cultures from old larch trees (L. decidua, L. kaempferi, L. sukaczewii, L. gmelinii, L. eurolepis). Serial subcultures without phytohormones (shoot tip propagation) led to the establishment of clone lines. After ten subcultures propagation velocity, shoot morphology and rooting behavior were similar to juvenile plant material. Serial subcultures which included a cytokinin induction led to the formation of adventitious shoot clusters (adventitious bud propagation). Adventitious shoots derived from male flowers of one L. kaempferi clone could be propagated via shoot tip propagation. Micrografting of meristems in vitro resulted in a regained rooting capacity of green cuttings from micrografts. Combining these in vitro techniques offers now the possibility to propagate selected mature larch trees for different breeding purposes. 23 refs, 5 figs, 2 tabs

  19. Tissue culture of adult larch as a tool for breeding purposes

    Energy Technology Data Exchange (ETDEWEB)

    Ewald, D; Kretzschmar, U [Federal Research Centre of Forestry and Forest Products, Waldsieversdorf (Germany). Inst. for Forest Tree Biology

    1996-12-31

    Aimed at the identical reproduction of genotypes which are considered superior different methods were tested to establish and to propagate tissue cultures from old larch trees (L. decidua, L. kaempferi, L. sukaczewii, L. gmelinii, L. eurolepis). Serial subcultures without phytohormones (shoot tip propagation) led to the establishment of clone lines. After ten subcultures propagation velocity, shoot morphology and rooting behavior were similar to juvenile plant material. Serial subcultures which included a cytokinin induction led to the formation of adventitious shoot clusters (adventitious bud propagation). Adventitious shoots derived from male flowers of one L. kaempferi clone could be propagated via shoot tip propagation. Micrografting of meristems in vitro resulted in a regained rooting capacity of green cuttings from micrografts. Combining these in vitro techniques offers now the possibility to propagate selected mature larch trees for different breeding purposes. 23 refs, 5 figs, 2 tabs

  20. Improved detection of Mycobacterium bovis infection in bovine lymph node tissue using immunomagnetic separation (IMS-based methods.

    Directory of Open Access Journals (Sweden)

    Linda D Stewart

    Full Text Available Immunomagnetic separation (IMS can selectively isolate and concentrate Mycobacterium bovis cells from lymph node tissue to facilitate subsequent detection by PCR (IMS-PCR or culture (IMS-MGIT. This study describes application of these novel IMS-based methods to test for M. bovis in a survey of 280 bovine lymph nodes (206 visibly lesioned (VL, 74 non-visibly lesioned (NVL collected at slaughter as part of the Northern Ireland bovine TB eradication programme. Their performance was evaluated relative to culture. Overall, 174 (62.1% lymph node samples tested positive by culture, 162 (57.8% by IMS-PCR (targeting IS6110, and 191 (68.2% by IMS-MGIT culture. Twelve (6.9% of the 174 culture positive lymph node samples were not detected by either of the IMS-based methods. However, an additional 79 M. bovis positive lymph node samples (27 (13.1% VL and 52 (70.3% NVL were detected by the IMS-based methods and not by culture. When low numbers of viable M. bovis are present in lymph nodes (e.g. in NVLs of skin test reactor cattle decontamination prior to culture may adversely affect viability, leading to false negative culture results. In contrast, IMS specifically captures whole M. bovis cells (live, dead or potentially dormant which are not subject to any deleterious treatment before detection by PCR or MGIT culture. During this study only 2.7% of NVL lymph nodes tested culture positive, whereas 70.3% of the same samples tested M. bovis positive by the IMS-based tests. Results clearly demonstrate that not only are the IMS-based methods more rapid but they have greater detection sensitivity than the culture approach currently used for the detection of M. bovis infection in cattle. Adoption of the IMS-based methods for lymph node testing would have the potential to improve M. bovis detection in clinical samples.

  1. Biomaterials and Culture Technologies for Regenerative Therapy of Liver Tissue.

    Science.gov (United States)

    Perez, Roman A; Jung, Cho-Rok; Kim, Hae-Won

    2017-01-01

    Regenerative approach has emerged to substitute the current extracorporeal technologies for the treatment of diseased and damaged liver tissue. This is based on the use of biomaterials that modulate the responses of hepatic cells through the unique matrix properties tuned to recapitulate regenerative functions. Cells in liver preserve their phenotype or differentiate through the interactions with extracellular matrix molecules. Therefore, the intrinsic properties of the engineered biomaterials, such as stiffness and surface topography, need to be tailored to induce appropriate cellular functions. The matrix physical stimuli can be combined with biochemical cues, such as immobilized functional groups or the delivered actions of signaling molecules. Furthermore, the external modulation of cells, through cocultures with nonparenchymal cells (e.g., endothelial cells) that can signal bioactive molecules, is another promising avenue to regenerate liver tissue. This review disseminates the recent approaches of regenerating liver tissue, with a focus on the development of biomaterials and the related culture technologies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Production of virus-free orchid Cymbidium aloifolium (L.) Sw. by various tissue culture techniques.

    Science.gov (United States)

    Pradhan, Shreeti; Regmi, Tripti; Ranjit, Mukunda; Pant, Bijaya

    2016-10-01

    Orchids are affected by many viruses resulting in poor growth, yield and quality, and an overall decline in population. Cymbidium mosaic virus (CymMV) is one of the common orchid viruses found in Cymbidium species but it infects different orchid genera. In this study Cymbidium aloifolium was propagated in vitro using MS medium at different strength (1.0, ½, and ¼) with or without 0.5 mg/l BAP (6-benzylaminopurine) and 0.5 mg/l NAA (Naphthalene acetic acid). To provide disease-free planting material, source plant for in vitro propagation needs to be screened for pathogenic viruses. In the present study, in vivo -grown source (mother) plants and tissue culture-derived plants of C. aloifolium were tested for CymMV virus using Double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA). All the tissue cultured plants were found to be 100% virus-free whereas the in vivo grown source plants were highly affected by CymMV virus (83.33%). The virus-free in vitro plantlets were multiplied in large scale and then acclimatized on earthen pot containing a mixture of cocopeat, litter and clay in the ratio of 3:2:1. Eighty five percent of acclimatized plantlets survived making this method an efficient mass production system for high quality virus-free C. aloifolium for commercial floriculture and germplasm preservation.

  3. Production of virus-free orchid Cymbidium aloifolium (L. Sw. by various tissue culture techniques

    Directory of Open Access Journals (Sweden)

    Shreeti Pradhan

    2016-10-01

    Full Text Available Orchids are affected by many viruses resulting in poor growth, yield and quality, and an overall decline in population. Cymbidium mosaic virus (CymMV is one of the common orchid viruses found in Cymbidium species but it infects different orchid genera. In this study Cymbidium aloifolium was propagated in vitro using MS medium at different strength (1.0, ½, and ¼ with or without 0.5 mg/l BAP (6-benzylaminopurine and 0.5 mg/l NAA (Naphthalene acetic acid. To provide disease-free planting material, source plant for in vitro propagation needs to be screened for pathogenic viruses. In the present study, in vivo-grown source (mother plants and tissue culture-derived plants of C. aloifolium were tested for CymMV virus using Double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA. All the tissue cultured plants were found to be 100% virus-free whereas the in vivo grown source plants were highly affected by CymMV virus (83.33%. The virus-free in vitro plantlets were multiplied in large scale and then acclimatized on earthen pot containing a mixture of cocopeat, litter and clay in the ratio of 3:2:1. Eighty five percent of acclimatized plantlets survived making this method an efficient mass production system for high quality virus-free C. aloifolium for commercial floriculture and germplasm preservation. Keywords: Biological sciences, Plant biology

  4. A novel method for isolation of epithelial cells from ovine esophagus for tissue engineering.

    Science.gov (United States)

    Macheiner, Tanja; Kuess, Anna; Dye, Julian; Saxena, Amulya K

    2014-01-01

    The yield of a critical number of basal epithelial cells with high mitotic rates from native tissue is a challenge in the field of tissue engineering. There are many protocols that use enzymatic methods for isolation of epithelial cells with unsatisfactory results for tissue engineering. This study aimed to develop a protocol for isolating a sufficient number of epithelial cells with a high Proliferating Index from ovine esophagus for tissue engineering applications. Esophageal mucosa was pretreated with dispase-collagenase solution and plated on collagen-coated culture dishes. Distinction of the various types of epithelial cells and developmental stages was done with specific primary antibodies to Cytokeratins and to Proliferating Cell Nuclear Antigen (PCNA). Up to approximately 8100 epithelial cells/mm2 of mucosa tissue were found after one week of migration. Cytokeratin 14 (CK 14) was positive identified in cells even after 83 days. At the same time the Proliferating Index was 71%. Our protocol for isolation of basal epithelial cells was successful to yield sufficient numbers of cells predominantly with proliferative character and without noteworthy negative enzymatic affection. The results at this study offer the possibility of generation critical cell numbers for tissue engineering applications.

  5. Exploring plant tissue culture in Withania somnifera (L.) Dunal: in vitro propagation and secondary metabolite production.

    Science.gov (United States)

    Shasmita; Rai, Manoj K; Naik, Soumendra K

    2017-12-26

    Withania somnifera (L.) Dunal (family: Solanaceae), commonly known as "Indian Ginseng", is a medicinally and industrially important plant of the Indian subcontinent and other warmer parts of the world. The plant has multi-use medicinal potential and has been listed among 36 important cultivated medicinal plants of India that are in high demand for trade due to its pharmaceutical uses. The medicinal importance of this plant is mainly due to the presence of different types of steroidal lactones- withanolides in the roots and leaves. Owing to low seed viability and poor germination, the conventional propagation of W. somnifera falls short to cater its commercial demands particularly for secondary metabolite production. Therefore, there is a great need to develop different biotechnological approaches through tissue and organ culture for seasonal independent production of plants in large scale which will provide sufficient raw materials of uniform quality for pharmaceutical purposes. During past years, a number of in vitro plant regeneration protocols via organogenesis and somatic embryogenesis and in vitro conservation through synthetic seed based encapsulation technology have been developed for W. somnifera. Several attempts have also been made to standardize the protocol of secondary metabolite production via tissue/organ cultures, cell suspension cultures, and Agrobacterium rhizogenes-mediated transformed hairy root cultures. Employment of plant tissue culture based techniques would provide means for rapid propagation and conservation of this plant species and also provide scope for enhanced production of different bioactive secondary metabolites. The present review provides a comprehensive report on research activities conducted in the area of tissue culture and secondary metabolite production in W. somnifera during the past years. It also discusses the unexplored areas which might be taken into consideration for future research so that the medicinal properties and

  6. Current status and future prospects for cultured limbal tissue transplants in Australia and New Zealand.

    Science.gov (United States)

    Harkin, Damien G; Apel, Andrew J; Di Girolamo, Nick; Watson, Stephanie; Brown, Karl; Daniell, Mark D; McGhee, J Jane; McGhee, Charles N J

    2013-04-01

    Cultured limbal tissue transplants have become widely used over the last decade as a treatment for limbal stem cell deficiency (LSCD). While the number of patients afflicted with LSCD in Australia and New Zealand is considered to be relatively low, the impact of this disease on quality of life is so severe that the potential efficacy of cultured transplants has necessitated investigation. We presently review the basic biology and experimental strategies associated with the use of cultured limbal tissue transplants in Australia and New Zealand. In doing so, we aim to encourage informed discussion on the issues required to advance the use of cultured limbal transplants in Australia and New Zealand. Moreover, we propose that a collaborative network could be established to maintain access to the technology in conjunction with a number of other existing and emerging treatments for eye diseases. © 2012 The Authors. Clinical and Experimental Ophthalmology © 2012 Royal Australian and New Zealand College of Ophthalmologists.

  7. Mass micropropagation of pineapple tissue culture using bioreactor technology

    International Nuclear Information System (INIS)

    Irwan Syafri; Amir Hamzah Harun; Rusli Ibrahim

    2005-01-01

    Pineapple (ananas comosus) is the most important fruit in terms of revenue earner in this country. The export of the canned pineapple is about 2 million standard cases annually valued at RM 60 million, while the export of fresh pineapple is about 40,000 tonnes worth about RM 10 million. The industry for canning is however, an ailing industry with production on the decline since the 70s. Scaling up the pineapple propagation using in vitro methods seems to be possible solutions for the lack of planting material. Temporary immersion system (TIS) has been described by Teisson and Alvard (1995) for plant tissue culture propagation. This system, also known as RITA, has been successfully used with embryogenic tissues of banana (Alvard et al 1993), coffee (Berthouly 1991), rubber (Etienne et al 1993) and sugarcane (Lorenzo et al 1998). In this study, the system has been set up with a potential capacity of 3 manifolds with 10 RITA each, to multiply meristem explants at different immersion periods. The system was compared with the conventional micropropagation system on solid medium. Both systems were treated with MS media containing 2.5 mg/l BAP and 0.1 NAA. In TIS the shoots were able to multiplied faster in comparison with solid media. The multiplication rates were increased up to 1:3 to 1:5 compared to normal propagation on solid media. The results show that TIS not only increase the propagation rates of pineapple but could also be adapted to reduce implementation costs to establish low-cost propagation systems. (Author)

  8. Metabolic aspects of growth in HU-treated crown-gall tissue cultures. I. Nicotiana tabacum

    Directory of Open Access Journals (Sweden)

    Aldona Rennert

    2015-01-01

    Full Text Available An influence of hydroxyurea (HU on the growth, DNA and RNA contents and protein synthesis in the tobacco tumour tissue culture was studied in comparison with a homologous callus tissue. In conformity with expectations considerable decrease of DNA level in both tissues is a primary effect of HU activity. This results in the growth inhibition and in the secondary metabolic effects; these effects depend not only on the concentration of inhibitor but also on the age of tissue. In spite of some common features the character of these changes shows a distinct differentiation depending on the tissue type. TMs points to specific modifications of the biochemical regulation of growth in a tumour.

  9. Tissue culture-induced genetic and epigenetic variation in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927) regenerants.

    Science.gov (United States)

    Machczyńska, Joanna; Zimny, Janusz; Bednarek, Piotr Tomasz

    2015-10-01

    Plant regeneration via in vitro culture can induce genetic and epigenetic variation; however, the extent of such changes in triticale is not yet understood. In the present study, metAFLP, a variation of methylation-sensitive amplified fragment length polymorphism analysis, was used to investigate tissue culture-induced variation in triticale regenerants derived from four distinct genotypes using androgenesis and somatic embryogenesis. The metAFLP technique enabled identification of both sequence and DNA methylation pattern changes in a single experiment. Moreover, it was possible to quantify subtle effects such as sequence variation, demethylation, and de novo methylation, which affected 19, 5.5, 4.5% of sites, respectively. Comparison of variation in different genotypes and with different in vitro regeneration approaches demonstrated that both the culture technique and genetic background of donor plants affected tissue culture-induced variation. The results showed that the metAFLP approach could be used for quantification of tissue culture-induced variation and provided direct evidence that in vitro plant regeneration could cause genetic and epigenetic variation.

  10. Generation of Functional Thyroid Tissue Using 3D-Based Culture of Embryonic Stem Cells.

    Science.gov (United States)

    Antonica, Francesco; Kasprzyk, Dominika Figini; Schiavo, Andrea Alex; Romitti, Mírian; Costagliola, Sabine

    2017-01-01

    During the last decade three-dimensional (3D) cultures of pluripotent stem cells have been intensively used to understand morphogenesis and molecular signaling important for the embryonic development of many tissues. In addition, pluripotent stem cells have been shown to be a valid tool for the in vitro modeling of several congenital or chronic human diseases, opening new possibilities to study their physiopathology without using animal models. Even more interestingly, 3D culture has proved to be a powerful and versatile tool to successfully generate functional tissues ex vivo. Using similar approaches, we here describe a protocol for the generation of functional thyroid tissue using mouse embryonic stem cells and give all the details and references for its characterization and analysis both in vitro and in vivo. This model is a valid approach to study the expression and the function of genes involved in the correct morphogenesis of thyroid gland, to elucidate the mechanisms of production and secretion of thyroid hormones and to test anti-thyroid drugs.

  11. Tissue engineering approaches to develop decellularized tendon matrices functionalized with progenitor cells cultured under undifferentiated and tenogenic conditions

    Directory of Open Access Journals (Sweden)

    Daniele D’Arrigo

    2017-11-01

    Full Text Available Tendon ruptures and retractions with an extensive tissue loss represent a major clinical problem and a great challenge in surgical reconstruction. Traditional approaches consist in autologous or allogeneic grafts, which still have some drawbacks. Hence, tissue engineering strategies aimed at developing functionalized tendon grafts. In this context, the use of xenogeneic tissues represents a promising perspective to obtain decellularized tendon grafts. This study is focused on the identification of suitable culture conditions for the generation of reseeded and functional decellularized constructs to be used as tendon grafts. Equine superficial digital flexor tendons were decellularized, reseeded with mesenchymal stem cells (MSCs from bone marrow and statically cultured in two different culture media to maintain undifferentiated cells (U-MSCs or to induce a terminal tenogenic differentiation (T-MSCs for 24 hours, 7 and 14 days. Cell viability, proliferation, morphology as well as matrix deposition and type I and III collagen production were assessed by means of histological, immunohistochemical and semi-quantitative analyses. Results showed that cell viability was not affected by any culture conditions and active proliferation was maintained 14 days after reseeding. However, seeded MSCs were not able to penetrate within the dense matrix of the decellularized tendons. Nevertheless, U-MSCs synthesized a greater amount of extracellular matrix rich in type I collagen compared to T-MSCs. In spite of the inability to deeply colonize the decellularized matrix in vitro, reseeding tendon matrices with U-MSCs could represent a suitable method for the functionalization of biological constructs, considering also any potential chemoattractant capability of the newly deposed extracellular matrix to recruit resident cells. This bioengineering approach can be exploited to produce functionalized tendon constructs for the substitution of large tendon defects.

  12. Metabolomics reveals the heterogeneous secretome of two entomopathogenic fungi to ex vivo cultured insect tissues.

    Directory of Open Access Journals (Sweden)

    Charissa de Bekker

    Full Text Available Fungal entomopathogens rely on cellular heterogeneity during the different stages of insect host infection. Their pathogenicity is exhibited through the secretion of secondary metabolites, which implies that the infection life history of this group of environmentally important fungi can be revealed using metabolomics. Here metabolomic analysis in combination with ex vivo insect tissue culturing shows that two generalist isolates of the genus Metarhizium and Beauveria, commonly used as biological pesticides, employ significantly different arrays of secondary metabolites during infectious and saprophytic growth. It also reveals that both fungi exhibit tissue specific strategies by a distinguishable metabolite secretion on the insect tissues tested in this study. In addition to showing the important heterogeneous nature of these two entomopathogens, this study also resulted in the discovery of several novel destruxins and beauverolides that have not been described before, most likely because previous surveys did not use insect tissues as a culturing system. While Beauveria secreted these cyclic depsipeptides when encountering live insect tissues, Metarhizium employed them primarily on dead tissue. This implies that, while these fungi employ comparable strategies when it comes to entomopathogenesis, there are most certainly significant differences at the molecular level that deserve to be studied.

  13. Nanotechnology, Cell Culture and Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Kazutoshi Haraguchi

    2011-01-01

    Full Text Available We have fabricated new types of polymer hydrogels and polymer nanocomposites, i.e., nanocomposite gels (NC gels and soft, polymer nanocomposites (M-NCs: solid, with novel organic/inorganic network structures. Both NC gels and M-NCs were synthesized by in-situ free-radical polymerization in the presence of exfoliated clay platelets in aqueous systems and were obtained in various forms such as film, sheet, tube, coating, etc. and sizes with a wide range of clay contents. Here, disk-like inorganic clay nanoparticles act as multi-functional crosslinkers to form new types of network systems. Both NC gels and M-NCs have extraordinary optical and mechanical properties including ultra-high reversible extensibility, as well as a number of new characteristics relating to optical anisotropy, polymer/clay morphology, biocompatibility, stimuli-sensitive surfaces, micro-patterning, etc. For examples, the biological testing of medical devices, comprised of a sensitization test, an irritation test, an intracutaneous test and an in vitro cytotoxicity test,was carried out for NC gels and M-NCs. The safety of NC gels and M-NCs was confirmed in all tests. Also, the interaction of living tissue with NC gel was investigated in vivo by implantation in live goats; neither inflammation nor concrescence occurred around the NC gels. Furthermore, it was found that both N-NC gels consisting of poly(N-isopropylacrylamide(PNIPA/clay network and M-NCs consisting of poly(2-methoxyethyacrylate(PMEA/clay network show characteristic cell culture and subsequent cell detachment on their surfaces, although it was almost impossible to culture cells on conventional, chemically-crosslinked PNIPA hydrogels and chemically crossslinked PMEA, regardless of their crosslinker concentration. Various kinds of cells, such ashumanhepatoma cells (HepG2, normal human dermal fibroblast (NHDF, and human umbilical vein endothelial cells (HUVEC, could be cultured to be confluent on the surfaces of N

  14. Study on rapid propagation of Zanhuang Chinese jujube by tissue culture

    International Nuclear Information System (INIS)

    Li Yun; Wang Yu; Tian Yanting

    2002-01-01

    Zanhuang jujube is a very precious and rare variety of Chinese jujube. Its development was restricted by the under-developed propagate technique in history. The rapid propagation by tissue culture was studied and the optimum media were screened out. Through studying the condition of initial, proliferating, acclimatizing and rooting culture, 4 media, MS +6-BA 0.5 mg/L+IBA 0.1 mg/L, MS+6-BA 1.5 mg/L+IBA 0.1-0.2 mg/L, MS+KT 0.5 mg/L+NAA 0.2 mg/L and 1/2 MS+IBA 0.6 mg/L+NAA 0.2-0.3 mg/L were selected respectively

  15. Comparison of mesencephalic free-floating tissue culture grafts and cell suspension grafts in the 6-hydroxydopamine-lesioned rat

    DEFF Research Database (Denmark)

    Meyer, Morten; Widmer, H R; Wagner, B

    1998-01-01

    of grafted dopaminergic neurons and to correlate that with the behavioral effects. Additional cultures and acutely prepared explants were also fixed and stored for histological investigation in order to estimate the loss of dopaminergic neurons in culture and after transplantation. Similar behavioral...... numbers of TH-immunoreactive (TH-ir) neurons in grafts of cultured tissue (775 +/- 98, mean +/- SEM) and grafts of fresh, dissociated cell suspension (806 +/- 105, mean +/- SEM). Cell counts in fresh explants, 7-day-old cultures, and grafted cultures revealed a 68.2% loss of TH-ir cells 7 days after......Ventral mesencephalon (VM) of fetal rat and human origin grown as free-floating roller-tube (FFRT) cultures can survive subsequent grafting to the adult rat striatum. To further explore the functional efficacy of such grafts, embryonic day 13 ventral mesencephalic tissue was grafted either after 7...

  16. Review of vascularised bone tissue-engineering strategies with a focus on co-culture systems.

    Science.gov (United States)

    Liu, Yuchun; Chan, Jerry K Y; Teoh, Swee-Hin

    2015-02-01

    Poor angiogenesis within tissue-engineered grafts has been identified as a main challenge limiting the clinical introduction of bone tissue-engineering (BTE) approaches for the repair of large bone defects. Thick BTE grafts often exhibit poor cellular viability particularly at the core, leading to graft failure and lack of integration with host tissues. Various BTE approaches have been explored for improving vascularisation in tissue-engineered constructs and are briefly discussed in this review. Recent investigations relating to co-culture systems of endothelial and osteoblast-like cells have shown evidence of BTE efficacy in increasing vascularization in thick constructs. This review provides an overview of key concepts related to bone formation and then focuses on the current state of engineered vascularized co-culture systems using bone repair as a model. It will also address key questions regarding the generation of clinically relevant vascularized bone constructs as well as potential directions and considerations for research with the objective of pursuing engineered co-culture systems in other disciplines of vascularized regenerative medicine. The final objective is to generate serious and functional long-lasting vessels for sustainable angiogenesis that will enable enhanced cellular survival within thick voluminous bone grafts, thereby aiding in bone formation and remodelling in the long term. However, more evidence about the quality of blood vessels formed and its associated functional improvement in bone formation as well as a mechanistic understanding of their interactions are necessary for designing better therapeutic strategies for translation to clinical settings. Copyright © 2012 John Wiley & Sons, Ltd.

  17. Antiandrogenic actions of medroxyprogesterone acetate on epithelial cells within normal human breast tissues cultured ex vivo.

    Science.gov (United States)

    Ochnik, Aleksandra M; Moore, Nicole L; Jankovic-Karasoulos, Tanja; Bianco-Miotto, Tina; Ryan, Natalie K; Thomas, Mervyn R; Birrell, Stephen N; Butler, Lisa M; Tilley, Wayne D; Hickey, Theresa E

    2014-01-01

    Medroxyprogesterone acetate (MPA), a component of combined estrogen-progestin therapy (EPT), has been associated with increased breast cancer risk in EPT users. MPA can bind to the androgen receptor (AR), and AR signaling inhibits cell growth in breast tissues. Therefore, the aim of this study was to investigate the potential of MPA to disrupt AR signaling in an ex vivo culture model of normal human breast tissue. Histologically normal breast tissues from women undergoing breast surgical operation were cultured in the presence or in the absence of the native AR ligand 5α-dihydrotestosterone (DHT), MPA, or the AR antagonist bicalutamide. Ki67, bromodeoxyuridine, B-cell CLL/lymphoma 2 (BCL2), AR, estrogen receptor α, and progesterone receptor were detected by immunohistochemistry. DHT inhibited the proliferation of breast epithelial cells in an AR-dependent manner within tissues from postmenopausal women, and MPA significantly antagonized this androgenic effect. These hormonal responses were not commonly observed in cultured tissues from premenopausal women. In tissues from postmenopausal women, DHT either induced or repressed BCL2 expression, and the antiandrogenic effect of MPA on BCL2 was variable. MPA significantly opposed the positive effect of DHT on AR stabilization, but these hormones had no significant effect on estrogen receptor α or progesterone receptor levels. In a subset of postmenopausal women, MPA exerts an antiandrogenic effect on breast epithelial cells that is associated with increased proliferation and destabilization of AR protein. This activity may contribute mechanistically to the increased risk of breast cancer in women taking MPA-containing EPT.

  18. Application of Synthetic Polymeric Scaffolds in Breast Cancer 3D Tissue Cultures and Animal Tumor Models

    Directory of Open Access Journals (Sweden)

    Girdhari Rijal

    2017-01-01

    Full Text Available Preparation of three-dimensional (3D porous scaffolds from synthetic polymers is a challenge to most laboratories conducting biomedical research. Here, we present a handy and cost-effective method to fabricate polymeric hydrogel and porous scaffolds using poly(lactic-co-glycolic acid (PLGA or polycaprolactone (PCL. Breast cancer cells grown on 3D polymeric scaffolds exhibited distinct survival, morphology, and proliferation compared to those on 2D polymeric surfaces. Mammary epithelial cells cultured on PLGA- or PCL-coated slides expressed extracellular matrix (ECM proteins and their receptors. Estrogen receptor- (ER- positive T47D breast cancer cells are less sensitive to 4-hydroxytamoxifen (4-HT treatment when cultured on the 3D porous scaffolds than in 2D cultures. Finally, cancer cell-laden polymeric scaffolds support consistent tumor formation in animals and biomarker expression as seen in human native tumors. Our data suggest that the porous synthetic polymer scaffolds satisfy the basic requirements for 3D tissue cultures both in vitro and in vivo. The scaffolding technology has appealing potentials to be applied in anticancer drug screening for a better control of the progression of human cancers.

  19. Participation of cob tissue in the transport of medium components into maize kernels cultured in vitro

    International Nuclear Information System (INIS)

    Felker, F.C.

    1990-01-01

    Maize (Zea mays L.) kernels cultured in vitro while still attached to cob pieces have been used as a model system to study the physiology of kernel development. In this study, the role of the cob tissue in uptake of medium components into kernels was examined. Cob tissue was essential for in vitro kernel growth, and better growth occurred with larger cob/kernel ratios. A symplastically transported fluorescent dye readily permeated the endosperm when supplied in the medium, while an apoplastic dye did not. Slicing the cob tissue to disrupt vascular connections, but not apoplastic continuity, greatly reduced [ 14 C]sucrose uptake into kernels. [ 14 C]Sucrose uptake by cob and kernel tissue was reduced 31% and 68%, respectively, by 5 mM PCMBS. L-[ 14 C]glucose was absorbed much more slowly than D-[ 14 C]glucose. These and other results indicate that phloem loading of sugars occurs in the cob tissue. Passage of medium components through the symplast cob tissue may be a prerequisite for uptake into the kernel. Simple diffusion from the medium to the kernels is unlikely. Therefore, the ability of substances to be transported into cob tissue cells should be considered in formulating culture medium

  20. Variation in primary and culture-expanded cells derived from connective tissue progenitors in human bone marrow space, bone trabecular surface and adipose tissue.

    Science.gov (United States)

    Qadan, Maha A; Piuzzi, Nicolas S; Boehm, Cynthia; Bova, Wesley; Moos, Malcolm; Midura, Ronald J; Hascall, Vincent C; Malcuit, Christopher; Muschler, George F

    2018-03-01

    Connective tissue progenitors (CTPs) embody the heterogeneous stem and progenitor cell populations present in native tissue. CTPs are essential to the formation and remodeling of connective tissue and represent key targets for tissue-engineering and cell-based therapies. To better understand and characterize CTPs, we aimed to compare the (i) concentration and prevalence, (ii) early in vitro biological behavior and (iii) expression of surface-markers and transcription factors among cells derived from marrow space (MS), trabecular surface (TS), and adipose tissues (AT). Cancellous-bone and subcutaneous-adipose tissues were collected from 8 patients. Cells were isolated and cultured. Colony formation was assayed using Colonyze software based on ASTM standards. Cell concentration ([Cell]), CTP concentration ([CTP]) and CTP prevalence (P CTP ) were determined. Attributes of culture-expanded cells were compared based on (i) effective proliferation rate and (ii) expression of surface-markers CD73, CD90, CD105, SSEA-4, SSEA-3, SSEA-1/CD15, Cripto-1, E-Cadherin/CD324, Ep-CAM/CD326, CD146, hyaluronan and transcription factors Oct3/4, Sox-2 and Nanog using flow cytometry. Mean [Cell], [CTP] and P CTP were significantly different between MS and TS samples (P = 0.03, P = 0.008 and P= 0.0003), respectively. AT-derived cells generated the highest mean total cell yield at day 6 of culture-4-fold greater than TS and more than 40-fold greater than MS per million cells plated. TS colonies grew with higher mean density than MS colonies (290 ± 11 versus 150 ± 11 cell per mm 2 ; P = 0.0002). Expression of classical-mesenchymal stromal cell (MSC) markers was consistently recorded (>95%) from all tissue sources, whereas all the other markers were highly variable. The prevalence and biological potential of CTPs are different between patients and tissue sources and lack variation in classical MSC markers. Other markers are more likely to discriminate differences

  1. A general native-state method for determination of proliferation capacity of human normal and tumor tissues in vitro

    International Nuclear Information System (INIS)

    Hoffman, R.M.; Connors, K.M.; Meerson-Monosov, A.Z.; Herrera, H.; Price, J.H.

    1989-01-01

    An important need in cancer research and treatment is a physiological means in vitro by which to assess the proliferation capacity of human tumors and corresponding normal tissue for comparison. The authors have recently developed a native-state, three-dimensional, gel-supported primary culture system that allows every type of human cancer to grow in vitro at more than 90% frequency, with maintenance of tissue architecture, tumor-stromal interaction, and differentiated functions. Here they demonstrate that the native-state culture system allows proliferation indices to be determined for all solid cancer types explanted directly from surgery into long-term culture. Normal tissues also proliferate readily in this system. The degree of resolution of measurement of cell proliferation by histological autoradiography within the cultured tissues is greatly enhanced with the use of epi-illumination polarization microscopy. The histological status of the cultured tissues can be assessed simultaneously with the proliferation status. Carcinomas generally have areas of high epithelial proliferation with quiescent stromal cells. Sarcomas have high proliferation of cells of mesenchymal organ. Normal tissues can also proliferate at high rates. An image analysis system has been developed to automate proliferation determination. The high-resolution physiological means described here to measure the proliferation capacity of tissues will be important in further understanding of the deregulation of cell proliferation in cancer as well as in cancer prognosis and treatment

  2. Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

    International Nuclear Information System (INIS)

    Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M.

    1990-01-01

    The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures

  3. Cryopreservation of testicular tissue before long-term testicular cell culture does not alter in vitro cell dynamics

    NARCIS (Netherlands)

    Baert, Yoni; Braye, Aude; Struijk, Robin B.; van Pelt, Ans M. M.; Goossens, Ellen

    2015-01-01

    To assess whether testicular cell dynamics are altered during long-term culture after testicular tissue cryopreservation. Experimental basic science study. Reproductive biology laboratory. Testicular tissue with normal spermatogenesis was obtained from six donors. None. Detection and comparison of

  4. Withania somnifera: Advances and Implementation of Molecular and Tissue Culture Techniques to Enhance Its Application

    Directory of Open Access Journals (Sweden)

    Vibha Pandey

    2017-08-01

    Full Text Available Withania somnifera, commonly known as Ashwagandha an important medicinal plant largely used in Ayurvedic and indigenous medicine for over 3,000 years. Being a medicinal plant, dried powder, crude extract as well as purified metabolies of the plant has shown promising therapeutic properties. Withanolides are the principal metabolites, responsible for the medicinal properties of the plant. Availability and amount of particular withanolides differ with tissue type and chemotype and its importance leads to identification characterization of several genes/ enzymes related to withanolide biosynthetic pathway. The modulation in withanolides can be achieved by controlling the environmental conditions like, different tissue culture techniques, altered media compositions, use of elicitors, etc. Among all the in vitro techniques, hairy root culture proved its importance at industrial scale, which also gets benefits due to more accumulation (amount and number of withanolides in roots tissues of W. somnifera. Use of media compostion and elicitors further enhances the amount of withanolides in hairy roots. Another important modern day technique used for accumulation of desired secondary metabolites is modulating the gene expression by altering environmental conditions (use of different media composition, elicitors, etc. or through genetic enginnering. Knowing the significance of the gene and the key enzymatic step of the pathway, modulation in withanolide contents can be achieved upto required amount in therapeutic industry. To accomplish maximum productivity through genetic enginnering different means of Withania transformation methods have been developed to obtain maximum transformation efficiency. These standardized transformation procedues have been used to overexpress/silence desired gene in W. somnifera to understand the outcome and succeed with enhanced metabolic production for the ultimate benefit of human race.

  5. Altered Loyalties of Neuronal Markers in Cultured Slices of Resected Human Brain Tissue

    NARCIS (Netherlands)

    Verwer, Ronald W. H.; Sluiter, Arja A.; Balesar, Rawien A.; Baayen, Johannes C.; Speijer, Dave; Idema, Sander; Swaab, Dick F.

    2016-01-01

    Organotypic cultures from normal neocortical tissue obtained at epilepsy surgery show a severe injury response. This response involves both neuronal degeneration and the proliferation of reactive cells. A salient feature of the reactive cells is the co-expression of microglial and astrocytic

  6. Diversity of endophytic bacteria of Dendrobium officinale based on culture-dependent and culture-independent methods

    Directory of Open Access Journals (Sweden)

    Cong Pei

    2017-01-01

    Full Text Available Culture-dependent and culture-independent methods were compared and evaluated in the study of the endophytic diversity of Dendrobium officinale. Culture-independent methods consisted of polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE and metagenome methods. According to the results, differences were found between the three methods. Three phyla, namely Firmicutes, Proteobacteria, and Actinobacteria, were detected using the culture-dependent method, and two phyla, Firmicutes and Proteobacteria, were detected by the DGGE method. Using the metagenome method, four major phyla were determined, including Proteobacteria (76.54%, Actinobacteria (18.56%, Firmicutes (2.27%, and Bacteroidetes (1.56%. A distinct trend was obtained at the genus level in terms of the method and the corresponding number of genera determined. There were 449 genera and 16 genera obtained from the metagenome and DGGE methods, respectively, and only 7 genera were obtained through the culture-dependent method. By comparison, all the genera from the culture-dependent and DGGE methods were contained in the members determined using the metagenome method. Overall, culture-dependent methods are limited to ‘finding’ endophytic bacteria in plants. DGGE is an alternative to investigating primary diversity patterns; however, the metagenome method is still the best choice for determining the endophytic profile in plants. It is essential to use multiphasic approaches to study cultured and uncultured microbes.

  7. Implementing oxygen control in chip-based cell and tissue culture systems.

    Science.gov (United States)

    Oomen, Pieter E; Skolimowski, Maciej D; Verpoorte, Elisabeth

    2016-09-21

    Oxygen is essential in the energy metabolism of cells, as well as being an important regulatory parameter influencing cell differentiation and function. Interest in precise oxygen control for in vitro cultures of tissues and cells continues to grow, especially with the emergence of the organ-on-a-chip and the desire to emulate in vivo conditions. This was recently discussed in this journal in a Critical Review by Brennan et al. (Lab Chip (2014). DOI: ). Microfluidics can be used to introduce flow to facilitate nutrient supply to and waste removal from in vitro culture systems. Well-defined oxygen gradients can also be established. However, cells can quickly alter the oxygen balance in their vicinity. In this Tutorial Review, we expand on the Brennan paper to focus on the implementation of oxygen analysis in these systems to achieve continuous monitoring. Both electrochemical and optical approaches for the integration of oxygen monitoring in microfluidic tissue and cell culture systems will be discussed. Differences in oxygen requirements from one organ to the next are a challenging problem, as oxygen delivery is limited by its uptake into medium. Hence, we discuss the factors determining oxygen concentrations in solutions and consider the possible use of artificial oxygen carriers to increase dissolved oxygen concentrations. The selection of device material for applications requiring precise oxygen control is discussed in detail, focusing on oxygen permeability. Lastly, a variety of devices is presented, showing the diversity of approaches that can be employed to control and monitor oxygen concentrations in in vitro experiments.

  8. Optimization of Ex Vivo Murine Bone Marrow Derived Immature Dendritic Cells: A Comparative Analysis of Flask Culture Method and Mouse CD11c Positive Selection Kit Method

    Directory of Open Access Journals (Sweden)

    Rahul Ashok Gosavi

    2018-01-01

    Full Text Available 12–14 days of culturing of bone marrow (BM cells containing various growth factors is widely used method for generating dendritic cells (DCs from suspended cell population. Here we compared flask culture method and commercially available CD11c Positive Selection kit method. Immature BMDCs’ purity of adherent as well as suspended cell population was generated in the decreasing concentration of recombinant-murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF in nontreated tissue culture flasks. The expression of CD11c, MHCII, CD40, and CD86 was measured by flow cytometry. We found significant difference (P<0.05 between the two methods in the adherent cells population but no significant difference was observed between the suspended cell populations with respect to CD11c+ count. However, CD11c+ was significantly higher in both adhered and suspended cell population by culture method but kit method gave more CD11c+ from suspended cells population only. On the other hand, using both methods, immature DC expressed moderate level of MHC class II molecules as well as low levels of CD40 and CD86. Our findings suggest that widely used culture method gives the best results in terms of yield, viability, and purity of BMDCs from both adherent and suspended cell population whereas kit method works well for suspended cell population.

  9. Morphological, biochemical and genetic influence of mutagen treatments on medicinal plant tissue cultures

    International Nuclear Information System (INIS)

    Onisei, T.; Toth, E.; Tesio, B.; Floria, F.

    1994-01-01

    Gamma rays and/or alkylant agents have been applied on callus tissue, young regenerants and cell suspension in order to establish their effect on morphogenesis, regeneration ability and biosynthetic potential. Growth dynamics, morpho-anatomic variables, secondary metabolite production, cell cytogenetics, enzyme specific activities, isoperoxidase and isoesterase patterns were analyzed in relation to the morphogenetic response of Atropa belladonna, Datura innoxia, Lavandula angustifolia, Chamomilla recutita, Digitalis lanata and Vinca minor tissue cultures. The effects of gamma-ray doses varied from one species to another; 10 to 20 Gy were generally able to stimulate growth and plant regeneration (via organogenesis and somatic embryogenesis), while 10 to 50 Gy enhanced secondary metabolite biosynthesis both in callus and cell suspension culture. Semnificative increase of secondary metabolite production was obtained when treatments with EMS (0.1-0.2%) have been applied to young regenerants. Many differences in biological features and biochemical behaviour were registered 20 days and one year, respectively, after treatment. (author)

  10. Comparative study of different seeding methods based on a multilayer SIS scaffold: Which is the optimal procedure for urethral tissue engineering?

    Science.gov (United States)

    Lv, Xiang-Guo; Feng, Chao; Fu, Qiang; Xie, Hong; Wang, Ying; Huang, Jian-Wen; Xie, Min-Kai; Atala, Anthony; Xu, Yue-Min; Zhao, Wei-Xin

    2016-08-01

    Seeding cells efficiently and uniformly onto three-dimensional scaffolds is key for engineering urological tissue with an ideal histological structure in vitro. Using an optimized seeding technology allows cells to cooperate positively with biomaterials, resulting in successful reconstructive surgery. In this study, we used four different types of seeding methods in a scaffold of small intestinal submucosa (SIS). The efficiency of the sandwich co-culture, layered co-culture, static-agitation seeding, and centrifugation seeding methods were compared. It was demonstrated that dynamic seeding methods, such as static-agitation and centrifugation seeding, had superior cell-matrix infiltration and mechanical properties. The seeding time could be reduced by 5-10 min using the centrifugation method. Furthermore, functional assessment of the barriers revealed that this function was better in the centrifugation seeding method than in any other method. Our study suggests that both the static-agitation and centrifugation methods are suitable for cell seeding on SIS. There is no significant change in surface area of SIS with different seeding methods. These methods reinforce the physiological and mechanical properties of biomaterials and allow for the future in vivo study of tissue-engineered urethral reconstruction. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1098-1108, 2016. © 2015 Wiley Periodicals, Inc.

  11. Toxicity and oxidative stress of canine mesenchymal stromal cells from adipose tissue in different culture passages

    Directory of Open Access Journals (Sweden)

    Arícia Gomes Sprada

    2015-12-01

    Full Text Available Abstract: Stem cells in regenerative therapy have received attention from researchers in recent decades. The culture of these cells allows studies about their behavior and metabolism. Thus, cell culture is the basis for cell therapy and tissue engineering researches. A major concern regarding the use of cultivated stem cell in human or veterinary clinical routine is the risk of carcinogenesis. Cellular activities require a balanced redox state. However, when there is an imbalance in this state, oxidative stress occurs. Oxidative stress contributes to cytotoxicity, which may result in cell death or genomic alterations, favoring the development of cancer cells. The aim of this study was to determine whether there are differences in the behavior of cultured mesenchymal stem cells from canine adipose tissue according to its site of collection (omentum and subcutaneous evaluating the rate of proliferation, viability, level of oxidative stress and cytotoxicity over six passages. For this experiment, two samples of adipose tissue from subcutaneous and omentum where taken from a female dog corpse, 13 years old, Pitbull. The results showed greater levels of oxidative stress in the first and last passages of both groups, favoring cytotoxicity and cell death.

  12. The release of bystander factor(s) from tissue explant cultures of rainbow trout (Onchorhynchus mykiss) after exposure to gamma radiation.

    Science.gov (United States)

    O'Dowd, Colm; Mothersill, Carmel E; Cairns, Michael T; Austin, Brian; McClean, Brendan; Lyng, Fiona M; Murphy, James E J

    2006-10-01

    The bystander response has been documented in cell lines and cell cultures derived from aquatic species over the past several years. However, little work has been undertaken to identify a similar bystander response in tissue explant cultures from fish. In this study, indirect effects of ionizing gamma radiation on tissue explant cultures of fish were investigated. Tissue explants in culture were exposed to 0.5 Gy and 5 Gy gamma radiation from a 60Co teletherapy unit. A bystander response in Epithelioma papulosum cyprini (EPC) cells exposed to gamma-irradiated tissue conditioned medium from rainbow trout explants was investigated, and the effects on cell survival were quantified by the clonogenic survival assay. Dichlorofluorescein and rhodamine 123 fluorescent dyes were used to identify alterations in reactive oxygen species (ROS) and mitochondrial membrane potential (MMP), respectively. Results indicate a different response for the three tissue types investigated. Clonogenic assay results vary from a decrease in cell survival (gill) to no effect (skin) to a stimulatory effect (spleen). Results from fluorescence assays of ROS and MMP show similarities to clonogenic assay results. This study identifies a useful model for further studies relating to the bystander effect in aquatic organisms in vivo and ex vivo.

  13. Three-Dimensional Culture Model of Skeletal Muscle Tissue with Atrophy Induced by Dexamethasone.

    Science.gov (United States)

    Shimizu, Kazunori; Genma, Riho; Gotou, Yuuki; Nagasaka, Sumire; Honda, Hiroyuki

    2017-06-15

    Drug screening systems for muscle atrophy based on the contractile force of cultured skeletal muscle tissues are required for the development of preventive or therapeutic drugs for atrophy. This study aims to develop a muscle atrophy model by inducing atrophy in normal muscle tissues constructed on microdevices capable of measuring the contractile force and to verify if this model is suitable for drug screening using the contractile force as an index. Tissue engineered skeletal muscles containing striated myotubes were prepared on the microdevices for the study. The addition of 100 µM dexamethasone (Dex), which is used as a muscle atrophy inducer, for 24 h reduced the contractile force significantly. An increase in the expression of Atrogin-1 and MuRF-1 in the tissues treated with Dex was established. A decrease in the number of striated myotubes was also observed in the tissues treated with Dex. Treatment with 8 ng/mL Insulin-like Growth Factor (IGF-I) for 24 h significantly increased the contractile force of the Dex-induced atrophic tissues. The same treatment, though, had no impact on the force of the normal tissues. Thus, it is envisaged that the atrophic skeletal muscle tissues induced by Dex can be used for drug screening against atrophy.

  14. Simultaneous separation and quantitation of amino acids and polyamines of forest tree tissues and cell cultures within a single high-performance liquid chromatography run using dansyl derivatization

    Science.gov (United States)

    Rakesh Minocha; Stephanie Long

    2004-01-01

    The objective of the present study was to develop a rapid HPLC method for simultaneous separation and quantitation of dansylated amino acids and common polyamines in the same matrix for analyzing forest tree tissues and cell cultures. The major modifications incorporated into this method as compared to previously published HPLC methods for separation of only dansyl...

  15. Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices.

    OpenAIRE

    Christensen, G D; Simpson, W A; Younger, J J; Baddour, L M; Barrett, F F; Melton, D M; Beachey, E H

    1985-01-01

    The adherence of coagulase-negative staphylococci to smooth surfaces was assayed by measuring the optical densities of stained bacterial films adherent to the floors of plastic tissue culture plates. The optical densities correlated with the weight of the adherent bacterial film (r = 0.906; P less than 0.01). The measurements also agreed with visual assessments of bacterial adherence to culture tubes, microtiter plates, and tissue culture plates. Selected clinical strains were passed through ...

  16. Tissue culture and associated biotechnological interventions for the improvement of coconut (Cocos nucifera L.): a review.

    Science.gov (United States)

    Nguyen, Quang Thien; Bandupriya, H D Dharshani; López-Villalobos, Arturo; Sisunandar, S; Foale, Mike; Adkins, Steve W

    2015-11-01

    The present review discusses not only advances in coconut tissue culture and associated biotechnological interventions but also future research directions toward the resilience of this important palm crop. Coconut (Cocos nucifera L.) is commonly known as the 'tree of life'. Every component of the palm can be used to produce items of value and many can be converted into industrial products. Coconut cultivation faces a number of acute problems that reduce its productivity and competitiveness. These problems include various biotic and abiotic challenges as well as an unstable market for its traditional oil-based products. Around 10 million small-holder farmers cultivate coconut palms worldwide on c. 12 million hectares of land, and many more people own a few coconut palms that contribute to their livelihoods. Inefficiency in the production of seedlings for replanting remains an issue; however, tissue culture and other biotechnological interventions are expected to provide pragmatic solutions. Over the past 60 years, much research has been directed towards developing and improving protocols for (i) embryo culture; (ii) clonal propagation via somatic embryogenesis; (iii) homozygote production via anther culture; (iv) germplasm conservation via cryopreservation; and (v) genetic transformation. Recently other advances have revealed possible new ways to improve these protocols. Although effective embryo culture and cryopreservation are now possible, the limited frequency of conversion of somatic embryos to ex vitro seedlings still prevents the large-scale clonal propagation of coconut. This review illustrates how our knowledge of tissue culture and associated biotechnological interventions in coconut has so far developed. Further improvement of protocols and their application to a wider range of germplasm will continue to open up new horizons for the collection, conservation, breeding and productivity of coconut.

  17. Metabolic aspects of growth in HU-treated crown-gall tissue cultures. II. Helianthus annuus

    Directory of Open Access Journals (Sweden)

    Aldona Rennert

    2015-01-01

    Full Text Available The dynamics of growth and changes in nucleic acid and protein contents in sunflower calluses and tumours cultured in hydroxyurea (HU containing media were examined. HU-induced changes in healthy tissues ran in parallel always in the same direction, in tumourous ones however an uncoupling between DNA synthesis and tissue growth on one hand and RNA and protein synthesis on the other took place. A detailed analysis of the results allows to suppose that the specific activity of HU on tumourous tissue could be an index of: 1 quantitative disturbances in its genes function (2 degree of the lass of sensitivity to the factors of regulation.

  18. A Robust Method to Generate Mechanically Anisotropic Vascular Smooth Muscle Cell Sheets for Vascular Tissue Engineering.

    Science.gov (United States)

    Backman, Daniel E; LeSavage, Bauer L; Shah, Shivem B; Wong, Joyce Y

    2017-06-01

    In arterial tissue engineering, mimicking native structure and mechanical properties is essential because compliance mismatch can lead to graft failure and further disease. With bottom-up tissue engineering approaches, designing tissue components with proper microscale mechanical properties is crucial to achieve the necessary macroscale properties in the final implant. This study develops a thermoresponsive cell culture platform for growing aligned vascular smooth muscle cell (VSMC) sheets by photografting N-isopropylacrylamide (NIPAAm) onto micropatterned poly(dimethysiloxane) (PDMS). The grafting process is experimentally and computationally optimized to produce PNIPAAm-PDMS substrates optimal for VSMC attachment. To allow long-term VSMC sheet culture and increase the rate of VSMC sheet formation, PNIPAAm-PDMS surfaces were further modified with 3-aminopropyltriethoxysilane yielding a robust, thermoresponsive cell culture platform for culturing VSMC sheets. VSMC cell sheets cultured on patterned thermoresponsive substrates exhibit cellular and collagen alignment in the direction of the micropattern. Mechanical characterization of patterned, single-layer VSMC sheets reveals increased stiffness in the aligned direction compared to the perpendicular direction whereas nonpatterned cell sheets exhibit no directional dependence. Structural and mechanical anisotropy of aligned, single-layer VSMC sheets makes this platform an attractive microstructural building block for engineering a vascular graft to match the in vivo mechanical properties of native arterial tissue. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Assessment of three types of spaceflight hardware for tissue culture studies: Comparison of skeletal tissue growth and differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Klement, B.J. [Space Medicine and Life Sciences Research Center Department of Anatomy Morehouse School of Medicine 720 Westview Dr. SW Atlanta, Georgia30310-1495 (United States); Spooner, B.S. [NASA Specialized Center of Research and Training Division of Biology Ackert Hall Kansas State University Manhattan, Kansas66506 (United States)

    1997-01-01

    Three different types of spaceflight hardware, the BioProcessing Module (BPM), the Materials Dispersion Apparatus (MDA), and the Fluid Processing Apparatus (FPA), were assessed for their ability to support pre-metatarsal growth and differentiation in experiments conducted on five space shuttle flights. BPM-cultured pre-metatarsal tissue showed no difference in flight and ground control lengths. Flight and ground controls cultured in the MDA grew 135 {mu}m and 141 {mu}m, respectively, in an 11 day experiment. Only five control rods and three flight rods mineralized. In another MDA experiment, pre-metatarsals were cultured at 4{degree}C (277K) or 20{degree}C (293K) for the 16 day mission, then cultured an additional 16 days in laboratory dishes at 37{degree}C (310K). The 20{degree}C (293K) cultures died post-flight. The 4{degree}C (277K) flight pre-metatarsals grew 417 {mu}m more than the 4{degree}C (277K) ground controls post-flight. In 5 and 6 day experiments done in FPAs, flight rods grew longer than ground control rods. In a 14 day experiment, ground control and flight rods also expanded in length, but there was no difference between them. The pre-metatarsals cultured in the FPAs did not mineralize, or terminally differentiate. These experiments demonstrate, that while supporting pre-metatarsal growth in length, the three types of hardware are not suitable to support routine differentiation. {copyright} {ital 1997 American Institute of Physics.}

  20. Products of cells from gliomas: VIII. Multiple-well immunoperoxidase assay of immunoreactivity of primary hybridoma supernatants with human glioma and brain tissue and cultured glioma cells.

    Science.gov (United States)

    McKeever, P E; Wahl, R L; Shakui, P; Jackson, G A; Letica, L H; Liebert, M; Taren, J A; Beierwaltes, W H; Hoff, J T

    1990-06-01

    To test the feasibility of primary screening of hybridoma supernatants against human glioma tissue, over 5000 combinations of hybridoma supernatants with glioma tissue, cultured glioma cells, and normal central neural tissue were screened with a new multiple-well (M-well) screening system. This is an immunoperoxidase assay system with visual endpoints for screening 20-30 hybridoma supernatants per single microscope slide. There were extensive differences between specificities to tissue and to cultured glioma cells when both were screened with M-wells and when cultured cells were screened with standard semi-automated fluorescence. Primary M-well screening with glioma tissue detected seven hybridoma supernatants that specifically identified parenchymal cells of glioma tissue and that were not detected with cultured cells. Immunoreactivities of individual supernatants for vascular components (nine supernatants), necrosis (five supernatants), and nuclei (three supernatants) were detected. Other supernatants bound multiple sites on glioma tissue and/or subpopulations of neurons and glia of normal tissue. The results show that primary screening with glioma tissue detects a number of different specificities of hybridoma supernatants to gliomas not detected by conventional screening with cultured cells. These are potentially applicable to diagnosis and therapy.

  1. Wagner classification and culture analysis of diabetic foot infection

    Directory of Open Access Journals (Sweden)

    Fatma Bozkurt

    2011-03-01

    Full Text Available The aim of this study was to determine the concordance ratio between microorganisms isolated from deep tissue culture and those from superficial culture in patients with diabetic foot according to Wagner’s wound classification method.Materials and methods: A total of 63 patients with Diabetic foot infection, who were admitted to Dicle University Hospital between October 2006 and November 2007, were included into the study. Wagner’s classification method was used for wound classification. For microbiologic studies superficial and deep tissue specimens were obtained from each patient, and were rapidly sent to laboratory for aerob and anaerob cultures. Microbiologic data were analyzed and interpreted in line with sensitivity and specifity formula.Results: Thirty-eight (60% of the patients were in Wagner’s classification ≤2, while 25 (40% patients were Wagner’s classification ≥3. According to our culture results, 66 (69% Gr (+ and 30 (31% Gr (- microorganisms grew in Wagner classification ≤2 patients. While in Wagner classification ≥3; 25 (35% Gr (+ and 46 (65% Gr (- microorganisms grew. Microorganisms grew in 89% of superficial cultures and 64% of the deep tissue cultures in patients with Wagner classification ≤2, while microorganism grew in 64% of Wagner classification ≥3.Conclusion: In ulcers of diabetic food infections, initial treatment should be started according to result of sterile superficial culture, but deep tissue culture should be taken, if unresponsive to initial treatment.

  2. Primary chondrocytes enhance cartilage tissue formation upon co-culture with expanded chondrocytes, dermal fibroblasts, 3T3 feeder cells and embryonic stem cells

    NARCIS (Netherlands)

    Hendriks, J.A.A.; Miclea, Razvan L.; Schotel, Roka; de Bruijn, Ewart; Moroni, Lorenzo; Karperien, Hermanus Bernardus Johannes; Riesle, J.U.; van Blitterswijk, Clemens

    2010-01-01

    Co-culture models have been increasingly used in tissue engineering applications to understand cell–cell interactions and consequently improve regenerative medicine strategies. Aiming at further elucidating cartilage tissue formation, we co-cultured bovine primary chondrocytes (BPCs) with human

  3. Comparison of Standard Culture-Based Method to Culture-Independent Method for Evaluation of Hygiene Effects on the Hand Microbiome.

    Science.gov (United States)

    Zapka, C; Leff, J; Henley, J; Tittl, J; De Nardo, E; Butler, M; Griggs, R; Fierer, N; Edmonds-Wilson, S

    2017-03-28

    Hands play a critical role in the transmission of microbiota on one's own body, between individuals, and on environmental surfaces. Effectively measuring the composition of the hand microbiome is important to hand hygiene science, which has implications for human health. Hand hygiene products are evaluated using standard culture-based methods, but standard test methods for culture-independent microbiome characterization are lacking. We sampled the hands of 50 participants using swab-based and glove-based methods prior to and following four hand hygiene treatments (using a nonantimicrobial hand wash, alcohol-based hand sanitizer [ABHS], a 70% ethanol solution, or tap water). We compared results among culture plate counts, 16S rRNA gene sequencing of DNA extracted directly from hands, and sequencing of DNA extracted from culture plates. Glove-based sampling yielded higher numbers of unique operational taxonomic units (OTUs) but had less diversity in bacterial community composition than swab-based sampling. We detected treatment-induced changes in diversity only by using swab-based samples ( P hand hygiene industry methods and for future hand microbiome studies. On the basis of our results and previously published studies, we propose recommendations for best practices in hand microbiome research. IMPORTANCE The hand microbiome is a critical area of research for diverse fields, such as public health and forensics. The suitability of culture-independent methods for assessing effects of hygiene products on microbiota has not been demonstrated. This is the first controlled laboratory clinical hand study to have compared traditional hand hygiene test methods with newer culture-independent characterization methods typically used by skin microbiologists. This study resulted in recommendations for hand hygiene product testing, development of methods, and future hand skin microbiome research. It also demonstrated the importance of inclusion of skin physiological metadata in

  4. Artificial urinary conduit construction using tissue engineering methods.

    Science.gov (United States)

    Kloskowski, Tomasz; Pokrywczyńska, Marta; Drewa, Tomasz

    2015-01-01

    Incontinent urinary diversion using an ileal conduit is the most popular method used by urologists after bladder cystectomy resulting from muscle invasive bladder cancer. The use of gastrointestinal tissue is related to a series of complications with the necessity of surgical procedure extension which increases the time of surgery. Regenerative medicine together with tissue engineering techniques gives hope for artificial urinary conduit construction de novo without affecting the ileum. In this review we analyzed history of urinary diversion together with current attempts in urinary conduit construction using tissue engineering methods. Based on literature and our own experience we presented future perspectives related to the artificial urinary conduit construction. A small number of papers in the field of tissue engineered urinary conduit construction indicates that this topic requires more attention. Three main factors can be distinguished to resolve this topic: proper scaffold construction along with proper regeneration of both the urothelium and smooth muscle layers. Artificial urinary conduit has a great chance to become the first commercially available product in urology constructed by regenerative medicine methods.

  5. Comparison of tumour age response to radiation for cells derived from tissue culture or solid tumours

    International Nuclear Information System (INIS)

    Keng, P.C.; Siemann, D.W.; Rochester Univ., NY; Rochester Univ., NY; Wheeler, K.T.

    1984-01-01

    Direct comparison of the cell age response of 9L and KHT tumour cells derived either from tissue culture or solid tumours was achieved. Cells from dissociated KHT and 9L tumours (the latter implanted either subcutaneously or intracerebrally) and cells from tissue culture were separated into homogenous sized populations by centrifugal elutriation. In both tumour models these homogeneous sized populations correspond to populations enriched at different stages of the cell cycle. The survival of these elutriated cell populations was measured after a single dose of Cs-137 gamma rays. For cells isolated from 9L solid tumours, there was little variation in radiosensitivity throughout the cell cycle; however, a very small but significant increase in resistance was found in late G 1 cells. This lack of a large variation in radiosensitivity through the cell cycle for 9L cells from solid tumours also was seen in 9L cells growing in monolayer tissue culture. When similar experiments were performed using the KHT sarcoma tumour model, the results showed that KHT cells in vitro exhibited a fairly conventional increase in radioresistance in both mid G 1 and late S. However, the cell age response of KHT cells from solid tumours was different; particularly in the late S and G 2 + M phases. (author)

  6. Allelopathy of small everlasting (Antennaria microphylla) : Phytotoxicity to leafy spurge (Euphorbia esula) in tissue culture.

    Science.gov (United States)

    Hogan, M E; Manners, G D

    1990-03-01

    Media and media extracts from callus cultures of small everlasting (Antennaria microphylla) inhibited leafy spurge (Euphorbia esula L.) callus tissue and suspension culture growth (50 and 70% of control, respectively) and were phytotoxic in lettuce and leafy spurge root elongation bioassays (64 and 77% of control, respectively). Hydroquinone, a phytotoxic compound previously isolated from small everlasting, was also biosynthesized by callus and suspension cultures of this species. Exogenously supplied hydroquinone (0.5 mM) was toxic to leafy spurge suspension culture cells and was only partially biotransformed to its nontoxic water-soluble monoglucoside, arbutin, by these cells. This report confirms the chronic involvement of hydroquinone in the allelopathic interaction between small everlasting and leafy spurge.

  7. Longitudinal Claudin Gene Expression Analyses in Canine Mammary Tissues and Thereof Derived Primary Cultures and Cell Lines

    Directory of Open Access Journals (Sweden)

    Susanne C. Hammer

    2016-09-01

    Full Text Available Human and canine mammary tumours show partial claudin expression deregulations. Further, claudins have been used for directed therapeutic approaches. However, the development of claudin targeting approaches requires stable claudin expressing cell lines. This study reports the establishment and characterisation of canine mammary tissue derived cell lines, analysing longitudinally the claudin-1, -3, -4 and -7 expressions in original tissue samples, primary cultures and developed cell lines. Primary cultures were derived from 17 canine mammary tissues: healthy, lobular hyperplasia, simple adenoma, complex adenoma, simple tubular carcinoma, complex carcinoma, carcinoma arising in a benign mixed tumour and benign mixed tissue. Cultivation was performed, if possible, until passage 30. Claudin mRNA and protein expressions were analysed by PCR, QuantiGene Plex Assay, immunocytochemistry and immunofluorescence. Further, cytokeratin expression was analysed immunocytochemically. Cultivation resulted in 11 established cell lines, eight showing epithelial character. In five of the early passages the claudin expressions decreased compared to the original tissues. In general, claudin expressions were diminished during cultivation. Three cell lines kept longitudinally claudin, as well as epithelial marker expressions, representing valuable tools for the development of claudin targeted anti-tumour therapies.

  8. Culture, Interface Design, and Design Methods for Mobile Devices

    Science.gov (United States)

    Lee, Kun-Pyo

    Aesthetic differences and similarities among cultures are obviously one of the very important issues in cultural design. However, ever since products became knowledge-supporting tools, the visible elements of products have become more universal so that the invisible parts of products such as interface and interaction are getting more important. Therefore, the cultural design should be extended to the invisible elements of culture like people's conceptual models beyond material and phenomenal culture. This chapter aims to explain how we address the invisible cultural elements in interface design and design methods by exploring the users' cognitive styles and communication patterns in different cultures. Regarding cultural interface design, we examined users' conceptual models while interacting with mobile phone and website interfaces, and observed cultural difference in performing tasks and viewing patterns, which appeared to agree with cultural cognitive styles known as Holistic thoughts vs. Analytic thoughts. Regarding design methods for culture, we explored how to localize design methods such as focus group interview and generative session for specific cultural groups, and the results of comparative experiments revealed cultural difference on participants' behaviors and performance in each design method and led us to suggest how to conduct them in East Asian culture. Mobile Observation Analyzer and Wi-Pro, user research tools we invented to capture user behaviors and needs especially in their mobile context, were also introduced.

  9. Propagation of jarrah (Eucalyptus marginata) by organ and tissue culture

    Energy Technology Data Exchange (ETDEWEB)

    Bennett, M.J.; McComb, J.A.

    1982-01-01

    Micropropagation methods are described for the production of clonal lines from Eucalyptus marginata (jarrah) seedlings. Nodal explants from mature trees can also yield shoot cultures, but a high frequency of contamination occurs among such explants. Uncontaminated callus cultures can be produced from mature trees by culturing stamen filaments and shoots can subsequently be regenerated from this callus. The rooting percentage of shoot cultures from either nodes or stamen callus of mature trees is low compared with that from seedling explants. Considerable variation was observed between trees in the ability of stamen callus to regenerate shoots and in the frequency of rooting. (Refs. 27)

  10. Repair of segmental bone defect using Totally Vitalized tissue engineered bone graft by a combined perfusion seeding and culture system.

    Directory of Open Access Journals (Sweden)

    Lin Wang

    Full Text Available BACKGROUND: The basic strategy to construct tissue engineered bone graft (TEBG is to combine osteoblastic cells with three dimensional (3D scaffold. Based on this strategy, we proposed the "Totally Vitalized TEBG" (TV-TEBG which was characterized by abundant and homogenously distributed cells with enhanced cell proliferation and differentiation and further investigated its biological performance in repairing segmental bone defect. METHODS: In this study, we constructed the TV-TEBG with the combination of customized flow perfusion seeding/culture system and β-tricalcium phosphate (β-TCP scaffold fabricated by Rapid Prototyping (RP technique. We systemically compared three kinds of TEBG constructed by perfusion seeding and perfusion culture (PSPC method, static seeding and perfusion culture (SSPC method, and static seeding and static culture (SSSC method for their in vitro performance and bone defect healing efficacy with a rabbit model. RESULTS: Our study has demonstrated that TEBG constructed by PSPC method exhibited better biological properties with higher daily D-glucose consumption, increased cell proliferation and differentiation, and better cell distribution, indicating the successful construction of TV-TEBG. After implanted into rabbit radius defects for 12 weeks, PSPC group exerted higher X-ray score close to autograft, much greater mechanical property evidenced by the biomechanical testing and significantly higher new bone formation as shown by histological analysis compared with the other two groups, and eventually obtained favorable healing efficacy of the segmental bone defect that was the closest to autograft transplantation. CONCLUSION: This study demonstrated the feasibility of TV-TEBG construction with combination of perfusion seeding, perfusion culture and RP technique which exerted excellent biological properties. The application of TV-TEBG may become a preferred candidate for segmental bone defect repair in orthopedic and

  11. Proteomic analysis reveals the mechanisms of Mycena dendrobii promoting transplantation survival and growth of tissue culture seedlings of Dendrobium officinale.

    Science.gov (United States)

    Xu, X B; Ma, X Y; Lei, H H; Song, H M; Ying, Q C; Xu, M J; Liu, S B; Wang, H Z

    2015-06-01

    Dendrobium officinale is an important traditional Chinese medicinal herb. Its seedlings generally show low survival and growth when transferred from in vitro tissue culture to a greenhouse or field environment. In this study, the effect of Mycena dendrobii on the survival and growth of D. officinale tissue culture seedlings and the mechanisms involved was explored. Mycena dendrobii were applied underneath the roots of D. officinale tissue culture seedlings. The seedling survival and growth were analysed. The root proteins induced by M. dendrobii were identified using two-dimensional (2-D) electrophoresis and matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF-MS). Mycena dendrobii treatment significantly enhanced survival and growth of D. officinale seedlings. Forty-one proteins induced by M. dendrobii were identified. Among them, 10 were involved in defence and stress response, two were involved in the formation of root or mycorrhizae, and three were related to the biosynthesis of bioactive constituents. These results suggest that enhancing stress tolerance and promoting new root formation induced by M. dendrobii may improve the survival and growth of D. officinale tissue culture seedlings. This study provides a foundation for future use of M. dendrobii in the large-scale cultivation of Dendrobiums. © 2015 The Society for Applied Microbiology.

  12. State-of-the-Art Methods for Brain Tissue Segmentation: A Review.

    Science.gov (United States)

    Dora, Lingraj; Agrawal, Sanjay; Panda, Rutuparna; Abraham, Ajith

    2017-01-01

    Brain tissue segmentation is one of the most sought after research areas in medical image processing. It provides detailed quantitative brain analysis for accurate disease diagnosis, detection, and classification of abnormalities. It plays an essential role in discriminating healthy tissues from lesion tissues. Therefore, accurate disease diagnosis and treatment planning depend merely on the performance of the segmentation method used. In this review, we have studied the recent advances in brain tissue segmentation methods and their state-of-the-art in neuroscience research. The review also highlights the major challenges faced during tissue segmentation of the brain. An effective comparison is made among state-of-the-art brain tissue segmentation methods. Moreover, a study of some of the validation measures to evaluate different segmentation methods is also discussed. The brain tissue segmentation, content in terms of methodologies, and experiments presented in this review are encouraging enough to attract researchers working in this field.

  13. Methylobacterium sp. resides in unculturable state in potato tissues in vitro and becomes culturable after induction by Pseudomonas fluorescens IMGB163.

    Science.gov (United States)

    Podolich, O; Laschevskyy, V; Ovcharenko, L; Kozyrovska, N; Pirttilä, A M

    2009-03-01

    To induce growth of endophytic bacteria residing in an unculturable state in tissues of in vitro-grown potato plantlets. To isolate and identify the induced bacteria and to localize the strains in tissues of in vitro-grown potato plantlets. The inoculation of in vitro-grown potato plants with Pseudomonas fluorescens IMBG163 led to induction of another bacterium, a pink-pigmented facultative methylotroph that was identified as Methylobacterium sp. using phylogenetic 16S rDNA approach. Two molecular methods were used for localizing methylobacteria in potato plantlets: PCR and in situ hybridization (ISH/FISH). A PCR product specific for the Methylobacterium genus was found in DNA isolated from the surface-sterilized plantlet leaves. Presence of Methylobacterium rRNA was detected by ISH/FISH in leaves and stems of inoculated as well as axenic potato plantlets although the bacterium cannot be isolated from the axenic plants. Methylobacterium sp. resides in unculturable state within tissues of in vitro-grown potato plants and becomes culturable after inoculation with P. fluorescens IMBG163. In order to develop endophytic biofertilizers and biocontrol agents, a detailed knowledge of the life-style of endophytes is essential. To our knowledge, this is the first report on increase of the culturability of endophytes in response to inoculation by nonpathogenic bacteria.

  14. Traditional and Modern Cell Culture in Virus Diagnosis.

    Science.gov (United States)

    Hematian, Ali; Sadeghifard, Nourkhoda; Mohebi, Reza; Taherikalani, Morovat; Nasrolahi, Abbas; Amraei, Mansour; Ghafourian, Sobhan

    2016-04-01

    Cell cultures are developed from tissue samples and then disaggregated by mechanical, chemical, and enzymatic methods to extract cells suitable for isolation of viruses. With the recent advances in technology, cell culture is considered a gold standard for virus isolation. This paper reviews the evolution of cell culture methods and demonstrates why cell culture is a preferred method for identification of viruses. In addition, the advantages and disadvantages of both traditional and modern cell culture methods for diagnosis of each type of virus are discussed. Detection of viruses by the novel cell culture methods is considered more accurate and sensitive. However, there is a need to include some more accurate methods such as molecular methods in cell culture for precise identification of viruses.

  15. Development of a rapid culture method to induce adipocyte differentiation of human bone marrow-derived mesenchymal stem cells

    International Nuclear Information System (INIS)

    Ninomiya, Yuichi; Sugahara-Yamashita, Yzumi; Nakachi, Yutaka; Tokuzawa, Yoshimi; Okazaki, Yasushi; Nishiyama, Masahiko

    2010-01-01

    Human mesenchymal stem cells (hMSCs) derived from bone marrow are multipotent stem cells that can regenerate mesenchymal tissues such as adipose, bone or muscle. It is thought that hMSCs can be utilized as a cell resource for tissue engineering and as human models to study cell differentiation mechanisms, such as adipogenesis, osteoblastogenesis and so on. Since it takes 2-3 weeks for hMSCs to differentiate into adipocytes using conventional culture methods, the development of methods to induce faster differentiation into adipocytes is required. In this study we optimized the culture conditions for adipocyte induction to achieve a shorter cultivation time for the induction of adipocyte differentiation in bone marrow-derived hMSCs. Briefly, we used a cocktail of dexamethasone, insulin, methylisobutylxanthine (DIM) plus a peroxisome proliferator-activated receptor γ agonist, rosiglitazone (DIMRo) as a new adipogenic differentiation medium. We successfully shortened the period of cultivation to 7-8 days from 2-3 weeks. We also found that rosiglitazone alone was unable to induce adipocyte differentiation from hMSCs in vitro. However, rosiglitazone appears to enhance hMSC adipogenesis in the presence of other hormones and/or compounds, such as DIM. Furthermore, the inhibitory activity of TGF-β1 on adipogenesis could be investigated using DIMRo-treated hMSCs. We conclude that our rapid new culture method is very useful in measuring the effect of molecules that affect adipogenesis in hMSCs.

  16. IN VITRO PROPAGATION OF DENDROBIUM AND PHALAENOPSIS THROUGH TISSUE CULTURE FOR CONSERVATION

    Directory of Open Access Journals (Sweden)

    Lita Soetopo

    2012-06-01

    Full Text Available The studies were focused on developing an efficient and effective propagation protocol for orchid species from genera Dendrobioum and Phalaenopsis through tissue culture. The Materials used were explants from adventive shoot tip, floral stalk buds and PLBs derived from seeds. The results indicated growth and development of adventive shoot tip explants of Dendrobium: a high survival percentage for explant with green color was shown by D. racianum, followed by D. laxiflorum, D. pseudo-conantum, D. strebloceras, D. lineale, and D. veratrifolium. However, plantlets regeneration occurred only on D. pseudoconantum, and D. strebloceras. Explant regeneration from seed derived protocorm-like bodies on D. spectabile occurred 40 days after inoculation transfer and subculture. High survival percentage of explant from floral stalk shoot was shown by P. amabilis. There were several plantlets surviving in acclimatisation. Explant regeneration from seed derived from protocorm-like bodies on P. hieroglypha occurred 40 days after inoculation and subculture. It was suggested that for ex situ conservation on certain species of Dendrobium and Phalaenopsis in the category of rare germplasms, tissue culture could be applied effectively and efficiently by using explant from adventive shoot tip, floral stalk buds and seed derived protocorm-like body explant for vegetative seed multiplication.

  17. Production of mutants by irradiation of in vitro-cultured tissues of coconut and banana and their mass propagation by the tissue culture technique

    International Nuclear Information System (INIS)

    Guzman, E.V. de; Rosario, A.G. del; Pagcaliwagan, P.C.

    1982-01-01

    Regeneration of buds/shoots as well as plantlets was induced from banana shoot tip explants cultured in highly modified Murashige and Skoog's medium supplemented with coconut water and benzyladenine. Initially shoot regeneration was sparse, but on further subculture became profuse. Gamma irradiation at low dosage (1.0 kR) was stimulating to explant growth and bud formation with the two types of explants used. With Bungulan stimulation was observed even at 2.5 kR. Several morphological aberrations were exhibited by shoots of 'irradiated' in vitro plants growing in potted soil. A highly and continuously proliferating tissue strain has been isolated from a subculture which was ultimately derived from an irradiated explant. Its continued proliferation is dependent on an external supply of coconut water and benzyladenine. In vitro-produced plants have been established under field conditions. The 'irradiated' plants are comparable with, and some seem to be better than, the unirradiated controls with respect to height, girth, sucker production and number of hands and fingers per bunch. Higher doses of irradiation are required to produce an adverse effect on growth of coconut embryos during the liquid culture than when growing in solid medium. (author)

  18. Hormonal effect on polyphenol accumulation in Cassia tissues cultured in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Shah, R R; Subbaiah, K V; Mehta, A R

    1976-06-01

    Effects of auxin and kinetin on growth and production of phenolic compounds in cultured Cassia fistula L. tissues were examined. Initiation of polyphenols was largely determined by the auxin concentration in the medium. Growth of the cells in relation to accumulation of polyphenols was studied at different auxin and kinetin concentrations. The accumulation of phenolic materials was essentially restricted to the most rapid phase of the growth cycle. Progressive changes in the pattern of peroxidase activity were followed and their relationship with polyphenol synthesis is examined.

  19. Royal Jelly Prevents Osteoporosis in Rats: Beneficial Effects in Ovariectomy Model and in Bone Tissue Culture Model

    Directory of Open Access Journals (Sweden)

    Saburo Hidaka

    2006-01-01

    Full Text Available Royal jelly (RJ has been used worldwide for many years as medical products, health foods and cosmetics. Since RJ contains testosterone and has steroid hormone-type activities, we hypothesized that it may have beneficial effects on osteoporosis. We used both an ovariectomized rat model and a tissue culture model. Rats were divided into eight groups as follows: sham-operated (Sham, ovariectomized (OVX, OVX given 0.5% (w/w raw RJ, OVX given 2.0% (w/w RJ, OVX given 0.5% (w/w protease-treated RJ (pRJ, OVX given 2.0% (w/w pRJ, OVX given 17β-estradiol and OVX given its vehicle, respectively. The Ovariectomy decreased tibial bone mineral density (BMD by 24%. Administration of 17β-estradiol to OVX rats recovered the tibial BMD decrease by 100%. Administration of 2.0% (w/w RJ and 0.5–2.0% (w/w pRJ to OVX rats recovered it by 85% or more. These results indicate that both RJ and pRJ are almost as effective as 17β-estradiol in preventing the development of bone loss induced by ovariectomy in rats. In tissue culture models, both RJ and pRJ increased calcium contents in femoral-diaphyseal and femoral-metaphyseal tissue cultures obtained from normal male rats. However, in a mouse marrow culture model, they neither inhibited the parathyroid hormone (PTH-induced calcium loss nor affected the formation of osteoclast-like cells induced by PTH in mouse marrow culture system. Therefore, our results suggest that both RJ and pRJ may prevent osteoporosis by enhancing intestinal calcium absorption, but not by directly antagonizing the action of PTH.

  20. Data on isolating mesenchymal stromal cells from human adipose tissue using a collagenase-free method

    Directory of Open Access Journals (Sweden)

    Wassim Shebaby

    2016-03-01

    Full Text Available The present dataset describes a detailed protocol to isolate mesenchymal cells from human fat without the use of collagenase. Human fat specimen, surgically cleaned from non-fat tissues (e.g., blood vessels and reduced into smaller fat pieces of around 1–3 mm size, is incubated in complete culture media for five to seven days. Then, cells started to spread out from the fat explants and to grow in cultures according to an exponential pattern. Our data showed that primary mesenchymal cells presenting heterogeneous morphology start to acquire more homogenous fibroblastic-like shape when cultured for longer duration or when subcultured into new flasks. Cell isolation efficiency as well as cell doubling time were also calculated throughout the culturing experimentations and illustrated in a separate figure thereafter. This paper contains data previously considered as an alternative protocol to isolate adipose-derived mesenchymal stem cell published in “Proliferation and differentiation of human adipose-derived mesenchymal stem cells (ASCs into osteoblastic lineage are passage dependent” [1]. Keywords: Adipose tissue, mesenchymal stromal cell, cell culture, doubling time

  1. Cardiac tissue engineering using perfusion bioreactor systems

    Science.gov (United States)

    Radisic, Milica; Marsano, Anna; Maidhof, Robert; Wang, Yadong; Vunjak-Novakovic, Gordana

    2009-01-01

    This protocol describes tissue engineering of synchronously contractile cardiac constructs by culturing cardiac cell populations on porous scaffolds (in some cases with an array of channels) and bioreactors with perfusion of culture medium (in some cases supplemented with an oxygen carrier). The overall approach is ‘biomimetic’ in nature as it tends to provide in vivo-like oxygen supply to cultured cells and thereby overcome inherent limitations of diffusional transport in conventional culture systems. In order to mimic the capillary network, cells are cultured on channeled elastomer scaffolds that are perfused with culture medium that can contain oxygen carriers. The overall protocol takes 2–4 weeks, including assembly of the perfusion systems, preparation of scaffolds, cell seeding and cultivation, and on-line and end-point assessment methods. This model is well suited for a wide range of cardiac tissue engineering applications, including the use of human stem cells, and high-fidelity models for biological research. PMID:18388955

  2. In vitro culture of functionally active buffalo hepatocytes isolated by using a simplified manual perfusion method.

    Directory of Open Access Journals (Sweden)

    Santanu Panda

    Full Text Available In farm animals, there is no suitable cell line available to understand liver-specific functions. This has limited our understanding of liver function and metabolism in farm animals. Culturing and maintenance of functionally active hepatocytes is difficult, since they survive no more than few days. Establishing primary culture of hepatocytes can help in studying cellular metabolism, drug toxicity, hepatocyte specific gene function and regulation. Here we provide a simple in vitro method for isolation and short-term culture of functionally active buffalo hepatocytes.Buffalo hepatocytes were isolated from caudate lobes by using manual enzymatic perfusion and mechanical disruption of liver tissue. Hepatocyte yield was (5.3 ± 0.66×107 cells per gram of liver tissue with a viability of 82.3 ± 3.5%. Freshly isolated hepatocytes were spherical with well contrasted border. After 24 hours of seeding onto fibroblast feeder layer and different extracellular matrices like dry collagen, matrigel and sandwich collagen coated plates, hepatocytes formed confluent monolayer with frequent clusters. Cultured hepatocytes exhibited typical cuboidal and polygonal shape with restored cellular polarity. Cells expressed hepatocyte-specific marker genes or proteins like albumin, hepatocyte nuclear factor 4α, glucose-6-phosphatase, tyrosine aminotransferase, cytochromes, cytokeratin and α1-antitrypsin. Hepatocytes could be immunostained with anti-cytokeratins, anti-albumin and anti α1-antitrypsin antibodies. Abundant lipid droplets were detected in the cytosol of hepatocytes using oil red stain. In vitro cultured hepatocytes could be grown for five days and maintained for up to nine days on buffalo skin fibroblast feeder layer. Cultured hepatocytes were viable for functional studies.We developed a convenient and cost effective technique for hepatocytes isolation for short-term culture that exhibited morphological and functional characteristics of active hepatocytes

  3. Negative-ion beam surface modification of tissue-culture polystyrene dishes for changing hydrophilic and cell-attachment properties

    International Nuclear Information System (INIS)

    Tsuji, H.; Satoh, H.; Ikeda, S.; Ikemura, S.; Gotoh, Y.; Ishikawa, J.

    1999-01-01

    Negative-silver-ion implantation into tissue-culture polystyrene (TCPS) dishes was investigated and it was found to modify hydrophilic and cell attachment properties of the dishes. Negative-ion implantation has an advantage of being almost free of surface charging, and is a suitable method for implantation into insulators such as polymers. Negative silver ions are used due to the antibacterial property of silver. Ag-implanted TCPS dishes had a contact angle larger than the normal value of 66 deg. of unimplanted dishes. The contact angle of water had a strong dependence on the ion energy rather than the dose. As a cell-culture experiment, human umbilical vascular endothelial cell (HUVEC) was used in unimplanted and Ag-implanted TCPS dishes, the implantation removed the cell-attachment property of the surface. In implantation with a mask with a striped pattern, most attached cells of HUVEC were in the unimplanted region aligned along a stripe direction

  4. Seismomorphogenesis: a novel approach to acclimatization of tissue culture regenerated plants.

    Science.gov (United States)

    Sarmast, Mostafa Khoshhal; Salehi, Hassan; Khosh-Khui, Morteza

    2014-12-01

    Plantlets under in vitro conditions transferred to ex vivo conditions are exposed to biotic and abiotic stresses. Furthermore, in vitro regenerated plants are typically frail and sometimes difficult to handle subsequently increasing their risk to damage and disease; hence acclimatization of these plantlets is the most important step in tissue culture techniques. An experiment was conducted under in vitro conditions to study the effects of shaking duration (twice daily at 6:00 a.m. and 9:00 p.m. for 2, 4, 8, and 16 min at 250 rpm for 14 days) on Sansevieria trifasciata L. as a model plant. Results showed that shaking improved handling, total plant height, and leaf characteristics of the model plant. Forty-eight hours after 14 days of shaking treatments with increasing shaking time, leaf length decreased but proline content of leaf increased. However, 6 months after starting the experiment different results were observed. In explants that received 16 min of shaking treatment, leaf length and area and photosynthesis rate were increased compared with control plantlets. Six months after starting the experiment, control plantlets had 12.5 % mortality; however, no mortality was observed in other treated explants. The results demonstrated that shaking improved the explants' root length and number and as a simple, cost-effective, and non-chemical novel approach may be substituted for other prevalent acclimatization techniques used for tissue culture regenerated plantlets. Further studies with sensitive plants are needed to establish this hypothesis.

  5. Screening Test of Greenhouse Seeding Exercise Matrix for Tissue Culture Seeding of Dendrobium Officinale Kimura et Migo

    Directory of Open Access Journals (Sweden)

    Zhou Yuan

    2015-01-01

    Full Text Available The Dendrobium officinale Kimura et Migo has a high demand on planting matrix, while its tissue culture seeding has much more demands on planting matrix. To find out a seeding exercise matrix to enhance the survival rate of tissue culture seeding of Dendrobium officinale Kimura et Migo more efficiently, this article carries out a screening test of greenhouse seeding exercise matrix material for tissue culture seeding of Dendrobium officinale Kimura et Migo. The test adopts full random test design, mainly for screening test of five matrix materials, namely pine bark, camphor tree bark, fern root, peanut shell and longan bark. Compare the impact of prepared seeding exercise matrix on the survival rate and growth trend (including plant height, growth rate and bud growth rate. The test result shows that: The seeding exercise matrix prepared by fern root is the most efficient, and the survival rate, plant height, growth rate and bud growth rate have achieved 100%, 4.5cm, 43.67% and 54.33% respectively. The main reason may be that the seeding exercise matrix C prepared by fern root is fairly loose and has a great water permeability, which is conducive to the growth of Dendrobium officinale Kimura et Migo.

  6. THE PLACE OF RETRACTION CORDS AMONG THE TISSUE DISPLACEMENT METHODS

    Directory of Open Access Journals (Sweden)

    Stoyan Yankov

    2017-12-01

    Full Text Available Gingival displacement is performed to create sufficient space between the finishing line and the gingival tissue, to allow the injection of the adequate bulk of the impression material into the expanded crevice. Control of moisture in the sulcus is also necessary. The variety of methods for tissue management can be broadly classified into surgical and non-surgical. Objective: To analyse the properties of tissue displacement methods, described in the literature for the last 4 years and display the prefered choices of the practitioners. Material and method: A time range from the last 4 years was set. Using the keywords “retraction cord” and “survey,” we found 64 from 115 articles in total, relevant to our topic. Patents, citations and books weren’t included in this review. Results from the overview of the properties of the different tissue management methods indicate that retraction cords take a significant place among them and can be recognised as a classical and well known method. Conclusions: The studies from the articles show adequate sulcal width right after retraction with most methods, sufficient haemostasis can also be obtained. Every each method, however, is accompanied by several drawbacks. Concidering all the quallities of the different tissue dispalcent methods, there is no specific evidence to promote the use of a single technique over any other. The selection of the method for gingival retraction primarily depends on each clinical case. However, the retraction cord technique remains to be the prefered method for gingival management due to its many advantages.

  7. Finite element study of scaffold architecture design and culture conditions for tissue engineering.

    Science.gov (United States)

    Olivares, Andy L; Marsal, Elia; Planell, Josep A; Lacroix, Damien

    2009-10-01

    Tissue engineering scaffolds provide temporary mechanical support for tissue regeneration and transfer global mechanical load to mechanical stimuli to cells through its architecture. In this study the interactions between scaffold pore morphology, mechanical stimuli developed at the cell microscopic level, and culture conditions applied at the macroscopic scale are studied on two regular scaffold structures. Gyroid and hexagonal scaffolds of 55% and 70% porosity were modeled in a finite element analysis and were submitted to an inlet fluid flow or compressive strain. A mechanoregulation theory based on scaffold shear strain and fluid shear stress was applied for determining the influence of each structures on the mechanical stimuli on initial conditions. Results indicate that the distribution of shear stress induced by fluid perfusion is very dependent on pore distribution within the scaffold. Gyroid architectures provide a better accessibility of the fluid than hexagonal structures. Based on the mechanoregulation theory, the differentiation process in these structures was more sensitive to inlet fluid flow than axial strain of the scaffold. This study provides a computational approach to determine the mechanical stimuli at the cellular level when cells are cultured in a bioreactor and to relate mechanical stimuli with cell differentiation.

  8. Direct DNA extraction method of an obligate parasitic fungus from infected plant tissue.

    Science.gov (United States)

    Liu, L; Wang, C L; Peng, W Y; Yang, J; Lan, M Q; Zhang, B; Li, J B; Zhu, Y Y; Li, C Y

    2015-12-28

    Powdery mildew and rust fungi are obligate parasites that cannot live without host organisms. They are difficult to culture in synthetic medium in the laboratory. Genomic DNA extraction is one of the basic molecular techniques used to study the genetic structure of populations. In this study, 2 different DNA extraction methods, Chelex-100 and cetyltrimethylammonium bromide (CTAB), were used to extract DNA from euonymus powdery mildew and Puccinia striiformis f. sp Tritici. Polymerase chain reaction was carried out with a race-specific-marker rDNA-internal transcribed spacer sequence. Both DNA extraction methods were compared and analyzed. The results showed that both Chelex-100 and CTAB were effective for extracting genomic DNA from infected plant tissue. However, less DNA was required for the Chelex-100 method than for the CTAB method, and the Chelex-100 method involved fewer steps, was simpler and safer, and did not require organic solvents compared to the CTAB method. DNA quality was evaluated by polymerase chain reaction, and the results showed that genomic DNA extracted using the Chelex-100 method was better than that using CTAB method, and was sufficient for studying the genetic structure of population.

  9. Tissue Engineering Using Transfected Growth-Factor Genes

    Science.gov (United States)

    Madry, Henning; Langer, Robert S.; Freed, Lisa E.; Trippel, Stephen; Vunjak-Novakovic, Gordana

    2005-01-01

    A method of growing bioengineered tissues includes, as a major component, the use of mammalian cells that have been transfected with genes for secretion of regulator and growth-factor substances. In a typical application, one either seeds the cells onto an artificial matrix made of a synthetic or natural biocompatible material, or else one cultures the cells until they secrete a desired amount of an extracellular matrix. If such a bioengineered tissue construct is to be used for surgical replacement of injured tissue, then the cells should preferably be the patient s own cells or, if not, at least cells matched to the patient s cells according to a human-leucocyteantigen (HLA) test. The bioengineered tissue construct is typically implanted in the patient's injured natural tissue, wherein the growth-factor genes enhance metabolic functions that promote the in vitro development of functional tissue constructs and their integration with native tissues. If the matrix is biodegradable, then one of the results of metabolism could be absorption of the matrix and replacement of the matrix with tissue formed at least partly by the transfected cells. The method was developed for articular chondrocytes but can (at least in principle) be extended to a variety of cell types and biocompatible matrix materials, including ones that have been exploited in prior tissue-engineering methods. Examples of cell types include chondrocytes, hepatocytes, islet cells, nerve cells, muscle cells, other organ cells, bone- and cartilage-forming cells, epithelial and endothelial cells, connective- tissue stem cells, mesodermal stem cells, and cells of the liver and the pancreas. Cells can be obtained from cell-line cultures, biopsies, and tissue banks. Genes, molecules, or nucleic acids that secrete factors that influence the growth of cells, the production of extracellular matrix material, and other cell functions can be inserted in cells by any of a variety of standard transfection techniques.

  10. Comparison of Standard Culture-Based Method to Culture-Independent Method for Evaluation of Hygiene Effects on the Hand Microbiome

    Science.gov (United States)

    Leff, J.; Henley, J.; Tittl, J.; De Nardo, E.; Butler, M.; Griggs, R.; Fierer, N.

    2017-01-01

    ABSTRACT Hands play a critical role in the transmission of microbiota on one’s own body, between individuals, and on environmental surfaces. Effectively measuring the composition of the hand microbiome is important to hand hygiene science, which has implications for human health. Hand hygiene products are evaluated using standard culture-based methods, but standard test methods for culture-independent microbiome characterization are lacking. We sampled the hands of 50 participants using swab-based and glove-based methods prior to and following four hand hygiene treatments (using a nonantimicrobial hand wash, alcohol-based hand sanitizer [ABHS], a 70% ethanol solution, or tap water). We compared results among culture plate counts, 16S rRNA gene sequencing of DNA extracted directly from hands, and sequencing of DNA extracted from culture plates. Glove-based sampling yielded higher numbers of unique operational taxonomic units (OTUs) but had less diversity in bacterial community composition than swab-based sampling. We detected treatment-induced changes in diversity only by using swab-based samples (P hand hygiene industry methods and for future hand microbiome studies. On the basis of our results and previously published studies, we propose recommendations for best practices in hand microbiome research. PMID:28351915

  11. Changing perspective on tissue processing - comparison of microwave histoprocessing method with the conventional method

    Directory of Open Access Journals (Sweden)

    G Shrestha

    2015-09-01

    Full Text Available Background: Histopathological examination of tissues requires sliver of formalin fixed tissue that has been chemically processed and then stained with Haematoxylin and Eosin. The time honored conventional method of tissue processing, which requires 12 to 13 hours for completion, is employed at majority of laboratories but is now seeing the

  12. Tissue culture media supplemented with 10% fetal calf serum contains a castrate level of testosterone.

    NARCIS (Netherlands)

    Sedelaar, J.P.M.; Isaacs, J.T.

    2009-01-01

    BACKGROUND: Human prostate cancer cells are routinely maintained in media supplemented with 10% Fetal Calf Serum (FCS) to provide androgen. In the present study, total and free testosterone levels in 10%FCS supplemented tissue culture media were determined and compared to levels in intact and

  13. Diagnostic accuracy of morphologic identification of filamentous fungi in paraffin embedded tissue sections: Correlation of histological and culture diagnosis

    Directory of Open Access Journals (Sweden)

    Sundaram Challa

    2014-01-01

    Full Text Available Aims and Objectives: The aim was to investigate the correlation between histological and culture diagnosis of filamentous fungi. Materials and Methods: Tissue sections from biopsy samples stained with Hematoxylin and Eosin and special stains from samples of chronic invasive/noninvasive sinusitis and intracranial space occupying lesions during 2005-2011 diagnosed to have infection due to filamentous fungi were reviewed. The histopathology and culture diagnoses were analyzed for correlation and discrepancy. Results: There were 125 samples positive for filamentous fungi on biopsy. Of these 76 (60.8% were submitted for culture and fungi grew in 30 (39.97% samples. There was a positive correlation between histological and culture diagnosis in 25 (83.33% samples that included Aspergillus species (16/19, Zygomycetes species (8/10 and dematiaceous fungi (1/1. The negative yield of fungi was more in Zygomycetes species (20/30 when compared to Aspergillus species (25/44. There was a discrepancy in diagnosis in 5/30 (16.67% samples which included probable dual infection in two, and dematiaceous fungi being interpreted as Aspergillus species in three samples. Conclusion: Histopathology plays a major role in the diagnosis of infection due to filamentous fungi, especially when cultures are not submitted or negative. The discrepancy between histological and culture diagnosis was either due to dematiaceous fungi being interpreted as Aspergillus species or probable dual infection.

  14. Applications of Biomaterials in Corneal Endothelial Tissue Engineering.

    Science.gov (United States)

    Wang, Tsung-Jen; Wang, I-Jong; Hu, Fung-Rong; Young, Tai-Horng

    2016-11-01

    When corneal endothelial cells (CECs) are diseased or injured, corneal endothelium can be surgically removed and tissue from a deceased donor can replace the original endothelium. Recent major innovations in corneal endothelial transplantation include replacement of diseased corneal endothelium with a thin lamellar posterior donor comprising a tissue-engineered endothelium carried or cultured on a thin substratum with an organized monolayer of cells. Repairing CECs is challenging because they have restricted proliferative ability in vivo. CECs can be cultivated in vitro and seeded successfully onto natural tissue materials or synthetic polymeric materials as grafts for transplantation. The optimal biomaterials for substrata of CEC growth are being investigated. Establishing a CEC culture system by tissue engineering might require multiple biomaterials to create a new scaffold that overcomes the disadvantages of single biomaterials. Chitosan and polycaprolactone are biodegradable biomaterials approved by the Food and Drug Administration that have superior biological, degradable, and mechanical properties for culturing substratum. We successfully hybridized chitosan and polycaprolactone into blended membranes, and demonstrated that CECs proliferated, developed normal morphology, and maintained their physiological phenotypes. The interaction between cells and biomaterials is important in tissue engineering of CECs. We are still optimizing culture methods for the maintenance and differentiation of CECs on biomaterials.

  15. Fusarium growth on culture media made of tissue juice from irradiated and unirradiated potato tubers

    International Nuclear Information System (INIS)

    Taczanowski, M.

    1994-01-01

    Fusarium Sulphureum Schlecht is one of the tuber pathogens causing potato storage disease knowing as dry rot. Because irradiation can disturb the tissue defence mechanism against the pathogen, it was decided to carry out experiments on influence of the treatment on subsequent tuber tissue reaction to a maceration process. The maceration as a physical stress was a substitute for the pathogen activity. Tubers of two potato varieties were tested: Mila -a resistant variety to Fusarium and Atol - susceptible one. Tubers of both varieties were irradiated with a dose of 105 kGy. Unirradiated tubers were taken as a control. A day after irradiation the cortex tissue was macerated using an ordinary rasper and the resulted tissue pulp was strained through medical gauze to obtain crude juice. The juice was clarified by centrifugation and then added to dissolved PDA. The volume ratio of juice to PDA was 1:1. The prepared media were dispensed into Petri dishes. Small pieces of the Fusarium culture were put on the surface of the medium at the centre of each Petri dish. Subsequent growth of the fungus was assessed by measurement of culture diameters every 24 hours. Linear functions of the Fusarium growth were obtained for Mila control and Atol control. In the case of Mila, the Fusarium found more favourable conditions for its growth in the presence of juice from irradiated tubers than from the control ones. Making the same comparison for Atol, no difference was detected. (author)

  16. The awareness of employees in safety culture through the improved nuclear safety culture evaluation method

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Young Ga; Sung, Chan Ho; Jung, Yeon Sub [KHNP Central Research Institute, Daejeon (Korea, Republic of)

    2012-10-15

    After the Chernobyl nuclear accident in 1986, nuclear safety culture terminology was at first introduced emphasizing the importance of employees' attitude and organizational safety. The concept of safety culture was spread by INSAG 4 published in 1991. From that time, IAEA had provided the service of ASCOT for the safety culture assessment. However, many people still are thinking that safety culture is abstract and is not clear. It is why the systematic and reliable assessment methodology was not developed. Assessing safety culture is to identify what is the basic assumption for any organization to accept unconsciously. Therefore, it is very difficult to reach a meaningful conclusion by a superficial investigation alone. KHNP had been doing the safety culture assessment which was based on ASCOT methodology every 2 years. And this result had contributed to improving safety culture. But this result could not represent the level of organization's safety culture due to the limitation of method. So, KHNP has improved the safety culture method by benchmarking the over sea assessment techniques in 2011. The effectiveness of this improved methodology was validated through a pilot assessment. In this paper, the level of employees' safety culture awareness was analyzed by the improved method and reviewed what is necessary for the completeness and objectivity of the nuclear safety culture assessment methodology.

  17. The awareness of employees in safety culture through the improved nuclear safety culture evaluation method

    International Nuclear Information System (INIS)

    Kim, Young Ga; Sung, Chan Ho; Jung, Yeon Sub

    2012-01-01

    After the Chernobyl nuclear accident in 1986, nuclear safety culture terminology was at first introduced emphasizing the importance of employees' attitude and organizational safety. The concept of safety culture was spread by INSAG 4 published in 1991. From that time, IAEA had provided the service of ASCOT for the safety culture assessment. However, many people still are thinking that safety culture is abstract and is not clear. It is why the systematic and reliable assessment methodology was not developed. Assessing safety culture is to identify what is the basic assumption for any organization to accept unconsciously. Therefore, it is very difficult to reach a meaningful conclusion by a superficial investigation alone. KHNP had been doing the safety culture assessment which was based on ASCOT methodology every 2 years. And this result had contributed to improving safety culture. But this result could not represent the level of organization's safety culture due to the limitation of method. So, KHNP has improved the safety culture method by benchmarking the over sea assessment techniques in 2011. The effectiveness of this improved methodology was validated through a pilot assessment. In this paper, the level of employees' safety culture awareness was analyzed by the improved method and reviewed what is necessary for the completeness and objectivity of the nuclear safety culture assessment methodology

  18. Cell culture density affects the proliferation activity of human adipose tissue stem cells.

    Science.gov (United States)

    Kim, Dae Seong; Lee, Myoung Woo; Ko, Young Jong; Chun, Yong Hoon; Kim, Hyung Joon; Sung, Ki Woong; Koo, Hong Hoe; Yoo, Keon Hee

    2016-01-01

    In this study, we investigated the effect of cell density on the proliferation activity of human mesenchymal stem cells (MSCs) derived from adipose tissue (AT-MSCs) over time in culture. Passage #4 (P4) and #12 (P12) AT-MSCs from two donors were plated at a density of 200 (culture condition 1, CC1) or 5000 (culture condition 2, CC2) cells cm(-2) . After 7 days of incubation, P4 and P12 AT-MSCs cultured in CC1 were thin and spindle-shaped, whereas those cultured in CC2 had extensive cell-to-cell contacts and an expanded cell volume. In addition, P4 and P12 AT-MSCs in CC1 divided more than three times, while those in CC2 divided less than once on average. Flow cytometric analysis using 5(6)-carboxyfluorescein diacetate N-succinimidyl ester dye showed that the fluorescence intensity of AT-MSCs was lower in CC1 than in CC2. Furthermore, expression of proliferation-associated genes, such as CDC45L, CDC20A and KIF20A, in P4 AT-MSCs was higher in CC1 than in CC2, and this difference was also observed in P12 AT-MSCs. These data demonstrated that cell culture density affects the proliferation activity of MSCs, suggesting that it is feasible to design a strategy to prepare suitable MSCs using specific culture conditions. Copyright © 2016 John Wiley & Sons, Ltd.

  19. Effects of cyclic compression on the mechanical properties and calcification process of immature chick bone tissue in culture.

    Science.gov (United States)

    Maeda, Eijiro; Nakagaki, Masashi; Ichikawa, Katsuhisa; Nagayama, Kazuaki; Matsumoto, Takeo

    2017-06-01

    Contribution of mechanical loading to tissue growth during both the development and post-natal maturation is of a particular interest, as its understanding would be important to strategies in bone tissue engineering and regenerative medicine. The present study has been performed to investigate how immature bone responds to mechanical loading using an ex vivo culture system. A slice of the tibia, with the thickness of 3 mm, was obtained from 0-day-old chick. For the ex vivo culture experiment in conjunction with cyclic compressive loading, we developed a custom-made, bioreactor system where both the load and the deformation applied to the specimen was recorded. Cyclic compression, with an amplitude of 0.3 N corresponding to 1 to 2% compressive strain, was applied to immature bone specimen during a 3-day culture period at an overall loading rate 3-4 cycles/min, in the presence of β-glycerol phosphate and dexamethasone in culture medium. The stress-strain relationship was obtained at the beginning and the end of the culture experiment. In addition, analyses for alkaline phosphate release, cell viability and tissue calcification were also performed. It was exhibited that elastic moduli of bone slices were significantly elevated at the end of the 3-day culture in the presence of cyclic compression, which was a similar phenomenon to significant elevation of the elastic moduli of bone tissue by the maturation from 0-day old to 3-day old. By contrast, no significant changes in the moduli were observed in the absence of cyclic compression or in deactivated, cell-free samples. The increases in the moduli were coincided with the increase in calcified area in the bone samples. It was confirmed that immature bone can respond to compressive loading in vitro and demonstrate the growth of bone matrix, similar to natural, in vivo maturation. The elevation of the elastic moduli was attributable to the increased calcified area and the realignment of collagen fibers parallel to

  20. Effects of cyclic compression on the mechanical properties and calcification process of immature chick bone tissue in culture

    Directory of Open Access Journals (Sweden)

    Eijiro Maeda

    2017-06-01

    Full Text Available Contribution of mechanical loading to tissue growth during both the development and post-natal maturation is of a particular interest, as its understanding would be important to strategies in bone tissue engineering and regenerative medicine. The present study has been performed to investigate how immature bone responds to mechanical loading using an ex vivo culture system. A slice of the tibia, with the thickness of 3 mm, was obtained from 0-day-old chick. For the ex vivo culture experiment in conjunction with cyclic compressive loading, we developed a custom-made, bioreactor system where both the load and the deformation applied to the specimen was recorded. Cyclic compression, with an amplitude of 0.3 N corresponding to 1 to 2% compressive strain, was applied to immature bone specimen during a 3-day culture period at an overall loading rate 3–4 cycles/min, in the presence of β-glycerol phosphate and dexamethasone in culture medium. The stress-strain relationship was obtained at the beginning and the end of the culture experiment. In addition, analyses for alkaline phosphate release, cell viability and tissue calcification were also performed. It was exhibited that elastic moduli of bone slices were significantly elevated at the end of the 3-day culture in the presence of cyclic compression, which was a similar phenomenon to significant elevation of the elastic moduli of bone tissue by the maturation from 0-day old to 3-day old. By contrast, no significant changes in the moduli were observed in the absence of cyclic compression or in deactivated, cell-free samples. The increases in the moduli were coincided with the increase in calcified area in the bone samples. It was confirmed that immature bone can respond to compressive loading in vitro and demonstrate the growth of bone matrix, similar to natural, in vivo maturation. The elevation of the elastic moduli was attributable to the increased calcified area and the realignment of collagen

  1. Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs.

    Science.gov (United States)

    Gnedeva, Ksenia; Hudspeth, A J; Segil, Neil

    2018-06-01

    The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

  2. Explaining the Effectiveness of the Contrast Culture Method for Managing Interpersonal Interactions across Cultures

    Science.gov (United States)

    Hiratsuka, Hiroyoshi; Suzuki, Hanako; Pusina, Alexis

    2016-01-01

    One of the current challenges in the field of intercultural education comes from the limited availability of training efficacy studies. The present study focused on explaining the effectiveness of the Contrast Culture Method (CCM) as an intercultural education method for managing interpersonal interactions across cultures between graduate…

  3. Cytokinesis-block micronucleus method in micro-blood cultures

    International Nuclear Information System (INIS)

    Liu Jinwen; Wang Lianzhi; Yang Cangzhen; Yao Yanyu

    1991-01-01

    This paper reports the cytokinesis-block micronucleus method in micro-blood cultures. The observations on detection induced micronuclei of different doses of 60 Co γ-rays irradiation and spontaneous micronucleus of different ages were performed with CB method in comporison with conventional micronucleus (CM) method. The results showed that with direct peripheral micro-blood cultures the cytoknesis-block micronuclei is also obtained. Using CB method, the micronuclei fequency of different ages was linear relationship, Y = 1.62 + 0.74 D, the spontaneous micronuclei frequency of different ages was 4.14%, the induced micronuclei also was a linear relationship, Y = 6.01 + 0.692 D. Using CM method, it showed that the induced micronuclei was a linear relationship, Y = 0.486 D - 1.968, but there is no significant difference between the micronuclei frequency of different ages. Comparison with CM and direct blood smear methods confirmed that the cytokinesis-block method of micro-blood cultures is more sensitive and precise

  4. Photoactivated methods for enabling cartilage-to-cartilage tissue fixation

    Science.gov (United States)

    Sitterle, Valerie B.; Roberts, David W.

    2003-06-01

    The present study investigates whether photoactivated attachment of cartilage can provide a viable method for more effective repair of damaged articular surfaces by providing an alternative to sutures, barbs, or fibrin glues for initial fixation. Unlike artificial materials, biological constructs do not possess the initial strength for press-fitting and are instead sutured or pinned in place, typically inducing even more tissue trauma. A possible alternative involves the application of a photosensitive material, which is then photoactivated with a laser source to attach the implant and host tissues together in either a photothermal or photochemical process. The photothermal version of this method shows potential, but has been almost entirely applied to vascularized tissues. Cartilage, however, exhibits several characteristics that produce appreciable differences between applying and refining these techniques when compared to previous efforts involving vascularized tissues. Preliminary investigations involving photochemical photosensitizers based on singlet oxygen and electron transfer mechanisms are discussed, and characterization of the photodynamic effects on bulk collagen gels as a simplified model system using FTIR is performed. Previous efforts using photothermal welding applied to cartilaginous tissues are reviewed.

  5. Strategies on process engineering of chondrocyte culture for cartilage tissue regeneration.

    Science.gov (United States)

    Mallick, Sarada Prasanna; Rastogi, Amit; Tripathi, Satyavrat; Srivastava, Pradeep

    2017-04-01

    The current work is an attempt to study the strategies for cartilage tissue regeneration using porous scaffold in wavy walled airlift bioreactor (ALBR). Novel chitosan, poly (L-lactide) and hyaluronic acid based composite scaffold were prepared. The scaffolds were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide and chondroitin sulfate to obtain interconnected 3D microstructure showing excellent biocompatibility, higher cellular differentiation and increased stability. The surface morphology and porosity of the scaffolds were analyzed using scanning electron microscopy (SEM) and mercury intrusion porosimeter and optimized for chondrocyte regeneration. The study shows that the scaffolds were highly porous with pore size ranging from 48 to 180 µm and the porosities in the range 80-92%. Swelling and in vitro degradation studies were performed for the composite scaffolds; by increasing the chitosan: HA ratio in the composite scaffolds, the swelling property increases and stabilizes after 24 h. There was controlled degradation of composite scaffolds for 4 weeks. The uniform chondrocyte distribution in the scaffold using various growth modes in the shake flask and ALBR was studied by glycosaminoglycans (GAG) quantification, MTT assay and mixing time evaluation. The cell culture studies demonstrated that efficient designing of ALBR increases the cartilage regeneration as compared to using a shake flask. The free chondrocyte microscopy and cell attachment were performed by inverted microscope and SEM, and from the study it was confirmed that the cells uniformly attached to the scaffold. This study focuses on optimizing strategies for the culture of chondrocyte using suitable scaffold for improved cartilage tissue regeneration.

  6. Effects of CO 2 concentration and moisture content of sugar-free media on the tissue-cultured plantlets in a large growth chamber

    Science.gov (United States)

    Qu, Y. H.; Lin, C.; Zhou, W.; Li, Y.; Chen, B.; Chen, G. Q.

    2009-01-01

    The dynamic fluctuations of CO 2 concentration in the tissue culture growth chamber after transplantation of petunia, chrysanthemum and tomato plantlets were recorded with a real-time control system to determine the critical CO 2 concentration levels of 35 μl l -1 at which CO 2 enrichment is needed. The experimental data showed that the tissue-cultured plantlets of petunia, chrysanthemum and tomato had the same CO 2 concentration dynamics. The results indicated that CO 2 enrichment was proper on the second day after transplantation. Petunia plantlets were used to conduct experiments under PPFD of 80 μmol m -2 s -1, and CO 2 concentrations of 350 ± 50 μl l -1, 650 ± 50 μl l -1 and 950 ± 50 μl l -1 as well as medium moisture contents of 60%, 70% and 80%, with the result that plantlets grew better under CO 2 concentration of 650 ± 50 μl l -1 than under the other two concentrations with all the different media water contents. Three media water contents under the same CO 2 concentration produced plantlets with the same quality. The impacts of CO 2 concentrations on plantlets are more important than those of the media water contents. Sugar-free tissue culture, as compared with the conventional culture, showed that CO 2 enrichment to 350 ± 50 μl l -1 can promote the growth of the cultured plantlets. Sugar-free tissue culture produced healthy plantlets with thick roots, almost equivalent to the common plantlets.

  7. Transformation and mass hyperplasia technique of the garden plant (lily) by radiation and so forth. Mass hyperplasia of the lily using tissue culture

    International Nuclear Information System (INIS)

    Shigematsu, Koji; Hamada, Yutaka

    1997-01-01

    For an aim of more uniform child bulb production and good quality kind conservation using tissue culture of the lily, some hyperplasia from organs over ground of the lily were tried. In particular, optimum culture media with higher hyperplasia rate of the child bulb, redifferentiation due to difference among kinds of the lilies, and difference of hyperplasia of the child bulbs were investigated. As a result, it was found that pollution due to various germs attached to used materials often occurs, that efficiency obtainable for initial child bulb by redifferentiation from the organs was low at 20%, and that pollution due to various germs was often found at 25degC of cultivation temperature, which was inferior to that at 20degC. And, when conducting mass hyperplasia of the lily using tissue culture, an optimum culture medium of formation and hyperplasia of child bulb could be obtained for its each kind. As a result of conducting some investigations on configuration of the lily nourished from its child bulb and flowered by the tissue culture, it was also found that cultured bulb had the same character as its parent bulb had. (G.K.)

  8. Prolonged hypoxic culture and trypsinization increase the pro-angiogenic potential of human adipose tissue-derived stem cells

    DEFF Research Database (Denmark)

    Rasmussen, Jeppe Grøndahl; Frøbert, Ole; Pilgaard, Linda

    2011-01-01

    Transplantation of mesenchymal stromal cells (MSC), including adipose tissue-derived stem cells (ASC), is a promising option in the treatment of vascular disease. Short-term hypoxic culture of MSC augments secretion of anti-apoptotic and angiogenic cytokines. We hypothesized that prolonged hypoxic...... (1% and 5% oxygen) culture and trypsinization would augment ASC expression of anti-apoptotic and angiogenic cytokines and increase the angiogenic potential of ASC-conditioned media....

  9. Induction of osteogenic differentiation of stem cells via a lyophilized microRNA reverse transfection formulation on a tissue culture plate

    Directory of Open Access Journals (Sweden)

    Wu K

    2013-05-01

    Full Text Available Kaimin Wu,1,* Jie Xu,2,* Mengyuan Liu,1 Wen Song,1 Jun Yan,1 Shan Gao,3 Lingzhou Zhao,2 Yumei Zhang1 1Department of Prosthetic Dentistry, 2Department of Periodontology and Oral Medicine, School of Stomatology, The Fourth Military Medical University, Xi’an, People’s Republic of China; 3The Interdisciplinary Nanoscience Center and Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark; School of Stomatology, Tianjin Medical University, Tianjin, People’s Republic of China*Both authors contributed equally to this workAbstract: MicroRNA (miRNA regulation is a novel approach to manipulating the fate of mesenchymal stem cells, but an easy, safe, and highly efficient method of transfection is required. In this study, we developed an miRNA reverse transfection formulation by lyophilizing Lipofectamine 2000-miRNA lipoplexes on a tissue culture plate. The lipoplexes can be immobilized on a tissue culture plate with an intact pseudospherical structure and lyophilization without any lyoprotectant. In this study, reverse transfection resulted in highly efficient cellular uptake of miRNA and enabled significant manipulation of the intracellular target miRNA level. Reverse transfection formulations containing Lipofectamine 2000 1 µL per well generated much higher transfection efficiency without obvious cytotoxicity compared with conventional and other transfection methods. Further, the transfection efficiency of the reverse transfection formulations did not deteriorate during 90 days of storage at 4°C and -20°C. We then assessed the efficiency of the miRNA reverse transfection formulation in promoting osteogenic differentiation of mesenchymal stem cells. We found that transfection with anti-miR-138 and miR-148b was efficient for enhancing osteogenic differentiation, as indicated by enhanced osteogenesis-related gene expression, amount of alkaline phosphatase present, production of collagen, and matrix mineralization. Overall

  10. Micropropagation of Pear Rootstock (Pyrus Communis) by using tissue culture technique and gamma irradiation

    International Nuclear Information System (INIS)

    El-Sharnouby, M.E.; ESSAM, E.R.; Ayoub, S.

    2006-01-01

    New growing shoots from healthy pear rootstock (Pyrus communis) trees were taken and sterilized 3 times in dipping water. Explants were subjected to antioxidant treatment, different media, different additives and different BAP and NAA concentrations. The obtained results showed that Murashig-Skoog (MS) supplemented with 1 mg/l BA was better than Gamborg medium. Adding antioxidant solution and adenine sulphate to the culture medium was preferred for maximizing explants development. Exposing the explants to gamma irradiation at different doses decreased tissue culture parameters with increasing gamma doses. However, the low dose of gamma rays (1 Krad) significantly increased the number of shoots than other gamma treatments. Adding of BAP at 2 mg/l to the culture medium increased number and length of shoots. However, addition of 1 mg/l NAA to the rooting medium led to increase the root formation

  11. Detection of Met-enkephalin: Development of a RIA and of an extraction method for studies on hypophyseal and brain tissues

    International Nuclear Information System (INIS)

    Holl, R.

    1982-01-01

    The thesis describes the development of a method of detecting Met-enkephalin, and the verification of the method's suitability to measurements in extracted tissue. The tissue extraction method reported has been the first step towards the goal of establishing the method for determining Met-enkephalin in cell and tissue extracts, in culture media, plasma and liquor samples. The difficulties involved in the development of the RIA for Met-enkephalin specifically arose from the following peculiarities of the substance: a) Due to the low molecular weight, (574), ME itself does not act as an antigen, antibodies can only be obtained by means of fixation to carrier molecules. b) Enkephalin very rapidly is decomposed by endogenic proteinases. c) The fact that normal physiological processes will produce peptides very similar in sequence requires the RIA to be extremely specific. The method has first been applied to screening measurements of Met-enkephalin concentrations in various brain sectors. The radioimmunological studies have been intended to supplement and verify the immunocytochemical results obtained, with the latter experiments having been made using the same antiserum, in order to improve the basis of comparison between the immunocytochemical results and the findings on antibody specifity obtained from the RIA. (orig./MG) [de

  12. Nondestructive mechanical characterization of developing biological tissues using inflation testing.

    Science.gov (United States)

    Oomen, P J A; van Kelle, M A J; Oomens, C W J; Bouten, C V C; Loerakker, S

    2017-10-01

    One of the hallmarks of biological soft tissues is their capacity to grow and remodel in response to changes in their environment. Although it is well-accepted that these processes occur at least partly to maintain a mechanical homeostasis, it remains unclear which mechanical constituent(s) determine(s) mechanical homeostasis. In the current study a nondestructive mechanical test and a two-step inverse analysis method were developed and validated to nondestructively estimate the mechanical properties of biological tissue during tissue culture. Nondestructive mechanical testing was achieved by performing an inflation test on tissues that were cultured inside a bioreactor, while the tissue displacement and thickness were nondestructively measured using ultrasound. The material parameters were estimated by an inverse finite element scheme, which was preceded by an analytical estimation step to rapidly obtain an initial estimate that already approximated the final solution. The efficiency and accuracy of the two-step inverse method was demonstrated on virtual experiments of several material types with known parameters. PDMS samples were used to demonstrate the method's feasibility, where it was shown that the proposed method yielded similar results to tensile testing. Finally, the method was applied to estimate the material properties of tissue-engineered constructs. Via this method, the evolution of mechanical properties during tissue growth and remodeling can now be monitored in a well-controlled system. The outcomes can be used to determine various mechanical constituents and to assess their contribution to mechanical homeostasis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Dynamic culture of a thermosensitive collagen hydrogel as an extracellular matrix improves the construction of tissue-engineered peripheral nerve.

    Science.gov (United States)

    Huang, Lanfeng; Li, Rui; Liu, Wanguo; Dai, Jin; Du, Zhenwu; Wang, Xiaonan; Ma, Jianchao; Zhao, Jinsong

    2014-07-15

    Tissue engineering technologies offer new treatment strategies for the repair of peripheral nerve injury, but cell loss between seeding and adhesion to the scaffold remains inevitable. A thermosensitive collagen hydrogel was used as an extracellular matrix in this study and combined with bone marrow mesenchymal stem cells to construct tissue-engineered peripheral nerve composites in vitro. Dynamic culture was performed at an oscillating frequency of 0.5 Hz and 35° swing angle above and below the horizontal plane. The results demonstrated that bone marrow mesenchymal stem cells formed membrane-like structures around the poly-L-lactic acid scaffolds and exhibited regular alignment on the composite surface. Collagen was used to fill in the pores, and seeded cells adhered onto the poly-L-lactic acid fibers. The DNA content of the bone marrow mesenchymal stem cells was higher in the composites constructed with a thermosensitive collagen hydrogel compared with that in collagen I scaffold controls. The cellular DNA content was also higher in the thermosensitive collagen hydrogel composites constructed with the thermosensitive collagen hydrogel in dynamic culture than that in static culture. These results indicate that tissue-engineered composites formed with thermosensitive collagen hydrogel in dynamic culture can maintain larger numbers of seeded cells by avoiding cell loss during the initial adhesion stage. Moreover, seeded cells were distributed throughout the material.

  14. ISSLS prize winner: integrating theoretical and experimental methods for functional tissue engineering of the annulus fibrosus.

    Science.gov (United States)

    Nerurkar, Nandan L; Mauck, Robert L; Elliott, Dawn M

    2008-12-01

    Integrating theoretical and experimental approaches for annulus fibrosus (AF) functional tissue engineering. Apply a hyperelastic constitutive model to characterize the evolution of engineered AF via scalar model parameters. Validate the model and predict the response of engineered constructs to physiologic loading scenarios. There is need for a tissue engineered replacement for degenerate AF. When evaluating engineered replacements for load-bearing tissues, it is necessary to evaluate mechanical function with respect to the native tissue, including nonlinearity and anisotropy. Aligned nanofibrous poly-epsilon-caprolactone scaffolds with prescribed fiber angles were seeded with bovine AF cells and analyzed over 8 weeks, using experimental (mechanical testing, biochemistry, histology) and theoretical methods (a hyperelastic fiber-reinforced constitutive model). The linear region modulus for phi = 0 degrees constructs increased by approximately 25 MPa, and for phi = 90 degrees by approximately 2 MPa from 1 day to 8 weeks in culture. Infiltration and proliferation of AF cells into the scaffold and abundant deposition of s-GAG and aligned collagen was observed. The constitutive model had excellent fits to experimental data to yield matrix and fiber parameters that increased with time in culture. Correlations were observed between biochemical measures and model parameters. The model was successfully validated and used to simulate time-varying responses of engineered AF under shear and biaxial loading. AF cells seeded on nanofibrous scaffolds elaborated an organized, anisotropic AF-like extracellular matrix, resulting in improved mechanical properties. A hyperelastic fiber-reinforced constitutive model characterized the functional evolution of engineered AF constructs, and was used to simulate physiologically relevant loading configurations. Model predictions demonstrated that fibers resist shear even when the shearing direction does not coincide with the fiber direction

  15. A novel semi-quantitative method for measuring tissue bleeding.

    Science.gov (United States)

    Vukcevic, G; Volarevic, V; Raicevic, S; Tanaskovic, I; Milicic, B; Vulovic, T; Arsenijevic, S

    2014-03-01

    In this study, we describe a new semi-quantitative method for measuring the extent of bleeding in pathohistological tissue samples. To test our novel method, we recruited 120 female patients in their first trimester of pregnancy and divided them into three groups of 40. Group I was the control group, in which no dilation was applied. Group II was an experimental group, in which dilation was performed using classical mechanical dilators. Group III was also an experimental group, in which dilation was performed using a hydraulic dilator. Tissue samples were taken from the patients' cervical canals using a Novak's probe via energetic single-step curettage prior to any dilation in Group I and after dilation in Groups II and III. After the tissue samples were prepared, light microscopy was used to obtain microphotographs at 100x magnification. The surfaces affected by bleeding were measured in the microphotographs using the Autodesk AutoCAD 2009 program and its "polylines" function. The lines were used to mark the area around the entire sample (marked A) and to create "polyline" areas around each bleeding area on the sample (marked B). The percentage of the total area affected by bleeding was calculated using the formula: N = Bt x 100 / At where N is the percentage (%) of the tissue sample surface affected by bleeding, At (A total) is the sum of the surfaces of all of the tissue samples and Bt (B total) is the sum of all the surfaces affected by bleeding in all of the tissue samples. This novel semi-quantitative method utilizes the Autodesk AutoCAD 2009 program, which is simple to use and widely available, thereby offering a new, objective and precise approach to estimate the extent of bleeding in tissue samples.

  16. Effect of gamma-radiation on callus initiation and oraganogenesis in the tissue culture of Nicotiana tabaccum L

    International Nuclear Information System (INIS)

    Shin, S. H.; Kim, J. G.; Song, H. S.

    2004-01-01

    It is generally agreed that ionizing radiations stimulate cell division, growth and development in various organisms including animals and plants. Differentiating tissues are the most sensitive to radiation. The present experiment was carried out to investigate the effects of ionizing radiation on callus initiation and organogenesis from the stem in the culture of Nicotiana tabaccum L. cv. When the stem segments were cultured on a Murashige and Skoog (MS) medium with 2 mg/L kinetin, with 1 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D), with 2 mg/L kinetin and 1 mg/L 2,4-D, the shoots and callus were differentiated 14 days after cultivation. Callus was especially formed on the MS medium with 2,4-D and/or kinetin and the formation was promoted by 1 Gy and 5 Gy of gamma radiation. The formation of the shoot clusters on the MS medium with 2 mg/L kinetin were prominent in the 5 Gy-irradiated groups. It is concluded that that gamma radiation enhanced the callus initiation and organogenesis in the tissue culture of Nicotiana tabaccum L

  17. Protein and Glycoprotein Patterns Related to Morphogenesis in Mammillaria gracillis Pfeiff. Tissue Culture

    Directory of Open Access Journals (Sweden)

    Biljana Balen

    2002-01-01

    Full Text Available As plants with Crassulacean Acid Metabolism (CAM, cacti are highly affected by artificial environmental conditions in tissue culture. Plants of Mammillaria gracillis Pfeiff. (Cactaceae propagated in vitro produced callus spontaneously. This habituated callus regenerated normal and hyperhydric shoots without the addition of growth regulators. In order to compare habituated callus with the tumorous one, cactus cells were transformed with two strains of Agrobacterium tumefaciens: the wild strain B6S3 (tumour line TW and the rooty mutant GV3101 (tumour line TR. Gene expression in cactus plants, habituated callus, regenerated shoots and two tumour lines was analysed at the level of cellular and extracellular protein and glycoprotein profiles. Proteins were separated by SDS-polyacrylamide gel electrophoresis and 2-D PAGE electrophoresis and silver stained. Concavalin A-peroxidase staining detected glycoproteins with D-manose in their glycan component on protein blots. Developmentally specific protein patterns of Mammillaria gracillis tissue lines were detected. The 2-D PAGE electrophoresis revealed some tissue specific protein groups. The cellular glycoprotein of 42 kDa detected by ConA was highly expressed in undifferentiated tissues (habituated callus, TW and TR tumours and in hyperhydric regenerants. Tumours produced extracellular proteins of 33, 23 and 22 kDa. The N glycosylation of cellular and extracellular proteins was related to specific developmental stage of cactus tissue.

  18. Increased adsorption of histidine-tagged proteins onto tissue culture polystyrene.

    Science.gov (United States)

    Holmberg, Maria; Hansen, Thomas Steen; Lind, Johan Ulrik; Hjortø, Gertrud Malene

    2012-04-01

    In this study we compare histidine-tagged and native proteins with regards to adsorption properties. We observe significantly increased adsorption of proteins with an incorporated polyhistidine amino acid motif (HIS-tag) onto tissue culture polystyrene (TCPS) compared to similar proteins without a HIS-tag. The effect is not observed on polystyrene (PS). Adsorption experiments have been performed at physiological pH (7.4) and the effect was only observed for the investigated proteins that have pI values below or around 7.4. Competitive adsorption experiments with imidazole and ethylenediaminetetraacetic acid (EDTA), as well as adsorption performed at different pH and ionic strength indicates that the high adsorption is caused by electrostatic interaction between negatively charged carboxylate groups on the TCPS surface and positively charged histidine residues in the proteins. Pre-adsorption of bovine serum albumin (BSA) does not decrease the adsorption of HIS-tagged proteins onto TCPS. Our findings identify a potential problem in using HIS-tagged signalling molecule in assays with cells cultured on TCPS, since the concentration of the molecule in solution might be affected and this could critically influence the assay outcome. Copyright © 2011 Elsevier B.V. All rights reserved.

  19. Selective enhancement of scopadulcic acid B production in the cultured tissues of Scoparia dulcis by methyl jasmonate.

    Science.gov (United States)

    Nkembo, Kasidimoko Marguerite; Lee, Jung-Bum; Hayashi, Toshimitsu

    2005-07-01

    The effects of methyl jasmonate (MeJA) on isoprenoid production were evaluated in cultured tissues of Scoparia dulcis. It was found that MeJA suppressed the accumulation of chlorophylls, carotenoids, phytol and beta-sitosterol in the tissues. MeJA, however, remarkably enhanced the production of scopadulcic acid B (SDB), with 10 microM being optimal observed concentration for stimulation of SDB production. The maximum concentration of SDB was observed 6 d after MeJA treatment.

  20. Polyamine patterns in haploid and diploid tobacco tissues and in vitro cultures

    Directory of Open Access Journals (Sweden)

    Sílvia Bicudo Carone

    2010-04-01

    Full Text Available The aim of this work was to determine PAs levels in pith tissues and callus cultures from haploid and diploid tobacco plants, explanted from the apical and basal regions of the stem. These explants were cultured in an RM-64 medium supplied with IAA and kinetin, under light or in the dark, during successive subcultures. PAs levels followed a basipetal decrease in diploid and an increase in haploid, pith tissues. A similar pattern of total PAs (free + conjugated was observed for the callus of diploid and haploid plants maintained in the light, and for the haploid callus in the dark, whereas the diploid callus in the dark showed a constant increase in total PAs levels until the end of culture. The PA increase in the diploid callus in the dark was related to free Put levels increase. The ploidy status of the plants could express different PA gradients together with the plant pith and in vitro callus cultures.O objetivo deste trabalho foi determinar os níveis de PAs em tecidos de medula e cultura de calos de plantas haplóides e diplóides de tabaco, obtidas da região apical e basal do caule. Estes explantes foram cultivados em meio RM-64 suplementado com AIA e cinetina, na luz e no escuro, durante vários subcultivos. Nos tecidos medulares, os níveis de PAs apresentam um decréscimo basípeto em diplóides e um aumento em haplóides.Um padrão similar nos níveis de PAs totais (livres+ conjugadas foi observado em calos haplóides e diplóides mantidos na luz, e haplóides no escuro, enquanto os diplóides cultivados no escuro mostraram um aumento constante até o final do cultivo. O aumento no conteúdo de PAs nos calos diplóides no escuro, foi devido ao aumento do conteúdo de Put livre. Foi observado que a ploidia da planta pode expressar diferentes gradientes de PA ao longo do tecido medular e nas culturas de calos in vitro.

  1. Biomimetic heterogenous elastic tissue development.

    Science.gov (United States)

    Tsai, Kai Jen; Dixon, Simon; Hale, Luke Richard; Darbyshire, Arnold; Martin, Daniel; de Mel, Achala

    2017-01-01

    There is an unmet need for artificial tissue to address current limitations with donor organs and problems with donor site morbidity. Despite the success with sophisticated tissue engineering endeavours, which employ cells as building blocks, they are limited to dedicated labs suitable for cell culture, with associated high costs and long tissue maturation times before available for clinical use. Direct 3D printing presents rapid, bespoke, acellular solutions for skull and bone repair or replacement, and can potentially address the need for elastic tissue, which is a major constituent of smooth muscle, cartilage, ligaments and connective tissue that support organs. Thermoplastic polyurethanes are one of the most versatile elastomeric polymers. Their segmented block copolymeric nature, comprising of hard and soft segments allows for an almost limitless potential to control physical properties and mechanical behaviour. Here we show direct 3D printing of biocompatible thermoplastic polyurethanes with Fused Deposition Modelling, with a view to presenting cell independent in-situ tissue substitutes. This method can expeditiously and economically produce heterogenous, biomimetic elastic tissue substitutes with controlled porosity to potentially facilitate vascularisation. The flexibility of this application is shown here with tubular constructs as exemplars. We demonstrate how these 3D printed constructs can be post-processed to incorporate bioactive molecules. This efficacious strategy, when combined with the privileges of digital healthcare, can be used to produce bespoke elastic tissue substitutes in-situ, independent of extensive cell culture and may be developed as a point-of-care therapy approach.

  2. Screenhouse and field persistence of nonpathogenic endophytic Fusarium oxysporum in Musa tissue culture plants.

    Science.gov (United States)

    Paparu, Pamela; Dubois, Thomas; Gold, Clifford S; Niere, Björn; Adipala, Ekwamu; Coyne, Daniel

    2008-04-01

    Two major biotic constraints to highland cooking banana (Musa spp., genome group AAA-EA) production in Uganda are the banana weevil Cosmopolites sordidus and the burrowing nematode Radopholus similis. Endophytic Fusarium oxysporum strains inoculated into tissue culture banana plantlets have shown control of the banana weevil and the nematode. We conducted screenhouse and field experiments to investigate persistence in the roots and rhizome of two endophytic Fusarium oxysporum strains, V2w2 and III4w1, inoculated into tissue-culture banana plantlets of highland cooking banana cultivars Kibuzi and Nabusa. Re-isolation of F. oxysporum showed that endophyte colonization decreased faster from the rhizomes than from the roots of inoculated plants, both in the screenhouse and in the field. Whereas rhizome colonization by F. oxysporum decreased in the screenhouse (4-16 weeks after inoculation), root colonization did not. However, in the field (17-33 weeks after inoculation), a decrease was observed in both rhizome and root colonization. The results show a better persistence in the roots than rhizomes of endophytic F. oxysporum strains V2w2 and III4w1.

  3. Genetic and anatomical analysis of normal and abnormal flowers of date palm cultivar barhy derived from offshoot and tissue culture

    International Nuclear Information System (INIS)

    Shair, O.H.

    2016-01-01

    Random Amplified Polymorphic DNA (RAPD) analysis between 6 normal flower producing offshoot derived and 6 abnormal multiple carpel, flower producing tissue culture (TC) derived trees of cultivar (cv.) Barhy, was performed with the objective to check genetic variation if any at DNA level. DNA samples were extracted from pollinated and un-pollinated flowers from both sets of plants. Amplified RAPD products were clearly detected with 30 primers used in this experiment but only 3 gave a few polymorphic bands which shows low level of genetic variation among the offshoot and TC derived plants. Cluster analysis by the unweighted paired group method of arithmetic means (UPGMA) showed close genomic similarity among the 12 DNA samples with the range of 0.486-0.904 Nei and Li's coefficient in the similarity matrix. The average similarity among the 12 DNA samples was more than 50%. Floral abnormalities in TC derived plants were also studied microscopically. Abnormalities like more than three carpel development, abnormal ovule development and deformities of style and stigma were observed. The results show that the composition and the abnormalities of flowers in TC derived plants of cultivar Barhy may be attributed to epigenetic changes that takes place at different stages of tissue culture and not due to major changes at DNA level. (author)

  4. A method for culturing human hair follicle cells.

    Science.gov (United States)

    Weterings, P J; Vermorken, A J; Bloemendal, H

    1981-01-01

    For the first time a method for culturing human hair follicle cells is described. The bovine eye lens capsule, a basement membrane-like structure, is used as the substrate for the cultures. In a culture medium supplemented with hydrocortisone and insulin about 70% of the original follicles will form growing colonies of diploid keratinocytes.

  5. Comparison of PCR method with the culture method for identification of gonococci from endocervical swabs

    Directory of Open Access Journals (Sweden)

    Alam A

    2002-01-01

    Full Text Available Gonococcal infection remains still a major cause of morbidity among sexually active individuals. Diagnosis of the infection in a female case is more difficult than that in a male. This was a prospective study among 269 female commercial sex workers (CSWs to screen them for gonococcal infection, comparing the rapid method of identification of gonococci by polymerase chain reaction (PCR with the selective culture method. A total of 92 (34.2% CSWs were identified positive for Neisseria gonorrhoeae by combination of the two methods. The PCR method identified 87 of the specimens to harbour cppB gene of N. gonorrhoeae, whereas culture method identified 83 specimens showing colonies of gonococci. Taking into consideration of the total positive cases (92, the PCR method showed a sensitivity of 94.57%, whereas sensitivity of culture method was 90.22%. The selective culture method appears to be the most applicable in the identification of gonococci from clinical specimens, particularly in the less resourceful countries like Bangladesh.

  6. A simple hanging drop cell culture protocol for generation of 3D spheroids.

    Science.gov (United States)

    Foty, Ramsey

    2011-05-06

    Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels or in biomaterial scaffolds. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of

  7. Continuous Nondestructive Monitoring Method Using the Reconstructed Three-Dimensional Conductivity Images via GREIT for Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Sujin Ahn

    2014-01-01

    Full Text Available A continuous Nondestructive monitoring method is required to apply proper feedback controls during tissue regeneration. Conductivity is one of valuable information to assess the physiological function and structural formation of regenerated tissues or cultured cells. However, conductivity imaging methods suffered from inherited ill-posed characteristics in image reconstruction, unknown boundary geometry, uncertainty in electrode position, and systematic artifacts. In order to overcome the limitation of microscopic electrical impedance tomography (micro-EIT, we applied a 3D-specific container with a fixed boundary geometry and electrode configuration to maximize the performance of Graz consensus reconstruction algorithm for EIT (GREIT. The separation of driving and sensing electrodes allows us to simplify the hardware complexity and obtain higher measurement accuracy from a large number of small sensing electrodes. We investigated the applicability of the GREIT to 3D micro-EIT images via numerical simulations and large-scale phantom experiments. We could reconstruct multiple objects regardless of the location. The resolution was 5 mm3 with 30 dB SNR and the position error was less than 2.54 mm. This shows that the new micro-EIT system integrated with GREIT is robust with the intended resolution. With further refinement and scaling down to a microscale container, it may be a continuous nondestructive monitoring tool for tissue engineering applications.

  8. Application of plant cell and tissue culture for the production of phytochemicals in medicinal plants.

    Science.gov (United States)

    Pant, Bijaya

    2014-01-01

    Approximately 80% of the world inhabitants depend on the medicinal plants in the form of traditional formulations for their primary health care system well as in the treatment of a number of diseases since the ancient time. Many commercially used drugs have come from the information of indigenous knowledge of plants and their folk uses. Linking of the indigenous knowledge of medicinal plants to modern research activities provides a new reliable approach, for the discovery of novel drugs much more effectively than with random collection. Increase in population and increasing demand of plant products along with illegal trade are causing depletion of medicinal plants and many are threatened in natural habitat. Plant tissue culture technique has proved potential alternative for the production of desirable bioactive components from plants, to produce the enough amounts of plant material that is needed and for the conservation of threatened species. Different plant tissue culture systems have been extensively studied to improve and enhance the production of plant chemicals in various medicinal plants.

  9. Mouse Pancreas Tissue Slice Culture Facilitates Long-Term Studies of Exocrine and Endocrine Cell Physiology in situ

    OpenAIRE

    Marciniak, Anja; Selck, Claudia; Friedrich, Betty; Speier, Stephan

    2013-01-01

    Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To ove...

  10. IN VITRO INOCULATION OF ASPARAGUS OFFICINALIS TISSUE CULTURE SHOOTS WITH FUSARIUM PROLIFERA TUM

    Directory of Open Access Journals (Sweden)

    A.K.MoHD OMAR

    1999-01-01

    Full Text Available Artificially inoculated asparagus tissue culture plantlets with a virulent fungus, Fusarium proliferatum showed signs of infection as early as 4 days after inoculat ion. Macroscopic observations revealed presence of early symptoms such as necrotic lesions at the affected area and light microscopic examinations clearly revealed the post-penetration events that took place including the destruction of surrounding cells. However, little is known of the hyphal activity or advancement on the host's surface at the initial stage after inoculation. Scanning electron microscopic examination clearly revealed the hyphal advancement on the surface and the mode of entrance into the host tissues beneath. Four days after inoculation, the fungi proceeded to spread out from the inoculation point onto the host surface which eventually developed into a sparse network of both aerial and non-aerial hyphae. Non-aerial hyphae form a network of mycelium that adheres to the surface and it's movement appeared to be oriented towards the stomata. Hyphal penetration occurs more often through the stomata, natural openings or wounds. In some cases, the hyphae crossed over the stomatal opening w ithout entering the host tissues. At places where the cuticle layer is absent or not well developed the hyphae successfully grew in between the epidermal cells into the tissues beneath.

  11. Equine ovarian tissue viability after cryopreservation and in vitro culture

    Science.gov (United States)

    The efficiency of several cryoprotective agents were compared using both slow-freezing and vitrification methods. Results indicate that the viability of ovarian tissue cells increases when DMSO (slow-freezing) and ethylene glycol (vitrification) are used....

  12. Comparison of Standard Culture-Based Method to Culture-Independent Method for Evaluation of Hygiene Effects on the Hand Microbiome

    Directory of Open Access Journals (Sweden)

    C. Zapka

    2017-03-01

    Full Text Available Hands play a critical role in the transmission of microbiota on one’s own body, between individuals, and on environmental surfaces. Effectively measuring the composition of the hand microbiome is important to hand hygiene science, which has implications for human health. Hand hygiene products are evaluated using standard culture-based methods, but standard test methods for culture-independent microbiome characterization are lacking. We sampled the hands of 50 participants using swab-based and glove-based methods prior to and following four hand hygiene treatments (using a nonantimicrobial hand wash, alcohol-based hand sanitizer [ABHS], a 70% ethanol solution, or tap water. We compared results among culture plate counts, 16S rRNA gene sequencing of DNA extracted directly from hands, and sequencing of DNA extracted from culture plates. Glove-based sampling yielded higher numbers of unique operational taxonomic units (OTUs but had less diversity in bacterial community composition than swab-based sampling. We detected treatment-induced changes in diversity only by using swab-based samples (P < 0.001; we were unable to detect changes with glove-based samples. Bacterial cell counts significantly decreased with use of the ABHS (P < 0.05 and ethanol control (P < 0.05. Skin hydration at baseline correlated with bacterial abundances, bacterial community composition, pH, and redness across subjects. The importance of the method choice was substantial. These findings are important to ensure improvement of hand hygiene industry methods and for future hand microbiome studies. On the basis of our results and previously published studies, we propose recommendations for best practices in hand microbiome research.

  13. Rose (Rosa hybrida L.) tissue culture mutagenesis for new mutants generation

    International Nuclear Information System (INIS)

    Salahbiah Abdul Majid; Rusli Ibrahim

    2004-01-01

    Tissue culture technique can be used to obtain complete regeneration of plant cells from shoots, rots, flowers, axillary buds and other parts of the plant. In this study, axillary buds from stem cuttings of Cutting Red, Christine Dior and Mini Rose varieties were used as the stating explants. Murashige and Skoog (1962) media supplemented with 6-Benzylaminopurine (BAP, at 4.44 - 8.88μM/l), Napthaleneacetic acid (NAA at 0.54μM/l),, nad 3% sucrose were used for plantlet initiation and regeneration. Cultured axillary buds were exposed to gamma ray (0.250 Gy/s) at 0, 15, 25, 35, 45, 55, 65 and 75 Gy for radiosensitivity test. From the dose respond curve, LD 5 0 the value for cutting red variety was 25 Gy, Christion Dior 30 Gy and Mini Rose 38 Gy, yet 22% of Mini Rose samples survived at 65 Gy and another 10% at 70 Gy. Screening of M3 plants of irradiated cultured shoots, 2 colour variations were obtained at 40 Gy for Cutting Red variety, while 3 colour variations for Mini Rose at 20 Gy. When 6 varieties of Fragrance Rose were irradiated at 40 Gy, 1 colour variation was obtained from 99 screened plants. This study suggests that the dose range of 20 to 45 can be considered for rose mutagenesis study to produce mutants. (Author)

  14. Cytokeratin expression of engrafted three-dimensional culture tissues using epithelial cells derived from porcine periodontal ligaments.

    Science.gov (United States)

    Yamada, Rie; Kitajima, Kayoko; Arai, Kyoko; Igarashi, Masaru

    2014-09-01

    This study investigated the differentiation and proliferation of epithelial cells derived from periodontal ligaments after three-dimensional culture using collagen gel with fibroblasts in vitro and in vivo. Epithelial cells and fibroblasts were derived from porcine periodontal ligaments. Epithelial cells were labeled using a fluorescent red membrane marker (PKH-26GL) and were seeded onto collagen gel with fibroblasts, followed by incubation in an air-liquid interface for 7 days. Three-dimensional cultures were grafted onto the backs of nude mice and removed at 1, 7, and 14 days after surgery (in vivo model). Unfixed sections (5 μm) were used to detect the presence of red fluorescent cells. Paraffin sections were analyzed histologically and immunohistochemically. Specimens were compared with three-dimensional culture tissues at 8, 14 and 21 days (in vitro model). Grafted three-dimensional cultures formed a stratified epithelial structure similar to skin in vivo. Epithelial cells were sequenced in basal-layer-like structures at 14 days in vivo. Immunohistochemical findings showed that the expression of cytokeratin was detected in the epithelial layer in in vitro and in vivo models. Ck8 + 18 + 19 was expressed in the upper epithelial layer in the in vitro model at 14 and 21 days, but not in vivo. Involucrin was expressed in the certified layers in vitro at 14 days, but not in vivo. Laminin was detected at the dermo-epidermal junction in vivo at 7 and 14 days, but not in vitro. These results suggest that differentiation of three-dimensional culture tissues differs in vivo and in vitro. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Integrating cultural control methods for tomato late blight (Phytophthora infestans) in Uganda

    NARCIS (Netherlands)

    Tumwine, J.; Frinking, H.D.; Jeger, M.J.

    2002-01-01

    Cultural control measures against tomato late blight (Phytophthora infestans) were evaluated in six field experiments over 3 years in Uganda. Each experiment included sanitation (removal of diseased plant tissues), fungicide (mancozeb) application, and an untreated control, as standard treatments.

  16. Design of biomimetic cellular scaffolds for co-culture system and their application

    Science.gov (United States)

    Kook, Yun-Min; Jeong, Yoon; Lee, Kangwon; Koh, Won-Gun

    2017-01-01

    The extracellular matrix of most natural tissues comprises various types of cells, including fibroblasts, stem cells, and endothelial cells, which communicate with each other directly or indirectly to regulate matrix production and cell functionality. To engineer multicellular interactions in vitro, co-culture systems have achieved tremendous success achieving a more realistic microenvironment of in vivo metabolism than monoculture system in the past several decades. Recently, the fields of tissue engineering and regenerative medicine have primarily focused on three-dimensional co-culture systems using cellular scaffolds, because of their physical and biological relevance to the extracellular matrix of actual tissues. This review discusses several materials and methods to create co-culture systems, including hydrogels, electrospun fibers, microfluidic devices, and patterning for biomimetic co-culture system and their applications for specific tissue regeneration. Consequently, we believe that culture systems with appropriate physical and biochemical properties should be developed, and direct or indirect cell–cell interactions in the remodeled tissue must be considered to obtain an optimal tissue-specific microenvironment. PMID:29081966

  17. Physical non-viral gene delivery methods for tissue engineering.

    Science.gov (United States)

    Mellott, Adam J; Forrest, M Laird; Detamore, Michael S

    2013-03-01

    The integration of gene therapy into tissue engineering to control differentiation and direct tissue formation is not a new concept; however, successful delivery of nucleic acids into primary cells, progenitor cells, and stem cells has proven exceptionally challenging. Viral vectors are generally highly effective at delivering nucleic acids to a variety of cell populations, both dividing and non-dividing, yet these viral vectors are marred by significant safety concerns. Non-viral vectors are preferred for gene therapy, despite lower transfection efficiencies, and possess many customizable attributes that are desirable for tissue engineering applications. However, there is no single non-viral gene delivery strategy that "fits-all" cell types and tissues. Thus, there is a compelling opportunity to examine different non-viral vectors, especially physical vectors, and compare their relative degrees of success. This review examines the advantages and disadvantages of physical non-viral methods (i.e., microinjection, ballistic gene delivery, electroporation, sonoporation, laser irradiation, magnetofection, and electric field-induced molecular vibration), with particular attention given to electroporation because of its versatility, with further special emphasis on Nucleofection™. In addition, attributes of cellular character that can be used to improve differentiation strategies are examined for tissue engineering applications. Ultimately, electroporation exhibits a high transfection efficiency in many cell types, which is highly desirable for tissue engineering applications, but electroporation and other physical non-viral gene delivery methods are still limited by poor cell viability. Overcoming the challenge of poor cell viability in highly efficient physical non-viral techniques is the key to using gene delivery to enhance tissue engineering applications.

  18. Physical non-viral gene delivery methods for tissue engineering

    Science.gov (United States)

    Mellott, Adam J.; Forrest, M. Laird; Detamore, Michael S.

    2016-01-01

    The integration of gene therapy into tissue engineering to control differentiation and direct tissue formation is not a new concept; however, successful delivery of nucleic acids into primary cells, progenitor cells, and stem cells has proven exceptionally challenging. Viral vectors are generally highly effective at delivering nucleic acids to a variety of cell populations, both dividing and non-dividing, yet these viral vectors are marred by significant safety concerns. Non-viral vectors are preferred for gene therapy, despite lower transfection efficiencies, and possess many customizable attributes that are desirable for tissue engineering applications. However, there is no single non-viral gene delivery strategy that “fits-all” cell types and tissues. Thus, there is a compelling opportunity to examine different non-viral vectors, especially physical vectors, and compare their relative degrees of success. This review examines the advantages and disadvantages of physical non-viral methods (i.e., microinjection, ballistic gene delivery, electroporation, sonoporation, laser irradiation, magnetofection, and electric field-induced molecular vibration), with particular attention given to electroporation because of its versatility, with further special emphasis on Nucleofection™. In addition, attributes of cellular character that can be used to improve differentiation strategies are examined for tissue engineering applications. Ultimately, electroporation exhibits a high transfection efficiency in many cell types, which is highly desirable for tissue engineering applications, but electroporation and other physical non-viral gene delivery methods are still limited by poor cell viability. Overcoming the challenge of poor cell viability in highly efficient physical non-viral techniques is the key to using gene delivery to enhance tissue engineering applications. PMID:23099792

  19. Evaluation of viability and proliferative activity of human urothelial cells cultured onto xenogenic tissue-engineered extracellular matrices.

    LENUS (Irish Health Repository)

    Davis, Niall F

    2011-04-01

    To evaluate the viability and proliferative activity of human urothelial cells (HUCs) cultured on tissue-engineered extracellular matrix scaffolds and to assess the potential of extracellular matrixes to support the growth of HUCs in their expected in vivo urine environment.

  20. Tooth Tissue Engineering: The Importance of Blood Products as a Supplement in Tissue Culture Medium for Human Pulp Dental Stem Cells.

    Science.gov (United States)

    Pisciolaro, Ricardo Luiz; Duailibi, Monica Talarico; Novo, Neil Ferreira; Juliano, Yara; Pallos, Debora; Yelick, Pamela Crotty; Vacanti, Joseph Phillip; Ferreira, Lydia Masako; Duailibi, Silvio Eduardo

    2015-11-01

    One of the goals in using cells for tissue engineering (TE) and cell therapy consists of optimizing the medium for cell culture. The present study compares three different blood product supplements for improved cell proliferation and protection against DNA damage in cultured human dental pulp stem cells for tooth TE applications. Human cells from dental pulp were first characterized as adult stem cells (ectomesenchymal mixed origin) by flow cytometry. Next, four different cell culture conditions were tested: I, supplement-free; II, supplemented with fetal bovine serum; III, allogeneic human serum; and IV, autologous human serum. Cultured cells were then characterized for cell proliferation, mineralized nodule formation, and colony-forming units (CFU) capability. After 28 days in culture, the comet assay was performed to assess possible damage in cellular DNA. Our results revealed that Protocol IV achieved higher cell proliferation than Protocol I (p = 0.0112). Protocols II and III resulted in higher cell proliferation than Protocol I, but no statistical differences were found relative to Protocol IV. The comet assay revealed less cell damage in cells cultured using Protocol IV as compared to Protocols II and III. The damage percentage observed on Protocol II was significantly higher than all other protocols. CFUs capability was highest using Protocol IV (p = 0.0018) and III, respectively, and the highest degree of mineralization was observed using Protocol IV as compared to Protocols II and III. Protocol IV resulted in significantly improved cell proliferation, and no cell damage was observed. These results demonstrate that human blood product supplements can be used as feasible supplements for culturing adult human dental stem cells.

  1. Cloning higher plants from aseptically cultured tissues and cells

    Science.gov (United States)

    Krikorian, A. D.

    1982-01-01

    A review of aseptic culture methods for higher plants is presented, which focuses on the existing problems that limit or prevent the full realization of cloning plants from free cells. It is shown that substantial progress in clonal multiplication has been made with explanted stem tips or lateral buds which can be stimulated to produce numerous precocious axillary branches. These branches can then be separated or subdivided and induced to root in order to yield populations of genetically and phenotypically uniorm plantlets. Similarly, undifferentiated calluses can sometimes be induced to form shoots and/or roots adventitiously. Although the cell culture techniques required to produce somatic embryos are presently rudimentary, steady advances are being made in learning how to stimulate formation of somatic or adventive embryos from totipotent cells grown in suspension cultures. It is concluded that many problems exist in the producing and growing of totipotent or morphogenetically competent cell suspensions, but the potential benefits are great.

  2. Changes in adipose tissue stromal-vascular cells in primary culture due to porcine sera

    International Nuclear Information System (INIS)

    Jewell, D.E.; Hausman, G.J.

    1986-01-01

    This study was conducted to determine the response of rat stromal-vascular cells to pig sea. Sera were collected from unselected contemporary (lean) and high backfat thickness selected (obese) pigs. Sera from obese pigs were collected either by exsanguination or cannulation. sera from lean pigs during the growing phase (45 kg) and the fattening phase (100-110 kg) were collected. Stromal-vascular cells derived rom rat inguinal tissue were cultured on either 25 cm 2 flasks, collagen-coated coverslips or petri dishes. Cell proliferation was measured by [ 3 H]-thymidine incorporation during the fourth day of culture. Coverslip cultures were used for histochemical analysis. Petri dish cultures were used for analysis of Sn-glycerol-3-phosphate dehydrogenase (GPDH) activity. All cells were plated for 24 hours in media containing 10 fetal bovine sera. Test media contained 2.5, 5.0, 10.0% sera. Sera from obese pigs increased GPDH activity and fat cell production when compared to the lean controls. The increased concentration of sera increased esterase activity and lipid as measured with oil red O. The sera from obese pigs collected at slaughter stimulated more fat cell production than obese sera collected by cannulation. These studies show there are adipogenic factors in obese pigs sera which promote fat cell development in primary cell culture

  3. Fabrication of bone marrow-like tissue in vitro from dispersed-state bone marrow cells

    Directory of Open Access Journals (Sweden)

    Kanae Sayo

    2016-03-01

    Full Text Available A three-dimensional (3D bone marrow (BM culture system may facilitate research into the molecular mechanisms involved in hematopoiesis and BM diseases. However, because >90% of BM cells are composed of non-adherent blood cells, it is difficult to organize the dispersed BM cells into 3D multicellular spheroids using conventional aggregation methods such as hanging drop, and rotary shaking culture. The objective of this study was to reproduce BM-like tissue. We reported successful formation of BM aggregates using a 3% methylcellulose (MC medium. This medium could aggregate even non-adherent materials. In MC medium, BM cells formed tissue-like aggregates within 24 h. Although the cell density of the BM-like tissue is slightly low, sections of the organoids resembled those of intact BM tissue. Cells of the BM-like tissue were approximately 70% viable after 7 days in culture. Staining for CD68, PDGFRα, and CXCL12 indicated that the BM-like tissue contained macrophages, and mesenchymal cells including CXCL12-abundant reticular cells. These results indicated that the method using MC medium effectively reconstitutes the BM-like tissue.

  4. Characterization of the Embryogenic Tissue of the Norway Spruce Including a Transition Layer between the Tissue and the Culture Medium by Magnetic Resonance Imaging

    Directory of Open Access Journals (Sweden)

    Kořínek R.

    2017-02-01

    Full Text Available The paper describes the visualization of the cells (ESEs and mucilage (ECMSN in an embryogenic tissue via magnetic resonance imaging (MRI relaxometry measurement combined with the subsequent multi-parametric segmentation. The computed relaxometry maps T1 and T2 show a thin layer (transition layer between the culture medium and the embryogenic tissue. The ESEs, mucilage, and transition layer differ in their relaxation times T1 and T2; thus, these times can be used to characterize the individual parts within the embryogenic tissue. The observed mean values of the relaxation times T1 and T2 of the ESEs, mucilage, and transition layer are as follows: 1469 ± 324 and 53 ± 10 ms, 1784 ± 124 and 74 ± 8 ms, 929 ± 164 and 32 ± 4.7 ms, respectively. The multi-parametric segmentation exploiting the T1 and T2 relaxation times as a classifier shows the distribution of the ESEs and mucilage within the embryogenic tissue. The discussed T1 and T2 indicators can be utilized to characterize both the growth-related changes in an embryogenic tissue and the effect of biotic/abiotic stresses, thus potentially becoming a distinctive indicator of the state of any examined embryogenic tissue.

  5. Reconstruction of Hyaline Cartilage Deep Layer Properties in 3-Dimensional Cultures of Human Articular Chondrocytes.

    Science.gov (United States)

    Nanduri, Vibudha; Tattikota, Surendra Mohan; T, Avinash Raj; Sriramagiri, Vijaya Rama Rao; Kantipudi, Suma; Pande, Gopal

    2014-06-01

    Articular cartilage (AC) injuries and malformations are commonly noticed because of trauma or age-related degeneration. Many methods have been adopted for replacing or repairing the damaged tissue. Currently available AC repair methods, in several cases, fail to yield good-quality long-lasting results, perhaps because the reconstructed tissue lacks the cellular and matrix properties seen in hyaline cartilage (HC). To reconstruct HC tissue from 2-dimensional (2D) and 3-dimensional (3D) cultures of AC-derived human chondrocytes that would specifically exhibit the cellular and biochemical properties of the deep layer of HC. Descriptive laboratory study. Two-dimensional cultures of human AC-derived chondrocytes were established in classical medium (CM) and newly defined medium (NDM) and maintained for a period of 6 weeks. These cells were suspended in 2 mm-thick collagen I gels, placed in 24-well culture inserts, and further cultured up to 30 days. Properties of chondrocytes, grown in 2D cultures and the reconstructed 3D cartilage tissue, were studied by optical and scanning electron microscopic techniques, immunohistochemistry, and cartilage-specific gene expression profiling by reverse transcription polymerase chain reaction and were compared with those of the deep layer of native human AC. Two-dimensional chondrocyte cultures grown in NDM, in comparison with those grown in CM, showed more chondrocyte-specific gene activity and matrix properties. The NDM-grown chondrocytes in 3D cultures also showed better reproduction of deep layer properties of HC, as confirmed by microscopic and gene expression analysis. The method used in this study can yield cartilage tissue up to approximately 1.6 cm in diameter and 2 mm in thickness that satisfies the very low cell density and matrix composition properties present in the deep layer of normal HC. This study presents a novel and reproducible method for long-term culture of AC-derived chondrocytes and reconstruction of cartilage

  6. Methods to Quantify Nickel in Soils and Plant Tissues

    Directory of Open Access Journals (Sweden)

    Bruna Wurr Rodak

    2015-06-01

    Full Text Available In comparison with other micronutrients, the levels of nickel (Ni available in soils and plant tissues are very low, making quantification very difficult. The objective of this paper is to present optimized determination methods of Ni availability in soils by extractants and total content in plant tissues for routine commercial laboratory analyses. Samples of natural and agricultural soils were processed and analyzed by Mehlich-1 extraction and by DTPA. To quantify Ni in the plant tissues, samples were digested with nitric acid in a closed system in a microwave oven. The measurement was performed by inductively coupled plasma/optical emission spectrometry (ICP-OES. There was a positive and significant correlation between the levels of available Ni in the soils subjected to Mehlich-1 and DTPA extraction, while for plant tissue samples the Ni levels recovered were high and similar to the reference materials. The availability of Ni in some of the natural soil and plant tissue samples were lower than the limits of quantification. Concentrations of this micronutrient were higher in the soil samples in which Ni had been applied. Nickel concentration differed in the plant parts analyzed, with highest levels in the grains of soybean. The grain, in comparison with the shoot and leaf concentrations, were better correlated with the soil available levels for both extractants. The methods described in this article were efficient in quantifying Ni and can be used for routine laboratory analysis of soils and plant tissues.

  7. Simple and convenient method for culturing anaerobic bacteria.

    OpenAIRE

    Behbehani, M J; Jordan, H V; Santoro, D L

    1982-01-01

    A simple and convenient method for culturing anaerobic bacteria is described. Cultures can be grown in commercially available flasks normally used for preparation of sterile external solutions. A special disposable rubber flask closure maintains anaerobic conditions in the flask after autoclaving. Growth of a variety of anaerobic oral bacteria was comparable to that obtained after anaerobic incubation of broth cultures in Brewer Anaerobic Jars.

  8. Prolonged hypoxic culture and trypsinization increase the pro-angiogenic potential of human adipose tissue-derived stem cells

    DEFF Research Database (Denmark)

    Rasmussen, Jeppe Grøndahl; Frøbert, Ole; Pilgaard, Linda

    2011-01-01

    Transplantation of mesenchymal stromal cells (MSC), including adipose tissue-derived stem cells (ASC), is a promising option in the treatment of vascular disease. Short-term hypoxic culture of MSC augments secretion of anti-apoptotic and angiogenic cytokines. We hypothesized that prolonged hypoxi...

  9. Porous PEOT/PBT scaffolds for bone tissue engineering: preparation, characterization, and in vitro bone marrow cell culturing

    NARCIS (Netherlands)

    Claase, M.B.; Grijpma, Dirk W.; Mendes, S.C.; Mendes, Sandra C.; de Bruijn, Joost Dick; Feijen, Jan

    2003-01-01

    The preparation, characterization, and in vitro bone marrow cell culturing on porous PEOT/PBT copolymer scaffolds are described. These scaffolds are meant for use in bone tissue engineering. Previous research has shown that PEOT/PBT copolymers showed in vivo degradation, calcification, and bone

  10. Method for organotypic tissue culture in the aged animal

    Directory of Open Access Journals (Sweden)

    Jared Schommer

    2017-01-01

    • Our method permits slices from mice as old as 16 months and rabbits as old as years of age to survive ex vivo up to 8 weeks [6–9]. Such a slice system may be relevant to investigating age-related brain diseases.

  11. Structural Analysis of Three-dimensional Human Neural Tissue derived from Induced Pluripotent Stem Cells

    DEFF Research Database (Denmark)

    Terrence Brooks, Patrick; Rasmussen, Mikkel Aabech; Hyttel, Poul

    2016-01-01

    Objective: The present study aimed at establishing a method for production of a three-dimensional (3D) human neural tissue derived from induced pluripotent stem cells (iPSCs) and analyzing the outcome by a combination of tissue ultrastructure and expression of neural markers. Methods: A two......-step cell culture procedure was implemented by subjecting human iPSCs to a 3D scaffoldbased neural differentiation protocol. First, neural fate-inducing small molecules were used to create a neuroepithelial monolayer. Second, the monolayer was trypsinized into single cells and seeded into a porous...... polystyrene scaffold and further cultured to produce a 3D neural tissue. The neural tissue was characterized by a combination of immunohistochemistry and transmission electron microscopy (TEM). Results: iPSCs developed into a 3D neural tissue expressing markers for neural progenitor cells, early neural...

  12. Evaluation of PCR methods for detection of Brucella strains from culture and tissues.

    Science.gov (United States)

    Çiftci, Alper; İça, Tuba; Savaşan, Serap; Sareyyüpoğlu, Barış; Akan, Mehmet; Diker, Kadir Serdar

    2017-04-01

    The genus Brucella causes significant economic losses due to infertility, abortion, stillbirth or weak calves, and neonatal mortality in livestock. Brucellosis is still a zoonosis of public health importance worldwide. The study was aimed to optimize and evaluate PCR assays used for the diagnosis of Brucella infections. For this aim, several primers and PCR protocols were performed and compared with Brucella cultures and biological material inoculated with Brucella. In PCR assays, genus- or species-specific oligonucleotide primers derived from 16S rRNA sequences (F4/R2, Ba148/928, IS711, BruP6-P7) and OMPs (JPF/JPR, 31ter/sd) of Brucella were used. All primers except for BruP6-P7 detected the DNA from reference Brucella strains and field isolates. In spiked blood, milk, and semen samples, F4-R2 primer-oriented PCR assays detected minimal numbers of Brucella. In spiked serum and fetal stomach content, Ba148/928 primer-oriented PCR assays detected minimal numbers of Brucella. Field samples collected from sheep and cattle were examined by bacteriological methods and optimized PCR assays. Overall, sensitivity of PCR assays was found superior to conventional bacteriological isolation. Brucella DNA was detected in 35.1, 1.1, 24.8, 5.0, and 8.0% of aborted fetus, blood, milk, semen, and serum samples by PCR assays, respectively. In conclusion, PCR assay in optimized conditions was found to be valuable in sensitive and specific detection of Brucella infections of animals.

  13. Chemical And Physiological Studies On Drought Stress Tolerance Of Irradiated Communis Pear Using Tissue Culture

    International Nuclear Information System (INIS)

    Zaied, N.S.; Ragab, E.A.

    2007-01-01

    The rooted in vitro irradiated pear rootstocks (Pyrus communis) were subjected to drought stress by using different concentrations of mannitol (20, 40, 60, 80 and 100 gm/l), polyethylene glycol (PEG) at concentrations 2, 4, 6, 8 and 10 % to culture medium and also agar at concentrations 6, 8, 10, 12 and 14 gm/l to study their effects on tissue culture and chemical analysis and their tolerance to drought stress. The obtained results showed that the number of shoots, shoot length and number of leaves were higher at 20 and 40 gm/l mannitol. Increasing mannitol concentration enhanced the increase of chlorophyll b, reducing sugars, total indoles and total phenols up to the highest level at 100 gm/l. Adding PEG at concentration 2% to the culture medium encouraged significant increases in the number of shoots and number of leaves and increase chlorophyll a, and non-reducing sugars as well as significant decrease in number of shoots, shoots length, number of leaves, root length and number of roots with increasing agar concentrations to the culture medium. However, decreasing agar concentration in the culture medium induced increase in chlorophyll A and non-reducing sugar

  14. Increased adsorption of histidine-tagged proteins onto tissue culture polystyrene

    DEFF Research Database (Denmark)

    Holmberg, Maria; Hansen, Thomas Steen; Lind, Johan Ulrik

    2012-01-01

    and ethylenediaminetetraacetic acid (EDTA), as well as adsorption performed at different pH and ionic strength indicates that the high adsorption is caused by electrostatic interaction between negatively charged carboxylate groups on the TCPS surface and positively charged histidine residues in the proteins. Pre......In this study we compare histidine-tagged and native proteins with regards to adsorption properties. We observe significantly increased adsorption of proteins with an incorporated polyhistidine amino acid motif (HIS-tag) onto tissue culture polystyrene (TCPS) compared to similar proteins without...... a HIS-tag. The effect is not observed on polystyrene (PS). Adsorption experiments have been performed at physiological pH (7.4) and the effect was only observed for the investigated proteins that have pI values below or around 7.4. Competitive adsorption experiments with imidazole...

  15. Generation of stomach tissue from mouse embryonic stem cells.

    Science.gov (United States)

    Noguchi, Taka-aki K; Ninomiya, Naoto; Sekine, Mari; Komazaki, Shinji; Wang, Pi-Chao; Asashima, Makoto; Kurisaki, Akira

    2015-08-01

    Successful pluripotent stem cell differentiation methods have been developed for several endoderm-derived cells, including hepatocytes, β-cells and intestinal cells. However, stomach lineage commitment from pluripotent stem cells has remained a challenge, and only antrum specification has been demonstrated. We established a method for stomach differentiation from embryonic stem cells by inducing mesenchymal Barx1, an essential gene for in vivo stomach specification from gut endoderm. Barx1-inducing culture conditions generated stomach primordium-like spheroids, which differentiated into mature stomach tissue cells in both the corpus and antrum by three-dimensional culture. This embryonic stem cell-derived stomach tissue (e-ST) shared a similar gene expression profile with adult stomach, and secreted pepsinogen as well as gastric acid. Furthermore, TGFA overexpression in e-ST caused hypertrophic mucus and gastric anacidity, which mimicked Ménétrier disease in vitro. Thus, in vitro stomach tissue derived from pluripotent stem cells mimics in vivo development and can be used for stomach disease models.

  16. Methods to induce primary and secondary traumatic damage in organotypic hippocampal slice cultures.

    Science.gov (United States)

    Adamchik, Y; Frantseva, M V; Weisspapir, M; Carlen, P L; Perez Velazquez, J L

    2000-04-01

    Organotypic brain slice cultures have been used in a variety of studies on neurodegenerative processes [K.M. Abdel-Hamid, M. Tymianski, Mechanisms and effects of intracellular calcium buffering on neuronal survival in organotypic hippocampal cultures exposed to anoxia/aglycemia or to excitotoxins, J. Neurosci. 17, 1997, pp. 3538-3553; D.W. Newell, A. Barth, V. Papermaster, A.T. Malouf, Glutamate and non-glutamate receptor mediated toxicity caused by oxygen and glucose deprivation in organotypic hippocampal cultures, J. Neurosci. 15, 1995, pp. 7702-7711; J.L. Perez Velazquez, M.V. Frantseva, P.L. Carlen, In vitro ischemia promotes glutamate mediated free radical generation and intracellular calcium accumulation in pyramidal neurons of cultured hippocampal slices, J. Neurosci. 23, 1997, pp. 9085-9094; L. Stoppini, L.A. Buchs, D. Muller, A simple method for organotypic cultures of nervous tissue, J. Neurosci. Methods 37, 1991, pp. 173-182; R.C. Tasker, J.T. Coyle, J.J. Vornov, The regional vulnerability to hypoglycemia induced neurotoxicity in organotypic hippocampal culture: protection by early tetrodotoxin or delayed MK 801, J. Neurosci. 12, 1992, pp. 4298-4308.]. We describe two methods to induce traumatic cell damage in hippocampal organotypic cultures. Primary trauma injury was achieved by rolling a stainless steel cylinder (0.9 g) on the organotypic slices. Secondary injury was followed after dropping a weight (0.137 g) on a localised area of the organotypic slice, from a height of 2 mm. The time course and extent of cell death were determined by measuring the fluorescence of the viability indicator propidium iodide (PI) at several time points after the injury. The initial localised impact damage spread 24 and 67 h after injury, cell death being 25% and 54%, respectively, when slices were kept at 37 degrees C. To validate these methods as models to assess neuroprotective strategies, similar insults were applied to slices at relatively low temperatures (30

  17. Development of a New Safety Culture Assessment Method for Nuclear Power Plants (NPPs) (A study to suggest a new safety culture assessment method in nuclear power plants)

    International Nuclear Information System (INIS)

    Han, Sang Min; Seong, Poong Hyun

    2014-01-01

    This study is conducted to suggest a new safety culture assessment method in nuclear power plants. Criteria with various existing safety culture analysis methods are united, and reliability analysis methods are applied. The concept of the most representative methods, Fault Tree Analysis (FTA) and Failure Mode and Effect Analysis (FMEA), are adopted to assess safety culture. Through this application, it is expected that the suggested method will bring results with convenience and objectiveness

  18. Development of a New Safety Culture Assessment Method for Nuclear Power Plants (NPPs) (A study to suggest a new safety culture assessment method in nuclear power plants)

    Energy Technology Data Exchange (ETDEWEB)

    Han, Sang Min; Seong, Poong Hyun [KAIST, Daejeon (Korea, Republic of)

    2014-08-15

    This study is conducted to suggest a new safety culture assessment method in nuclear power plants. Criteria with various existing safety culture analysis methods are united, and reliability analysis methods are applied. The concept of the most representative methods, Fault Tree Analysis (FTA) and Failure Mode and Effect Analysis (FMEA), are adopted to assess safety culture. Through this application, it is expected that the suggested method will bring results with convenience and objectiveness.

  19. Implementation of a new rapid tissue processing method--advantages and challenges

    DEFF Research Database (Denmark)

    Munkholm, Julie; Talman, Maj-Lis; Hasselager, Thomas

    2008-01-01

    Conventional tissue processing of histologic specimens has been carried out in the same manner for many years. It is a time-consuming process involving batch production, resulting in a 1-day delay of the diagnosis. Microwave-assisted tissue processing enables a continuous high flow of histologic...... specimens through the processor with a processing time of as low as 1h. In this article, we present the effects of the automated microwave-assisted tissue processor on the histomorphologic quality and the turnaround time (TAT) for histopathology reports. We present a blind comparative study regarding...... the histomorphologic quality of microwave-processed and conventionally processed tissue samples. A total of 333 specimens were included. The microwave-assisted processing method showed a histomorphologic quality comparable to the conventional method for a number of tissue types, including skin and specimens from...

  20. Sensing in tissue bioreactors

    Science.gov (United States)

    Rolfe, P.

    2006-03-01

    Specialized sensing and measurement instruments are under development to aid the controlled culture of cells in bioreactors for the fabrication of biological tissues. Precisely defined physical and chemical conditions are needed for the correct culture of the many cell-tissue types now being studied, including chondrocytes (cartilage), vascular endothelial cells and smooth muscle cells (blood vessels), fibroblasts, hepatocytes (liver) and receptor neurones. Cell and tissue culture processes are dynamic and therefore, optimal control requires monitoring of the key process variables. Chemical and physical sensing is approached in this paper with the aim of enabling automatic optimal control, based on classical cell growth models, to be achieved. Non-invasive sensing is performed via the bioreactor wall, invasive sensing with probes placed inside the cell culture chamber and indirect monitoring using analysis within a shunt or a sampling chamber. Electroanalytical and photonics-based systems are described. Chemical sensing for gases, ions, metabolites, certain hormones and proteins, is under development. Spectroscopic analysis of the culture medium is used for measurement of glucose and for proteins that are markers of cell biosynthetic behaviour. Optical interrogation of cells and tissues is also investigated for structural analysis based on scatter.

  1. Comparison of Biocompatibility and Adsorption Properties of Different Plastics for Advanced Microfluidic Cell and Tissue Culture Models

    NARCIS (Netherlands)

    van Midwoud, Paul M.; Janse, Arnout; Merema, M.T.; Groothuis, Geny M. M.; Verpoorte, Elisabeth

    2012-01-01

    Microfluidic technology is providing new routes toward advanced cell and tissue culture models to better understand human biology and disease. Many advanced devices have been made from poly(dimethylsiloxane) (PDMS) to enable experiments, for example, to study drug metabolism by use of precision cut

  2. Comparison of tissue processing methods for microvascular visualization in axolotls.

    Science.gov (United States)

    Montoro, Rodrigo; Dickie, Renee

    2017-01-01

    The vascular system, the pipeline for oxygen and nutrient delivery to tissues, is essential for vertebrate development, growth, injury repair, and regeneration. With their capacity to regenerate entire appendages throughout their lifespan, axolotls are an unparalleled model for vertebrate regeneration, but they lack many of the molecular tools that facilitate vascular imaging in other animal models. The determination of vascular metrics requires high quality image data for the discrimination of vessels from background tissue. Quantification of the vasculature using perfused, cleared specimens is well-established in mammalian systems, but has not been widely employed in amphibians. The objective of this study was to optimize tissue preparation methods for the visualization of the microvascular network in axolotls, providing a basis for the quantification of regenerative angiogenesis. To accomplish this aim, we performed intracardiac perfusion of pigment-based contrast agents and evaluated aqueous and non-aqueous clearing techniques. The methods were verified by comparing the quality of the vascular images and the observable vascular density across treatment groups. Simple and inexpensive, these tissue processing techniques will be of use in studies assessing vascular growth and remodeling within the context of regeneration. Advantages of this method include: •Higher contrast of the vasculature within the 3D context of the surrounding tissue •Enhanced detection of microvasculature facilitating vascular quantification •Compatibility with other labeling techniques.

  3. In vitro culture method of powdery mildew (Oidium heveae ...

    African Journals Online (AJOL)

    ELOHO

    2012-08-23

    Aug 23, 2012 ... A method for culturing powdery mildew (Oidium heveae) from isolated leaves of ... solution; d, Colour phase leaves with nutrient solution in culture dish; e, in vitro ... Plant and fungus materials .... same change trend during whole culture period. ... consistent with the field resistance identification results of.

  4. Multizone Paper Platform for 3D Cell Cultures

    Science.gov (United States)

    Derda, Ratmir; Hong, Estrella; Mwangi, Martin; Mammoto, Akiko; Ingber, Donald E.; Whitesides, George M.

    2011-01-01

    In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other environmental factors (temperature, mechanical stress, etc). The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously (“cells-in-gels-in-paper” or CiGiP), this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM) gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers) containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D culture, by peeling apart the sheets of paper, “sections” all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures. PMID:21573103

  5. Evaluation of the effects of different culture media on the myogenic differentiation potential of adipose tissue- or bone marrow-derived human mesenchymal stem cells.

    Science.gov (United States)

    Stern-Straeter, Jens; Bonaterra, Gabriel Alejandro; Juritz, Stephanie; Birk, Richard; Goessler, Ulrich Reinhart; Bieback, Karen; Bugert, Peter; Schultz, Johannes; Hörmann, Karl; Kinscherf, Ralf; Faber, Anne

    2014-01-01

    The creation of functional muscles/muscle tissue from human stem cells is a major goal of skeletal muscle tissue engineering. Mesenchymal stem cells (MSCs) from fat/adipose tissue (AT-MSCs), as well as bone marrow (BM-MSCs) have been shown to bear myogenic potential, which makes them candidate stem cells for skeletal muscle tissue engineering applications. The aim of this study was to analyse the myogenic differentiation potential of human AT-MSCs and BM-MSCs cultured in six different cell culture media containing different mixtures of growth factors. The following cell culture media were used in our experiments: mesenchymal stem cell growth medium (MSCGM)™ as growth medium, MSCGM + 5-azacytidine (5-Aza), skeletal muscle myoblast cell growth medium (SkGM)-2 BulletKit™, and 5, 30 and 50% conditioned cell culture media, i.e., supernatant of human satellite cell cultures after three days in cell culture mixed with MSCGM. Following the incubation of human AT-MSCs or BM-MSCs for 0, 4, 8, 11, 16 or 21 days with each of the cell culture media, cell proliferation was measured using the alamarBlue® assay. Myogenic differentiation was evaluated by quantitative gene expression analyses, using quantitative RT-PCR (qRT-PCR) and immunocytochemical staining (ICC), using well-defined skeletal markers, such as desmin (DES), myogenic factor 5 (MYF5), myosin, heavy chain 8, skeletal muscle, perinatal (MYH8), myosin, heavy chain 1, skeletal muscle, adult (MYH1) and skeletal muscle actin-α1 (ACTA1). The highest proliferation rates were observed in the AT-MSCs and BM-MSCs cultured with SkGM-2 BulletKit medium. The average proliferation rate was higher in the AT-MSCs than in the BM-MSCs, taking all six culture media into account. qRT-PCR revealed the expression levels of the myogenic markers, ACTA1, MYH1 and MYH8, in the AT-MSC cell cultures, but not in the BM-MSC cultures. The muscle-specific intermediate filament, DES, was only detected (by ICC) in the AT-MSCs, but not in the BM

  6. Vibrational Micro-Spectroscopy of Human Tissues Analysis: Review.

    Science.gov (United States)

    Bunaciu, Andrei A; Hoang, Vu Dang; Aboul-Enein, Hassan Y

    2017-05-04

    Vibrational spectroscopy (Infrared (IR) and Raman) and, in particular, micro-spectroscopy and micro-spectroscopic imaging have been used to characterize developmental changes in tissues, to monitor these changes in cell cultures and to detect disease and drug-induced modifications. The conventional methods for biochemical and histophatological tissue characterization necessitate complex and "time-consuming" sample manipulations and the results are rarely quantifiable. The spectroscopy of molecular vibrations using mid-IR or Raman techniques has been applied to samples of human tissue. This article reviews the application of these vibrational spectroscopic techniques for analysis of biological tissue published between 2005 and 2015.

  7. Influence of Cryopreservation Solution on the In Vitro Culture of Skin Tissues Derived from Collared Peccary (Pecari tajacu Linnaeus, 1758).

    Science.gov (United States)

    Borges, Alana A; Lira, Gabriela P O; Nascimento, Lucas E; Queiroz Neta, Luiza B; Santos, Maria V O; Oliveira, Moacir F; Silva, Alexandre R; Pereira, Alexsandra F

    2018-04-01

    Skin vitrification is a promising and alternative tool for the conservation of biodiversity, especially for wild mammals, such as collared peccaries. Several factors can affect the success of this procedure, such as the cryoprotectant solution used. Therefore, this study was carried out to compare the efficiency of various vitrification solutions for recovery of viable cells after in vitro culture of cryopreserved skin tissues derived from the collared peccary, aiming to study the application in biobanking, where cellular use is not immediately required. Then, Dulbecco's modified Eagle's medium (DMEM) composed of 2.2 g/L sodium bicarbonate and 10% fetal bovine serum (FBS) was supplemented with 3.0 M ethylene glycol (EG) or 3.0 M dimethyl sulfoxide (DMSO) or 1.5 M EG plus 1.5 M DMSO with or without sucrose (SUC; 0.25 M) to produce six solutions for solid-surface vitrification. After warming, skin tissues were cultured in vitro and recovered cells were analyzed for morphology, adhesion, subconfluence, and proliferative activity for developing the growth curve and determining the population doubling time (PDT), and viability by Trypan Blue. The vitrification did not alter the ability of the tissues to adhere to the culture dish, as well as the day of all explants with cell growth, subconfluence samples, subconfluence total time, and PDT (p > 0.05). Moreover, independent of the cryoprotectant solution used, the vitrification altered the day of all attached explants (p  0.05). Additionally, for viability after the third passage, only the EG-SUC group maintained the cell quality (88.3%), when compared with the nonvitrified (97.8%, p > 0.05). In conclusion, DMEM with 10% FBS, 3.0 M EG, and 0.25 M sucrose was the most efficient solution for vitrifying collared peccary skin tissues, leading to the in vitro culture of viable cells.

  8. Impact of dental implant insertion method on the peri-implant bone tissue: Experimental study

    Directory of Open Access Journals (Sweden)

    Stamatović Novak

    2013-01-01

    Full Text Available Background/Aim. The function of dental implants depends on their stability in bone tissue over extended period of time, i.e. on osseointegration. The process through which osseointegration is achieved depends on several factors, surgical insertion method being one of them. The aim of this study was to histopathologically compare the impact of the surgical method of implant insertion on the peri-implant bone tissue. Methods. The experiment was performed on 9 dogs. Eight weeks following the extraction of lower premolars implants were inserted using the one-stage method on the right mandibular side and two-stage method on the left side. Three months after implantation the animals were sacrificed. Three distinct regions of bone tissue were histopathologically analyzed, the results were scored and compared. Results. In the specimens of one-stage implants increased amount of collagen fibers was found in 5 specimens where tissue necrosis was also observed. Only moderate osteoblastic activity was found in 3 sections. The analysis of bone-to-implant contact region revealed statistically significantly better results regarding the amount of collagen tissue fibers for the implants inserted in the two-stage method (Wa = 59 105, α = 0.05. No necrosis and osteoblastic activity were observed. Conclusion. Better results were achieved by the two-stage method in bone-to-implant contact region regarding the amount of collagen tissue, while the results were identical regarding the osteoblastic activity and bone tissue necrosis. There was no difference between the methods in the bone-implant interface region. In the bone tissue adjacent to the implant the results were identical regarding the amount of collagen tissue, osteoblastic reaction and bone tissue necrosis, while better results were achieved by the two-stage method regarding the number of osteocytes.

  9. Factors affecting callus and protoplast production and regeneration of plants from garlic tissue cultures

    International Nuclear Information System (INIS)

    Al-Safadi, B.; Nabulsi, I.

    2001-08-01

    Five cultivars of garlic, two explants, six callusing media, six regeneration media, two kinds of light and several doses of gamma irradiation were used to determine the best conditions for callus induction and plant regeneration from garlic tissue cultures. Also, some experiments were conducted to study the possibility to isolate protoplast and regenerate plants. The experiment showed that medium MS9 was good for regenerating plant directly from basal plate without going through callus phase. ANOVA exhibited significant differences among used cultivars in their ability to form callus. No significant difference was observed between 16 hr light and complete darkness in callus growth. However, appearance of callus was generally better on darkness. Cultivar varied in their ability to regenerate and interaction between cultivars and media was observed. Cultivar kisswany was the best in regeneration (38%) and medium MS47 was the best among used media (35%). Light type played a significant role in regeneration of plants where red light was much better than white light in inducing regeneration (68% vs 36%). ANOVA revealed significant effect of low doses of gamma irradiation on stimulation regeneration of plant whereas high doses prevented regeneration. Many experiments were conducted to isolate protoplast and regenerate plants. The best method for culturing was the droplet and the best conditions for incubation were complete darkness at 25 Degreed centigrade. This lead to formation of cell wall but no cell division was observed (author)

  10. Properties of Dental Pulp-derived Mesenchymal Stem Cells and the Effects of Culture Conditions.

    Science.gov (United States)

    Kawashima, Nobuyuki; Noda, Sonoko; Yamamoto, Mioko; Okiji, Takashi

    2017-09-01

    Dental pulp mesenchymal stem cells (DPMSCs) highly express mesenchymal stem cell markers and possess the potential to differentiate into neural cells, osteoblasts, adipocytes, and chondrocytes. Thus, DPMSCs are considered suitable for tissue regeneration. The colony isolation method has commonly been used to collect relatively large amounts of heterogeneous DPMSCs. Homogenous DPMSCs can be isolated by fluorescence-activated cell sorting using antibodies against mesenchymal stem cell markers, although this method yields a limited number of cells. Both quality and quantity of DPMSCs are critical to regenerative therapy, and cell culture methods need to be improved. We thus investigated the properties of DPMSCs cultured with different methods. DPMSCs in a three-dimensional spheroid culture system, which is similar to the hanging drop culture for differentiation of embryonic stem cells, showed upregulation of odonto-/osteoblastic markers and mineralized nodule formation. This suggests that this three-dimensional spheroid culturing system for DPMSCs may be suitable for inducing hard tissues. We further examined the effect of cell culture density on the properties of DPMSCs because the properties of stem cells can be altered depending on the cell density. DPMSCs cultured under the confluent cell density condition showed slight downregulation of some mesenchymal stem cell markers compared with those under the sparse condition. The ability of DPMSCs to differentiate into hard tissue-forming cells was found to be enhanced in the confluent condition, suggesting that the confluent culture condition may not be suitable for maintaining the stemness of DPMSCs. When DPMSCs are to be used for hard tissue regeneration, dense followed by sparse cell culture conditions may be a better alternative strategy. Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  11. Culture, Method, and the Content of Self-Concepts: Testing Trait, Individual-Self-Primacy, and Cultural Psychology Perspectives.

    Science.gov (United States)

    Del Prado, Alicia M; Church, A Timothy; Katigbak, Marcia S; Miramontes, Lilia G; Whitty, Monica; Curtis, Guy J; de Jesús Vargas-Flores, José; Ibáñez-Reyes, Joselina; Ortiz, Fernando A; Reyes, Jose Alberto S

    2007-12-01

    Three theoretical perspectives on cultural universals and differences in the content of self-concepts were tested in individualistic (United States, n = 178; Australia, n = 112) and collectivistic (Mexico, n = 157; Philippines, n = 138) cultures, using three methods of self-concept assessment. Support was found for both trait perspectives and the individual-self-primacy hypothesis. In contrast, support for cultural psychology hypotheses was limited because traits and other personal attributes were not more salient, or social attributes less salient, in individualistic cultures than collectivistic cultures. The salience of some aspects of self-concept depended on the method of assessment, calling into question conclusions based on monomethod studies.

  12. Detection of Bacteria by Fluorescence in Situ Hybridization in Culture-Negative Soft Tissue Filler Lesions

    DEFF Research Database (Denmark)

    Bjarnsholt, Thomas; Tolker-Nielsen, Tim; Givskov, Michael

    2009-01-01

    BACKGROUND Adverse reactions to polyacrylamide gel occur as swellings or nodules, and controversy exists whether these are due to bacterial infection or an autoimmune reaction to the filler. OBJECTIVES Biopsies from culture-negative long-lasting nodules after injection with different types...... of polyacrylamide gel were examined with a combination of Gram stain and fluorescence in situ hybridization. RESULTS Bacteria were detected in biopsies from seven of eight patients. They inhabited gel and intervening tissue and tended to lie in aggregates. CONCLUSION This study supports the assumption...... that infection with bacteria in aggregates causes culture-negative late adverse reactions to polyacrylamide gel, suggesting a biofilm environment. The authors have indicated no significant interest with commercial supporters....

  13. Dialectical Method and the Critical Political Economy of Culture

    Directory of Open Access Journals (Sweden)

    Brice Nixon

    2012-05-01

    Full Text Available This article argues that the quality that defines critical political economy is its critical method. Definitions of the critical political economy of culture are considered and shown to focus on specific theoretical concerns while not fully addressing the fundamental issue of method. Method is here discussed in terms of the way human reason is used to produce knowledge. A critical method for Marx is a historical materialist dialectical method, thus this paper argues for a deeper consideration of the Marxist dialectical method in relation to critical political-economic theorizing. Sources for methodological consideration from Marx to 20th-century Western Marxists are outlined. The potential contribution of the Marxist dialectical method in the continued development of the critical political economy of culture is demonstrated by showing the possibility of developing a complementary critical political economy of consciousness. Smythe’s theorizing of audiences as workers is considered as a useful starting point, and its potential development through incorporation of the work of other critical scholars of media and culture is outlined.

  14. Improvement of tissue culture, genetic transformation, and applications of biotechnology to Brassica.

    Science.gov (United States)

    Ravanfar, Seyed Ali; Orbovic, Vladimir; Moradpour, Mahdi; Abdul Aziz, Maheran; Karan, Ratna; Wallace, Simon; Parajuli, Saroj

    2017-04-01

    Development of in vitro plant regeneration method from Brassica explants via organogenesis and somatic embryogenesis is influenced by many factors such as culture environment, culture medium composition, explant sources, and genotypes which are reviewed in this study. An efficient in vitro regeneration system to allow genetic transformation of Brassica is a crucial tool for improving its economical value. Methods to optimize transformation protocols for the efficient introduction of desirable traits, and a comparative analysis of these methods are also reviewed. Hence, binary vectors, selectable marker genes, minimum inhibitory concentration of selection agents, reporter marker genes, preculture media, Agrobacterium concentration and regeneration ability of putative transformants for improvement of Agrobacterium-mediated transformation of Brassica are discussed.

  15. Electroactive Tissue Scaffolds with Aligned Pores as Instructive Platforms for Biomimetic Tissue Engineering.

    Science.gov (United States)

    Hardy, John G; Cornelison, R Chase; Sukhavasi, Rushi C; Saballos, Richard J; Vu, Philip; Kaplan, David L; Schmidt, Christine E

    2015-01-14

    Tissues in the body are hierarchically structured composite materials with tissue-specific chemical and topographical properties. Here we report the preparation of tissue scaffolds with macroscopic pores generated via the dissolution of a sacrificial supramolecular polymer-based crystal template (urea) from a biodegradable polymer-based scaffold (polycaprolactone, PCL). Furthermore, we report a method of aligning the supramolecular polymer-based crystals within the PCL, and that the dissolution of the sacrificial urea yields scaffolds with macroscopic pores that are aligned over long, clinically-relevant distances ( i.e ., centimeter scale). The pores act as topographical cues to which rat Schwann cells respond by aligning with the long axis of the pores. Generation of an interpenetrating network of polypyrrole (PPy) and poly(styrene sulfonate) (PSS) in the scaffolds yields electroactive tissue scaffolds that allow the electrical stimulation of Schwann cells cultured on the scaffolds which increases the production of nerve growth factor (NGF).

  16. Analyses of Tissue Culture Adaptation of Human Herpesvirus-6A by Whole Genome Deep Sequencing Redefines the Reference Sequence and Identifies Virus Entry Complex Changes.

    Science.gov (United States)

    Tweedy, Joshua G; Escriva, Eric; Topf, Maya; Gompels, Ursula A

    2017-12-31

    Tissue-culture adaptation of viruses can modulate infection. Laboratory passage and bacterial artificial chromosome (BAC)mid cloning of human cytomegalovirus, HCMV, resulted in genomic deletions and rearrangements altering genes encoding the virus entry complex, which affected cellular tropism, virulence, and vaccine development. Here, we analyse these effects on the reference genome for related betaherpesviruses, Roseolovirus, human herpesvirus 6A (HHV-6A) strain U1102. This virus is also naturally "cloned" by germline subtelomeric chromosomal-integration in approximately 1% of human populations, and accurate references are key to understanding pathological relationships between exogenous and endogenous virus. Using whole genome next-generation deep-sequencing Illumina-based methods, we compared the original isolate to tissue-culture passaged and the BACmid-cloned virus. This re-defined the reference genome showing 32 corrections and 5 polymorphisms. Furthermore, minor variant analyses of passaged and BACmid virus identified emerging populations of a further 32 single nucleotide polymorphisms (SNPs) in 10 loci, half non-synonymous indicating cell-culture selection. Analyses of the BAC-virus genome showed deletion of the BAC cassette via loxP recombination removing green fluorescent protein (GFP)-based selection. As shown for HCMV culture effects, select HHV-6A SNPs mapped to genes encoding mediators of virus cellular entry, including virus envelope glycoprotein genes gB and the gH/gL complex. Comparative models suggest stabilisation of the post-fusion conformation. These SNPs are essential to consider in vaccine-design, antimicrobial-resistance, and pathogenesis.

  17. Characterization of aldehyde dehydrogenase isozymes in ovarian cancer tissues and sphere cultures

    Directory of Open Access Journals (Sweden)

    Saw Yu-Ting

    2012-08-01

    Full Text Available Abstract Background Aldehyde dehydrogenases belong to a superfamily of detoxifying enzymes that protect cells from carcinogenic aldehydes. Of the superfamily, ALDH1A1 has gained most attention because current studies have shown that its expression is associated with human cancer stem cells. However, ALDH1A1 is only one of the 19 human ALDH subfamilies currently known. The purpose of the present study was to determine if the expression and activities of other major ALDH isozymes are associated with human ovarian cancer and ovarian cancer sphere cultures. Methods Immunohistochemistry was used to delineate ALDH isozyme localization in clinical ovarian tissues. Western Blot analyses were performed on lysates prepared from cancer cell lines and ovarian cancer spheres to confirm the immunohistochemistry findings. Quantitative reverse transcription-polymerase chain reactions were used to measure the mRNA expression levels. The Aldefluor® assay was used to measure ALDH activity in cancer cells from the four tumor subtypes. Results Immunohistochemical staining showed significant overexpression of ALDH1A3, ALDH3A2, and ALDH7A1 isozymes in ovarian tumors relative to normal ovarian tissues. The expression and activity of ALDH1A1 is tumor type-dependent, as seen from immunohistochemisty, Western blot analysis, and the Aldefluor® assay. The expression was elevated in the mucinous and endometrioid ovarian epithelial tumors than in serous and clear cell tumors. In some serous and most clear cell tumors, ALDH1A1 expression was found in the stromal fibroblasts. RNA expression of all studied ALDH isozymes also showed higher expression in endometrioid and mucinous tumors than in the serous and clear cell subtypes. The expression of ALDH enzymes showed tumor type-dependent induction in ovarian cancer cells growing as sphere suspensions in serum-free medium. Conclusions The results of our study indicate that ALDH enzyme expression and activity may be associated

  18. Cultural adaptation and translation of measures: an integrated method.

    Science.gov (United States)

    Sidani, Souraya; Guruge, Sepali; Miranda, Joyal; Ford-Gilboe, Marilyn; Varcoe, Colleen

    2010-04-01

    Differences in the conceptualization and operationalization of health-related concepts may exist across cultures. Such differences underscore the importance of examining conceptual equivalence when adapting and translating instruments. In this article, we describe an integrated method for exploring conceptual equivalence within the process of adapting and translating measures. The integrated method involves five phases including selection of instruments for cultural adaptation and translation; assessment of conceptual equivalence, leading to the generation of a set of items deemed to be culturally and linguistically appropriate to assess the concept of interest in the target community; forward translation; back translation (optional); and pre-testing of the set of items. Strengths and limitations of the proposed integrated method are discussed. (c) 2010 Wiley Periodicals, Inc.

  19. Genetic programming based models in plant tissue culture: An addendum to traditional statistical approach.

    Science.gov (United States)

    Mridula, Meenu R; Nair, Ashalatha S; Kumar, K Satheesh

    2018-02-01

    In this paper, we compared the efficacy of observation based modeling approach using a genetic algorithm with the regular statistical analysis as an alternative methodology in plant research. Preliminary experimental data on in vitro rooting was taken for this study with an aim to understand the effect of charcoal and naphthalene acetic acid (NAA) on successful rooting and also to optimize the two variables for maximum result. Observation-based modelling, as well as traditional approach, could identify NAA as a critical factor in rooting of the plantlets under the experimental conditions employed. Symbolic regression analysis using the software deployed here optimised the treatments studied and was successful in identifying the complex non-linear interaction among the variables, with minimalistic preliminary data. The presence of charcoal in the culture medium has a significant impact on root generation by reducing basal callus mass formation. Such an approach is advantageous for establishing in vitro culture protocols as these models will have significant potential for saving time and expenditure in plant tissue culture laboratories, and it further reduces the need for specialised background.

  20. Culture of insect tissues

    International Nuclear Information System (INIS)

    Cestari, A.N.; Simoes, L.C.G.

    1978-01-01

    Several aspects are discussed related to the behavior of politenic chromosomes from Rhyncosciara salivary glands kept in culture during different periods of time, without interference of insect hormones. Nucleic acid-and protein synthesis in isolated nuclei and chromosomes are also investigated. Autoradiographic techniques and radioactive precursors for nucleic acids and proteins are used in the research. (M.A.) [pt

  1. The discussion on the qualitative and quantitative evaluation methods for safety culture

    International Nuclear Information System (INIS)

    Gao Kefu

    2005-01-01

    The fundamental methods for safely culture evaluation are described. Combining with the practice of the quantitative evaluation of safety culture in Daya Bay NPP, the quantitative evaluation method for safety culture are discussed. (author)

  2. Survival and growth of isolated pre-antral follicles from human ovarian medulla tissue during long-term 3D culture

    DEFF Research Database (Denmark)

    Yin, H L; Kristensen, S G; Jiang, H

    2016-01-01

    during long-term culture has received only little attention. STUDY DESIGN, SIZE, DURATION: Two to ten human pre-antral follicles were encapsulated together within an alginate bead and cultured with or without ovarian interstitial tissue for either 7 days or >30 days. Follicles were cultured in either 20...... interregional project ReproHigh are thanked for having funded this study; and the Key Program of Medical Science and Technology Innovation of Nanjing Military Area Command in China (14ZX06; 11Z010). They had no role in the study design, collection and analysis of data, data interpretation or in writing...

  3. In vivo studies of peritendinous tissue in exercise

    DEFF Research Database (Denmark)

    Kjaer, M; Langberg, Henning; Skovgaard, D

    2000-01-01

    Soft tissue injury of tendons represents a major problem within sports medicine. Although several animal and cell culture studies have addressed this, human experiments have been limited in their ability to follow changes in specific tissue directly in response to interventions. Recently, methods...... have allowed for in vivo determination of tissue concentrations and release rates of substances involved in metabolism, inflammation and collagen synthesis, together with the measurement of tissue blood flow and oxygenation in the peritendinous region around the Achilles tendon in humans during...... exercise. This coincides with a surprisingly marked drop in tissue pressure during contraction. With regards to both circulation, metabolism and collagen formation, peritendinous tissue represents a dynamic, responsive region that adapts markedly to acute muscular activity....

  4. NEW METHOD FOR DETERMINATION OF ACTINIDES AND STRONTIUM IN ANIMAL TISSUE

    Energy Technology Data Exchange (ETDEWEB)

    Maxwell, S; Jay Hutchison, J; Don Faison, D

    2007-05-07

    The analysis of actinides in animal tissue samples is very important for environmental monitoring. There is a need to measure actinide isotopes with very low detection limits in animal tissue samples, including fish, deer, hogs, beef and shellfish. A new, rapid actinide separation method has been developed and implemented that allows the measurement of plutonium, neptunium, uranium, americium, curium and strontium isotopes in large animal tissue samples (100-200 g) with high chemical recoveries and effective removal of matrix interferences. This method uses stacked TEVA Resin{reg_sign}, TRU Resin{reg_sign} and DGA-Resin{reg_sign} cartridges from Eichrom Technologies (Darien, IL, USA) that allows the rapid separation of plutonium (Pu), neptunium (Np), uranium (U), americium (Am), and curium (Cm) using a single multi-stage column combined with alpha spectrometry. Sr-90 is collected on Sr Resin{reg_sign} from Eichrom Technologies (Darien, IL, USA). After acid digestion and furnace heating of the animal tissue samples, the actinides and Sr-89/90 are separated using column extraction chromatography. This method has been shown to be effective over a wide range of animal tissue matrices. By using vacuum box cartridge technology with rapid flow rates, sample preparation time is minimized.

  5. Tissue culture as a plant production technique for horticultural crops

    African Journals Online (AJOL)

    STORAGESEVER

    2009-08-18

    Aug 18, 2009 ... Recovery of regenerants from transformed cells. - Cell culture .... methods. Micropropagation techniques. Micropropagation is a simple concept. The basic pro- tocols were well established by the 1960s and a whole research field and ... the environment are naturally contaminated on their sur- faces (and ...

  6. Nuclear morphology, polyploidy, and chromatin elimination in tissue culture of Allium fistulosum L.

    Directory of Open Access Journals (Sweden)

    Andrzej Joachimiak

    2011-01-01

    Full Text Available The morphology of cell nuclei in callus obtained from root-tip meristems of Allium fistulosum L. (Monocotyledoneae, Alliaceae was analysed. The most interesting phenomena observed in long-term callus culture were the different mechanisms of cell polyploidization, enlargement of telomeric segments of heterochromatin, and extensive chromatin elimination, associated with instability of nuclei size and DNA content. Protruding heterochromatin "spikes" were observed on the surface of some di- and polyploid nuclei. The presence of these spikes was connected with the formation of small heterochromatic micronuclei frequently found in the cytoplasm. It is suggested that these micronuclei are produced by direct elimination of heterochromatin from the interphase nuclei. Polyploid cells accumulated with each successive cell collection. The ploidy level attained by highly polyploid cells was 15C-220C. The shape of the nuclei and heterochromatin distribution suggest that polyploid nuclei in A. fistulosum tissue culture are produced by endoreduplication and by restitution cycles.

  7. Methods for culturing retinal pigment epithelial cells: a review of current protocols and future recommendations

    Directory of Open Access Journals (Sweden)

    Aaron H Fronk

    2016-07-01

    Full Text Available The retinal pigment epithelium is an important part of the vertebrate eye, particularly in studying the causes and possible treatment of age-related macular degeneration. The retinal pigment epithelium is difficult to access in vivo due to its location at the back of the eye, making experimentation with age-related macular degeneration treatments problematic. An alternative to in vivo experimentation is cultivating the retinal pigment epithelium in vitro, a practice that has been going on since the 1970s, providing a wide range of retinal pigment epithelial culture protocols, each producing cells and tissue of varying degrees of similarity to natural retinal pigment epithelium. The purpose of this review is to provide researchers with a ready list of retinal pigment epithelial protocols, their effects on cultured tissue, and their specific possible applications. Protocols using human and animal retinal pigment epithelium cells, derived from tissue or cell lines, are discussed, and recommendations for future researchers included.

  8. Characterization of connective tissue growth factor expression in primary cultures of human tubular epithelial cells: modulation by hypoxia

    NARCIS (Netherlands)

    Kroening, Sven; Neubauer, Emily; Wullich, Bernd; Aten, Jan; Goppelt-Struebe, Margarete

    2010-01-01

    Kroening S, Neubauer E, Wullich B, Aten J, Goppelt-Struebe M. Characterization of connective tissue growth factor expression in primary cultures of human tubular epithelial cells: modulation by hypoxia. Am J Physiol Renal Physiol 298:F796-F806, 2010. First published December 23, 2009;

  9. Unusual 4-hydroxybenzaldehyde synthase activity from tissue cultures of the vanilla orchid Vanilla planifolia.

    Science.gov (United States)

    Podstolski, Andrzej; Havkin-Frenkel, Daphna; Malinowski, Jacek; Blount, Jack W; Kourteva, Galina; Dixon, Richard A

    2002-11-01

    Tissue cultures of the vanilla orchid, Vanilla planifolia, produce the flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde) and vanillin precursors such as 4-hydroxybenzaldehyde. A constitutively expressed enzyme activity catalyzing chain shortening of a hydroxycinnamic acid, believed to be the first reaction specific for formation of vanilla flavor compounds, was identified in these cultures. The enzyme converts 4-coumaric acid non-oxidatively to 4-hydroxybenzaldehyde in the presence of a thiol reagent but with no co-factor requirement. Several forms of this 4-hydroxybenzaldehyde synthase (4HBS) were resolved and partially purified by a combination of hydrophobic interaction, ion exchange and gel filtration chromatography. These forms appear to be interconvertible. The unusual properties of the 4HBS, and its appearance in different protein fractions, raise questions as to its physiological role in vanillin biosynthesis in vivo.

  10. Suggestions on the Development of Safety Culture Assessment Method

    International Nuclear Information System (INIS)

    Choi, Young Sung; Choi, Kwang Sik; Kim, Woong Sik

    2006-01-01

    Several efforts have been made to assess safety culture of organization that operates nuclear power plants in Korea. The MOST and KINS played a major role to develop assessment methods and KHNP applied them to its NPPs. This paper explains the two methods developed by KINS briefly and presents the insights obtained from the two different applications. It concludes with some suggestions for safety culture assessment based on the insights

  11. Electroactive Tissue Scaffolds with Aligned Pores as Instructive Platforms for Biomimetic Tissue Engineering

    Directory of Open Access Journals (Sweden)

    John G. Hardy

    2015-01-01

    Full Text Available Tissues in the body are hierarchically structured composite materials with tissue-specific chemical and topographical properties. Here we report the preparation of tissue scaffolds with macroscopic pores generated via the dissolution of a sacrificial supramolecular polymer-based crystal template (urea from a biodegradable polymer-based scaffold (polycaprolactone, PCL. Furthermore, we report a method of aligning the supramolecular polymer-based crystals within the PCL, and that the dissolution of the sacrificial urea yields scaffolds with macroscopic pores that are aligned over long, clinically-relevant distances (i.e., centimeter scale. The pores act as topographical cues to which rat Schwann cells respond by aligning with the long axis of the pores. Generation of an interpenetrating network of polypyrrole (PPy and poly(styrene sulfonate (PSS in the scaffolds yields electroactive tissue scaffolds that allow the electrical stimulation of Schwann cells cultured on the scaffolds which increases the production of nerve growth factor (NGF.

  12. Cardiac tissue engineering

    Directory of Open Access Journals (Sweden)

    MILICA RADISIC

    2005-03-01

    Full Text Available We hypothesized that clinically sized (1-5 mm thick,compact cardiac constructs containing physiologically high density of viable cells (~108 cells/cm3 can be engineered in vitro by using biomimetic culture systems capable of providing oxygen transport and electrical stimulation, designed to mimic those in native heart. This hypothesis was tested by culturing rat heart cells on polymer scaffolds, either with perfusion of culture medium (physiologic interstitial velocity, supplementation of perfluorocarbons, or with electrical stimulation (continuous application of biphasic pulses, 2 ms, 5 V, 1 Hz. Tissue constructs cultured without perfusion or electrical stimulation served as controls. Medium perfusion and addition of perfluorocarbons resulted in compact, thick constructs containing physiologic density of viable, electromechanically coupled cells, in contrast to control constructs which had only a ~100 mm thick peripheral region with functionally connected cells. Electrical stimulation of cultured constructs resulted in markedly improved contractile properties, increased amounts of cardiac proteins, and remarkably well developed ultrastructure (similar to that of native heart as compared to non-stimulated controls. We discuss here the state of the art of cardiac tissue engineering, in light of the biomimetic approach that reproduces in vitro some of the conditions present during normal tissue development.

  13. Application of cell and biomaterial-based tissue engineering methods in the treatment of cartilage, menisci and ligament injuries.

    Science.gov (United States)

    Trzeciak, Tomasz; Richter, Magdalena; Suchorska, Wiktoria; Augustyniak, Ewelina; Lach, Michał; Kaczmarek, Małgorzata; Kaczmarczyk, Jacek

    2016-03-01

    Over 20 years ago it was realized that the traditional methods of the treatment of injuries to joint components: cartilage, menisci and ligaments, did not give satisfactory results and so there is a need of employing novel, more effective therapeutic techniques. Recent advances in molecular biology, biotechnology and polymer science have led to both the experimental and clinical application of various cell types, adapting their culture conditions in order to ensure a directed differentiation of the cells into a desired cell type, and employing non-toxic and non-immunogenic biomaterial in the treatment of knee joint injuries. In the present review the current state of knowledge regarding novel cell sources, in vitro conditions of cell culture and major important biomaterials, both natural and synthetic, used in cartilage, meniscus and ligament repair by tissue engineering techniques are described, and the assets and drawbacks of their clinical application are critically evaluated.

  14. Effects of estradiol and medroxyprogesterone acetate on morphology, proliferation and apoptosis of human breast tissue in organ cultures

    International Nuclear Information System (INIS)

    Eigėlienė, Natalija; Härkönen, Pirkko; Erkkola, Risto

    2006-01-01

    Human breast tissue undergoes phases of proliferation, differentiation and regression regulated by changes of the levels of circulating sex hormones during the menstrual cycle or aging. Ovarian hormones also likely play a key role in the etiology and biology of breast cancer. Reports concerning the proliferative effects of steroid hormones on the normal epithelium of human breast have been conflicting. Some studies have shown that steroid hormones may predispose breast epithelial cells to malignant changes by stimulating their proliferation, which is known to be regulated tightly by stromal cells. The aim of this study was to investigate the effects of 17β-estradiol and medroxyprogesterone acetate on proliferation, apoptosis, expression of differentiation markers and steroid hormone receptors in breast epithelium using an in vitro model of freshly isolated human breast tissue, in which a proper interaction of breast epithelium and stroma has been maintained. Human breast tissues were obtained from women undergoing surgery for breast tumours. Peritumoral tissues were excised and explants were cultured for 3 weeks in medium supplemented with E 2 or MPA or with E 2 +MPA. Endpoints included histopathological, histomorphometric and immunohistochemical assessment of the breast explants. Culture of breast explants for 14 or 21 days with steroid hormones increased proliferative activity and the thickness of acinar and ductal epithelium. E 2 -treatment led to hyperplastic epithelial morphology, MPA to hypersecretory single-layered epithelium and E 2 +MPA to multilayered but organised epithelium. The proliferative response to E 2 in comparison to control (p < 0.001) was more pronounced than to MPA (p < 0.05) or E 2 +MPA (p < 0.05) at 7 and 14 days for Ki-67 and PCNA. E 2 treatment also decreased the proportion of apoptotic cells after 7 (p < 0.01) and 14 (p < 0.01) days. In addition, the relative number of ERα, ERβ and PR positive epithelial cells was decreased by all

  15. Rapid authentication of different ages of tissue-cultured and wild Dendrobium huoshanense as well as wild Dendrobium henanense using FTIR and 2D-COS IR

    Science.gov (United States)

    Chen, Nai-Dong; Chen, Nai-Fu; Li, Jun; Cao, Cai-Yun; Wang, Jin-Mei

    2015-12-01

    The accumulating of pharmaceutical chemicals in medicinal plants would greatly be affected by their ages and establishing a fast quality-identification method to evaluate the similarity of medicinal herbs at different cultivated ages is a critical step for assurance of quality and safety in the TCM industry. In this work, tri-step IR macro-fingerprinting and 2D-COS IR spectrum techniques combined with statistical pattern recognition were applied for discrimination and similarity evaluation of different ages of tissue-cultured and wild Dendrobium huoshanense C. Z. Tang et S. J. Cheng as well as Dendrobium henanense J.L.Lu et L.X Gao. Both tissue-cultured and wild D. huoshanense were easily differentiated from D. henanense by FTIR and SD-IR spectra, while it's quite difficult to discriminate different cultivated years of the three investigated Dendrobiums. In 2D-COS IR spectra, 1-5 auto-peaks with different indensity and positions were located in the region 1160-1030 cm-1 of the twelve Dendrobium samples and thus could be used to identify Dendrobium samples at different ages. Principle component analysis (PCA) of synchronous 2D-COS data showed that the twelve samples were effectively identified and evaluated. The results indicated that the tri-step infrared macro-fingerprinting combined with PCA method was suitable to differentiate the cultivated ages of Dendrobiums with species and orgins rapidly and nondestructively.

  16. Determination of the cause of the symptoms on yellow yam (Dioscorea cayenensis Lam.) leaf tissue and their eradication, enriching the culture medium and using techniques of meristem culture, thermo and chemotherapy on in vitro conditions

    International Nuclear Information System (INIS)

    Brenes Huertas, Mauricio

    2010-01-01

    Yams (Dioscorea spp) has been cultivated for exportation in Costa Rica, in North Huetar region. In vitro culture technique has been used for multiplying planting material for many advantages. However, cleaning of viruses that affect has been ineffective. Viruses such as: the potyvirus, potexvirus, cucumovirus . Methods like meristem culture, chemotherapy, thermotherapy and combinations of these have been used for the elimination of virus in plant species. The plants were evaluated in indexing assays, observing symptoms, serological methods and electron microscopy, among others. Other problems that have been affecting in vitro plant are deficient culture media in some nutrient. The presence of some abnormal characteristics in leaf tissue was determined whether have been caused by a virus or a nutritional deficiency in the culture medium. The presence of the virus has tried to find using ELISA and electron microscopy. Tests meristem culture, thermotherapy and chemotherapy have been made for the eradication of a possible virus; which have been assessed by observation of symptomatology and ELISA. The efficiency of the culture medium was evaluated to enrich it with nitrogen or excess iron. None of the suspected virus found in ELISA tests. Filaments are presumably viral particles were found through analysis of ultrastructure, as well as alterations in chloroplasts which indicated the presence of a pathogen or toxicity. Thermotherapy and chemotherapy with the concentration of 40 mg/L of ribavirin have been the most effective for the elimination of symptoms in virus eradication treatments. Assessments nutrient concentrations have shown that the differences between the various treatments used were undetectable. The symptoms presented were caused, according to the conclusions, by a virus which should preferably deal with thermotherapy. (author) [es

  17. [Differential diagnosis for detection of hyphae in tissue].

    Science.gov (United States)

    Tintelnot, K

    2013-11-01

    Usually the detection of hyphae in tissue is unmistakable evidence of a deep mycosis requiring antimycotic treatment. Micromorphology alone rarely allows a specific diagnosis, thus confusion is possible between Candida, Aspergillus, Alternaria and Fusarium species or several other fungal agents. If broad, nearly non-septated hyphae are detected histologically mucormycosis can be suspected. Detection of hyphae in tissue is always a cause for concern because therapeutic consequences must follow. Because therapeutic strategies may differ depending on the specific fungal agent, a suspected diagnosis should be supplemented by other methods, e.g. culture of unfixed specimens, by immunohistology or molecular biological methods.

  18. Soft tissue deformation estimation by spatio-temporal Kalman filter finite element method.

    Science.gov (United States)

    Yarahmadian, Mehran; Zhong, Yongmin; Gu, Chengfan; Shin, Jaehyun

    2018-01-01

    Soft tissue modeling plays an important role in the development of surgical training simulators as well as in robot-assisted minimally invasive surgeries. It has been known that while the traditional Finite Element Method (FEM) promises the accurate modeling of soft tissue deformation, it still suffers from a slow computational process. This paper presents a Kalman filter finite element method to model soft tissue deformation in real time without sacrificing the traditional FEM accuracy. The proposed method employs the FEM equilibrium equation and formulates it as a filtering process to estimate soft tissue behavior using real-time measurement data. The model is temporally discretized using the Newmark method and further formulated as the system state equation. Simulation results demonstrate that the computational time of KF-FEM is approximately 10 times shorter than the traditional FEM and it is still as accurate as the traditional FEM. The normalized root-mean-square error of the proposed KF-FEM in reference to the traditional FEM is computed as 0.0116. It is concluded that the proposed method significantly improves the computational performance of the traditional FEM without sacrificing FEM accuracy. The proposed method also filters noises involved in system state and measurement data.

  19. Introgression of genetic material from Zea mays ssp. Mexicana into cultivated maize was facilitated by tissue culture

    International Nuclear Information System (INIS)

    Wang, L.; Gu, X.; Qu, M.; Luan, J.; Zhang, J.

    2012-01-01

    Zea mays ssp. mexicana, a wild relative of cultivated maize (Z. mays ssp. mays), is a useful gene resource for maize breeding. In this study, two populations were generated by conventional breeding scheme (population I) or tissue culture regime (population II), respectively, to introgress genetic material of Z. mays ssp. mexicana into maize. Karyotype analysis showed that the arm ratios of 10 pairs of chromosomes in parent maize Ye515 and derivative lines from 2 different populations with 26% and 38% chromosome variation frequencies, respectively. Alien chromatin was detected in the root tip cells of progeny plants through genomic in situ hybridization (GISH). There were 3.3 chromosomes carrying alien chromatin on average in population I and 6.5 in population II. The hybridization signals were located mainly at the terminal or sub terminal regions of the chromosomes and the sizes were notably variant among lines. Based on those results, it is concluded that the introgression of genetic material from Z. mays ssp. mexicana into cultivated maize was facilitated by tissue culture, and subsequently some excellent materials for maize breeding were created. (author)

  20. Extracellular matrix production by human osteoblasts cultured on biodegradable polymers applicable for tissue engineering.

    Science.gov (United States)

    El-Amin, S F; Lu, H H; Khan, Y; Burems, J; Mitchell, J; Tuan, R S; Laurencin, C T

    2003-03-01

    The nature of the extracellular matrix (ECM) is crucial in regulating cell functions via cell-matrix interactions, cytoskeletal organization, and integrin-mediated signaling. In bone, the ECM is composed of proteins such as collagen (CO), fibronectin (FN), laminin (LM), vitronectin (VN), osteopontin (OP) and osteonectin (ON). For bone tissue engineering, the ECM should also be considered in terms of its function in mediating cell adhesion to biomaterials. This study examined ECM production, cytoskeletal organization, and adhesion of primary human osteoblastic cells on biodegradable matrices applicable for tissue engineering, namely polylactic-co-glycolic acid 50:50 (PLAGA) and polylactic acid (PLA). We hypothesized that the osteocompatible, biodegradable polymer surfaces promote the production of bone-specific ECM proteins in a manner dependent on polymer composition. We first examined whether the PLAGA and PLA matrices could support human osteoblastic cell growth by measuring cell adhesion at 3, 6 and 12h post-plating. Adhesion on PLAGA was consistently higher than on PLA throughout the duration of the experiment, and comparable to tissue culture polystyrene (TCPS). ECM components, including CO, FN, LM, ON, OP and VN, produced on the surface of the polymers were quantified by ELISA and localized by immunofluorescence staining. All of these proteins were present at significantly higher levels on PLAGA compared to PLA or TCPS surfaces. On PLAGA, OP and ON were the most abundant ECM components, followed by CO, FN, VN and LN. Immunofluorescence revealed an extracellular distribution for CO and FN, whereas OP and ON were found both intracellularly as well as extracellularly on the polymer. In addition, the actin cytoskeletal network was more extensive in osteoblasts cultured on PLAGA than on PLA or TCPS. In summary, we found that osteoblasts plated on PLAGA adhered better to the substrate, produced higher levels of ECM molecules, and showed greater cytoskeletal

  1. Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.

    Science.gov (United States)

    Rodriguez-Canales, Jaime; Hanson, Jeffrey C; Hipp, Jason D; Balis, Ulysses J; Tangrea, Michael A; Emmert-Buck, Michael R; Bova, G Steven

    2013-01-01

    Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This updated chapter reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high-quality, appropriately anatomically tagged scientific results. Improvement in this area will significantly increase life science quality and productivity. The chapter is divided into introduction, materials, protocols, and notes subheadings. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this chapter, readers are advised to read through the entire

  2. Determination of lactic microflora of kefir grains and kefir beverage by using culture-dependent and culture-independent methods.

    Science.gov (United States)

    Kesmen, Zülal; Kacmaz, Nazife

    2011-01-01

    In this study, we investigated the bacterial compositions of kefir grains and kefir beverages collected from different regions of Turkey by using culture-independent and culture-dependent methods. In the culture-independent detection, 10 different species of bacteria were detected in total by using the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of the 16S rRNA gene V3 region. Among these species, Lactobacillus kefiranofaciens was the most dominant one in the kefir grains, while Lactococcus lactis was found to be significantly prevalent in the kefir beverages. In the culture-dependent detection, the primary differentiation and grouping of the isolates from kefir beverages and kefir grains were performed using repetitive sequence-based PCR (rep-PCR) fingerprinting, and the results were validated by 16S rDNA full-length sequencing. According to the results of culture-dependent methods, the most frequently isolated species were L. lactis, Leuconostoc mesenteroides, and Lactobacillus kefiri, respectively. Only 3 species, which are L. lactis, Lactobacillus acidophilus, and Streptococcus thermophilus, were detected with both culture-dependent and culture-independent methods. This study showed that the combination of both methods is necessary for a detailed and reliable investigation of microbial communities in kefir grains and kefir beverages. Due to their artisan- and region-dependent microflora, kefir products can be a source of interesting lactic acid bacteria, either new taxa or strains with specific functional properties, which might be used for the development of new starter cultures and innovative food products. Therefore, an increasing demand exists for new strains that show desirable effects on the product characteristics Artisan dairy products are a candidate source of such microorganisms. For this reason, in this study, the bacterial compositions of kefir grains and kefir beverages obtained from

  3. Increased Pathogen Identification in Vascular Graft Infections by the Combined Use of Tissue Cultures and 16S rRNA Gene Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Evelyne Ajdler-Schaeffler

    2018-06-01

    Full Text Available Background: Vascular graft infections (VGI are difficult to diagnose and treat, and despite redo surgery combined with antimicrobial treatment, outcomes are often poor. VGI diagnosis is based on a combination of clinical, radiological, laboratory and microbiological criteria. However, as many of the VGI patients are already under antimicrobial treatment at the time of redo surgery, microbiological identification is often difficult and bacterial cultures often remain negative rendering targeted treatment impossible. We aimed to assess the benefit of 16S rRNA gene polymerase chain reaction (broad-range PCR for better microbiological identification in patients with VGI.Methods: We prospectively analyzed the clinical, microbiological, and treatment data of patients enrolled in the observational Vascular Graft Cohort Study (VASGRA, University Hospital Zurich, Switzerland. The routine diagnostic work-up involved microbiological cultures of minced tissue samples, and the use of molecular techniques in parallel. Patient-related and microbiological data were assessed in descriptive analyses, and we calculated sensitivity, specificity, negative and positive predictive value for broad-range 16S rRNA gene PCR versus culture (considered as gold standard.Results: We investigated 60 patients (median age 66 years (Interquartile range [IQR] 59–75 with confirmed VGI between May 2013 and July 2017. The prevalence of antimicrobial pretreatment at the time of sampling was high [91%; median days of antibiotics 7 days (IQR 1–18]. We investigated 226 microbiological specimens. Thereof, 176 (78% were culture-negative and 50 (22% were culture-positive. There was a concordance of 70% (158/226 between conventional culture and broad-range PCR (sensitivity 58% (95% CI 43–72; specificity 74% (67–80%. Among the group of 176 culture-negative specimens, 46 specimens were broad-range PCR-positive resulting in identification of overall 69 species. Among the culture and

  4. An Evaluation Method for Team Competencies to Enhance Nuclear Safety Culture

    International Nuclear Information System (INIS)

    Hang, S. M.; Seong, P. H.; Kim, A. R.

    2016-01-01

    Safety culture has received attention in safety-critical industries, including nuclear power plants (NPPs), due to various prominent accidents such as concealment of a Station Blackout (SBO) of Kori NPP unit 1 in 2012, the Sewol ferry accident in 2014, and the Chernobyl accident in 1986. Analysis reports have pointed out that one of the major contributors to the cause of the accidents is ‘the lack of safety culture’. The term, nuclear safety culture, was firstly defined after the Chernobyl accident by the IAEA in INSAG report no. 4, as follows “Safety culture is that assembly of characteristics and attitudes in organizations and individuals which establishes that, as an overriding priority, nuclear plant safety issues receive the attention warranted their significance.” Afterwards, a wide consensus grew among researchers and nuclear-related organizations, that safety culture should be evaluated and managed in a certain manner. Consequently, each nuclear-related organization defined and developed their own safety culture definitions and assessment methods. However, none of these methods provides a way for an individual or a team to enhance the safety culture of an organization. Especially for a team, which is the smallest working unit in NPPs, team members easily overlook their required practices to improve nuclear safety culture. Therefore in this study, we suggested a method to estimate nuclear safety culture of a team, by approaching with the ‘competency’ point of view. The competency is commonly focused on individuals, and defined as, “underlying characteristics of an individual that are causally related to effective or superior performance in a job.” Similar to safety culture, the definition of competency focuses on characteristics and attitudes of individuals. Thus, we defined ‘safety culture competency’ as “underlying characteristics and outward attitudes of individuals that are causally related to a healthy and strong nuclear safety

  5. Rapid real-time PCR assay for culture and tissue identification of Geomyces destructans: the etiologic agent of bat geomycosis (white nose syndrome).

    Science.gov (United States)

    Chaturvedi, Sudha; Rudd, Robert J; Davis, April; Victor, Tanya R; Li, Xiaojiang; Appler, Kim A; Rajkumar, Sunanda S; Chaturvedi, Vishnu

    2011-10-01

    Geomyces destructans is the etiologic agent of bat geomycosis, commonly referred to as white nose syndrome (WNS). This infection has caused severe morbidity and mortality in little brown bats (Myotis lucifugus) and has also spread to other bat species with significant decline in the populations. Currently, G. destructans infection is identified by culture, ITS-PCR, and histopathology. We hypothesized that a real-time PCR assay would considerably improve detection of G. destructans in bats. The 100 bp sequence of the Alpha-L-Rhamnosidase gene was validated as a target for real-time PCR. The assay sensitivity was determined from serial dilution of DNA extracted from G. destructans conidia (5 × 10(-1)-5 × 10(7)), and the specificity was tested using DNA from 30 closely and distantly related fungi and 5 common bacterial pathogens. The real-time PCR assay was highly sensitive with detection limit of two G. destructans conidia per reaction at 40 PCR cycles. The assay was also highly specific as none of the other fungal or bacterial DNA cross-reacted in the real-time PCR assay. One hundred and forty-seven bat tissue samples, suspected of infection with G. destructans, were used to compare the real-time PCR assay to other methods employed for the detection of G. destructans. Real-time PCR was highly sensitive with 80 of 147 (55%) samples testing positive for G. destructans DNA. In comparison, histopathology examination revealed 64/147 (44%) positive samples. The internal transcribed spacer (ITS)-PCR yielded positive amplicon for G. destructans from 37 tissue samples (25%). The least sensitive assay was the fungal culture with only 17 tissue samples (12%) yielding G. destructans in culture. The data suggested that the real-time PCR assay is highly promising for rapid, sensitive, and specific identification of G. destructans. Further trials and inter-laboratory comparisons of this novel assay are recommended to improve the diagnosis of bat geomycosis.

  6. Isolation and propagation of mutations in dahlia by in vitro culture

    International Nuclear Information System (INIS)

    Asahira, T.; Yamagata, H.; Inagaki, M.; Osuga, S.

    1975-01-01

    The present study was undertaken to search for a successful method for in vitro culture of mutated tissues in dahlia. Preceding the objective, the features of induced mutations and the effects of cutting propagation in dahlia were investigated, and the tissues easily regenerating plantlets in vitro were searched following the examination of effective condition of medium. Induction of mutations: Tuberous roots of two cultivars, 'Kosei' and 'Sunlight', were irradiated with 1,000 - 2,000 R of X-rays. Chlorophyll and flower-color mutations were successfully induced in both cultivars, but the frequency differed with genotypic constitution. The maximum frequency was observed at leaves and shoots on or from the fourth to fifth nodes from the base of plant. The use of M 1 tuberous roots seemed a way for isolating mutations though not so much efficient. Tissue culture: In vitro cultured basal parts of ray florets, exactly the ovary, differentiated shoots. No shoot formation occurred in receptacle and leaf cultures, while roots were differentiated in leaf culture. Supplements of auxin and adenine to the medium besides cytokinin appeared to be necessary for inducing shoots. It is a serious problem in the tissue culture of dahlia that a large number of explants are endogenously comtaminated with bacteria. Taking into consideration low rates of surviving and regenerating explants, it seems difficult at present for dahlia to conclude whether or not the tissue culture may become efficient in mutation breeding as compared with cutting propagation. (author)

  7. Effect of MELT method on thoracolumbar connective tissue: The full study.

    Science.gov (United States)

    Sanjana, Faria; Chaudhry, Hans; Findley, Thomas

    2017-01-01

    Altered connective tissue structure has been identified in adults with chronic low back pain (LBP). A self-care treatment for managing LBP is the MELT method. The MELT method is a hands-off, self-treatment that is said to alleviate chronic pain, release tension and restore mobility, utilizing specialized soft treatments balls, soft body roller and techniques mimicking manual therapy. The objective of this study was to determine whether thickness of thoracolumbar connective tissue and biomechanical and viscoelastic properties of myofascial tissue in the low back region change in subjects with chronic LBP as a result of MELT. This study was designed using a quasi experimental pre-post- design that analyzed data from subjects who performed MELT. Using ultrasound imaging and an algorithm developed in MATLAB, thickness of thoracolumbar connective tissue was analyzed in 22 subjects. A hand-held digital palpation device, called the MyotonPRO, was used to assess biomechanical properties such as stiffness, elasticity, tone and mechanical stress relaxation time of the thoracolumbar myofascial tissue. A forward bending test assessing flexibility and pain scale was added to see if MELT affected subjects with chronic LBP. A significant decrease in connective tissue thickness and pain was observed in participants. Significant increase in flexibility was also recorded. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Flowering of Woody Bamboo in Tissue Culture Systems

    Directory of Open Access Journals (Sweden)

    Jin-Ling Yuan

    2017-09-01

    Full Text Available Flowering and subsequent seed set are not only normal activities in the life of most plants, but constitute the very reason for their existence. Woody bamboos can take a long time to flower, even over 100 years. This makes it difficult to breed bamboo, since flowering time cannot be predicted and passing through each generation takes too long. Another unique characteristic of woody bamboo is that a bamboo stand will often flower synchronously, both disrupting the supply chain within the bamboo industry and affecting local ecology. Therefore, an understanding of the mechanism that initiates bamboo flowering is important not only for biology research, but also for the bamboo industry. Induction of flowering in vitro is an effective way to both shorten the flowering period and control the flowering time, and has been shown for several species of bamboo. The use of controlled tissue culture systems allows investigation into the mechanism of bamboo flowering and facilitates selective breeding. Here, after a brief introduction of flowering in bamboo, we review the research on in vitro flowering of bamboo, including our current understanding of the effects of plant growth regulators and medium components on flower induction and how in vitro bamboo flowers can be used in research.

  9. Use of fibroblast growth factor 2 for expansion of chondrocytes and tissue engineering

    Science.gov (United States)

    Vunjak-Novakovic, Gordana (Inventor); Martin, Ivan (Inventor); Freed, Lisa E. (Inventor); Langer, Robert (Inventor)

    2003-01-01

    The present invention provides an improved method for expanding cells for use in tissue engineering. In particular the method provides specific biochemical factors to supplement cell culture medium during the expansion process in order to reproduce events occurring during embryonic development with the goal of regenerating tissue equivalents that resemble natural tissues both structurally and functionally. These specific biochemical factors improve proliferation of the cells and are capable of de-differentiation mature cells isolated from tissue so that the differentiation potential of the cells is preserved. The bioactive molecules also maintain the responsiveness of the cells to other bioactive molecules. Specifically, the invention provides methods for expanding chondrocytes in the presence of fibroblast growth factor 2 for use in regeneration of cartilage tissue.

  10. Numerical study of water diffusion in biological tissues using an improved finite difference method

    International Nuclear Information System (INIS)

    Xu Junzhong; Does, Mark D; Gore, John C

    2007-01-01

    An improved finite difference (FD) method has been developed in order to calculate the behaviour of the nuclear magnetic resonance signal variations caused by water diffusion in biological tissues more accurately and efficiently. The algorithm converts the conventional image-based finite difference method into a convenient matrix-based approach and includes a revised periodic boundary condition which eliminates the edge effects caused by artificial boundaries in conventional FD methods. Simulated results for some modelled tissues are consistent with analytical solutions for commonly used diffusion-weighted pulse sequences, whereas the improved FD method shows improved efficiency and accuracy. A tightly coupled parallel computing approach was also developed to implement the FD methods to enable large-scale simulations of realistic biological tissues. The potential applications of the improved FD method for understanding diffusion in tissues are also discussed. (note)

  11. Organotypic hippocampal slice culture from the adult mouse brain: a versatile tool for translational neuropsychopharmacology.

    Science.gov (United States)

    Kim, Hyunjeong; Kim, Eosu; Park, Minsun; Lee, Eun; Namkoong, Kee

    2013-03-05

    One of the most significant barriers towards translational neuropsychiatry would be an unavailability of living brain tissues. Although organotypic brain tissue culture could be a useful alternative enabling observation of temporal changes induced by various drugs in living brain tissues, a proper method to establish a stable organotypic brain slice culture system using adult (rather than neonatal) hippocampus has been still elusive. In this study, we evaluated our simple method using the serum-free culture medium for successful adult organotypic hippocampal slice culture. Several tens of hippocampal slices from a single adult mouse (3-5 months old) were cultured in serum-free versus serum-containing conventional culture medium for 30 days and underwent various experiments to validate the effects of the existence of serum in the culture medium. Neither the excessive regression of neuronal viability nor metabolic deficiency was observed in the serum-free medium culture in contrast to the serum-containing medium culture. Despite such viability, newly generated immature neurons were scarcely detected in the serum-free culture, suggesting that the original neurons in the brain slice persist rather than being replaced by neurogenesis. Key structural features of in vivo neural tissue constituting astrocytes, neural processes, and pre- and post-synapses were also well preserved in the serum-free culture. In conclusion, using the serum-free culture medium, the adult hippocampal slice culture system will serve as a promising ex vivo tool for various fields of neuroscience, especially for studies on aging-related neuropsychiatric disorders or for high throughput screening of potential agents working against such disorders. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Tissue non-specific alkaline phosphatase production by human dental pulp stromal cells is enhanced by high density cell culture.

    Science.gov (United States)

    Tomlinson, Matthew J; Dennis, Caitriona; Yang, Xuebin B; Kirkham, Jennifer

    2015-08-01

    The cell surface hydrolase tissue non-specific alkaline phosphatase (TNAP) (also known as MSCA-1) is used to identify a sub-population of bone marrow stromal cells (BMSCs) with high mineralising potential and is found on subsets of cells within the dental pulp. We aim to determine whether TNAP is co-expressed by human dental pulp stromal cells (hDPSCs) alongside a range of BMSC markers, whether this is an active form of the enzyme and the effects of culture duration and cell density on its expression. Cells from primary dental pulp and culture expanded hDPSCs expressed TNAP. Subsequent analyses revealed persistent TNAP expression and co-expression with BMSC markers such as CD73 and CD90. Flow cytometry and biochemical assays showed that increased culture durations and cell densities enhanced TNAP expression by hDPSCs. Arresting the hDPSC cell cycle also increased TNAP expression. These data confirm that TNAP is co-expressed by hDPSCs together with other BMSC markers and show that cell density affects TNAP expression levels. We conclude that TNAP is a potentially useful marker for hDPSC selection especially for uses in mineralised tissue regenerative therapies.

  13. Hydrogel microfabrication technology toward three dimensional tissue engineering

    Directory of Open Access Journals (Sweden)

    Fumiki Yanagawa

    2016-03-01

    Full Text Available The development of biologically relevant three-dimensional (3D tissue constructs is essential for the alternative methods of organ transplantation in regenerative medicine, as well as the development of improved drug discovery assays. Recent technological advances in hydrogel microfabrication, such as micromolding, 3D bioprinting, photolithography, and stereolithography, have led to the production of 3D tissue constructs that exhibit biological functions with precise 3D microstructures. Furthermore, microfluidics technology has enabled the development of the perfusion culture of 3D tissue constructs with vascular networks. In this review, we present these hydrogel microfabrication technologies for the in vitro reconstruction and cultivation of 3D tissues. Additionally, we discuss current challenges and future perspectives of 3D tissue engineering.

  14. Comparative in silico profiling of epigenetic modifiers in human tissues.

    Science.gov (United States)

    Son, Mi-Young; Jung, Cho-Rok; Kim, Dae-Soo; Cho, Hyun-Soo

    2018-04-06

    The technology of tissue differentiation from human pluripotent stem cells has attracted attention as a useful resource for regenerative medicine, disease modeling and drug development. Recent studies have suggested various key factors and specific culture methods to improve the successful tissue differentiation and efficient generation of human induced pluripotent stem cells. Among these methods, epigenetic regulation and epigenetic signatures are regarded as an important hurdle to overcome during reprogramming and differentiation. Thus, in this study, we developed an in silico epigenetic panel and performed a comparative analysis of epigenetic modifiers in the RNA-seq results of 32 human tissues. We demonstrated that an in silico epigenetic panel can identify epigenetic modifiers in order to overcome epigenetic barriers to tissue-specific differentiation.

  15. Growing Arabidopsis in vitro: cell suspensions, in vitro culture, and regeneration.

    Science.gov (United States)

    Barkla, Bronwyn J; Vera-Estrella, Rosario; Pantoja, Omar

    2014-01-01

    An understanding of basic methods in Arabidopsis tissue culture is beneficial for any laboratory working on this model plant. Tissue culture refers to the aseptic growth of cells, organs, or plants in a controlled environment, in which physical, nutrient, and hormonal conditions can all be easily manipulated and monitored. The methodology facilitates the production of a large number of plants that are genetically identical over a relatively short growth period. Techniques, including callus production, cell suspension cultures, and plant regeneration, are all indispensable tools for the study of cellular biochemical and molecular processes. Plant regeneration is a key technology for successful stable plant transformation, while cell suspension cultures can be exploited for metabolite profiling and mining. In this chapter we report methods for the successful and highly efficient in vitro regeneration of plants and production of stable cell suspension lines from leaf explants of both Arabidopsis thaliana and Arabidopsis halleri.

  16. Studies on salt and drought tolerance of lavender using tissue culture and gamma rays

    International Nuclear Information System (INIS)

    Essam, K.E.; El-Sharnoby, M.E.

    2005-01-01

    The present study was carried out to investigate the propagation and chemical composition of Lavandula spica using different cytokinins (BA, Ki and 2ip) on MS medium,, besides medium strength. Also, GA3 concentrations, auxin types, gamma irradiation (0, 2, 4, 6 and 8 Krad), different concentrations of mannitol (10, 20, 40, 80 and 100 mg/l) as well as different salinity concentrations of CaCl 2 or NaCl or both at levels 250, 500, 750 and 1000 ppm were used in the study. Maximum proliferation parameter was produced on MS medium supplemented with 1 mg/l BA than other cytokinin types. Growing the explant of Lavandula. spica on MS medium, containing 3 mg/l BA, gave the highest proliferation, growth and greening parameters. However, using MS medium at half strength supplemented with 3 mg/l BA resulted in significant increase in both shoot elongation and greening parameters as compared with the other medium strengths, while 4 mg/l GA induced the best shoot elongation. Adding 1 mg/l IBA enhanced rooting and also the obtained results showed that increasing gamma irradiation decreased growth and proliferation parameters. Moreover, increasing drought stress induced an adverse effect on tissue culture parameter while some chemical analysis parameters were increased to maximum degree of their tolerance to drought stress. Also, using different salinity treatment by NaCl, CaCl 2 and their combinations showed adverse effects on tissue culture parameters chemical composition

  17. A high-resolution method for the localization of proanthocyanidins in plant tissues

    Directory of Open Access Journals (Sweden)

    Panter Stephen

    2011-05-01

    Full Text Available Abstract Background Histochemical staining of plant tissues with 4-dimethylaminocinnamaldehyde (DMACA or vanillin-HCl is widely used to characterize spatial patterns of proanthocyanidin accumulation in plant tissues. These methods are limited in their ability to allow high-resolution imaging of proanthocyanidin deposits. Results Tissue embedding techniques were used in combination with DMACA staining to analyze the accumulation of proanthocyanidins in Lotus corniculatus (L. and Trifolium repens (L. tissues. Embedding of plant tissues in LR White or paraffin matrices, with or without DMACA staining, preserved the physical integrity of the plant tissues, allowing high-resolution imaging that facilitated cell-specific localization of proanthocyanidins. A brown coloration was seen in proanthocyanidin-producing cells when plant tissues were embedded without DMACA staining and this was likely to have been due to non-enzymatic oxidation of proanthocyanidins and the formation of colored semiquinones and quinones. Conclusions This paper presents a simple, high-resolution method for analysis of proanthocyanidin accumulation in organs, tissues and cells of two plant species with different patterns of proanthocyanidin accumulation, namely Lotus corniculatus (birdsfoot trefoil and Trifolium repens (white clover. This technique was used to characterize cell type-specific patterns of proanthocyanidin accumulation in white clover flowers at different stages of development.

  18. Are specific gene expressions of extracellular matrix and nucleus pulposus affected by primary cell cultures prepared from intact or degenerative intervertebral disc tissues?

    Science.gov (United States)

    Karaarslan, Numan; Yilmaz, Ibrahim; Ozbek, Hanefi; Sirin Yasar, Duygu; Kaplan, Necati; Akyuva, Yener; Gonultas, Aylin; Ates, Ozkan

    2018-01-22

    In this scientific research project, the researchers aimed to determine the gene expression patterns of nucleus pulposus (NP) in cell cultures obtained from degenerated or intact tissues. Whereas 12 of the cases were diagnosed with lumbar disc hernia and had undergone lumbar microdiscectomy, 12 cases had undergone traumatic intervertebral discectomy and corpectomy, along with discectomy after spinal trauma. NP-specific markers and gene expressions of the reagents of the extracellular matrix in the experimental setup were tested at the 0th, 24th, and 48th hours by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Visual evaluations were simultaneously made in all samples using invert and fluorescence microscopy. Vitality and proliferation analyses were evaluated by UV spectrophotometer. As a method of statistical evaluation, Spearman was used for categorical variants, and the Pearson correlation was used for variants with numerical and plain distribution. No association was found either between the tissue type and times (r=0.000; p=1.000) or between the region that the tissue was obtained from and hypoxia transcription factor-1 alpha (HIF-1α) gene expression (r=0.098; p=0.245). There was no correlation between cell proliferation and chondroadherin (CHAD) expression or between type II collagen (COL2A1) and CHAD gene expressions. It was found that CHAD and HIF-1α gene expressions and HIF-1α and COL2A1 gene expressions affected cell proliferation. Cell culture setups are of paramount importance because they may influence the pattern of changes in the gene expressions of the cells used in these setups.

  19. Establishment of primary bovine intestinal epithelial cell culture and clone method.

    Science.gov (United States)

    Zhan, Kang; Lin, Miao; Liu, Ming-Mei; Sui, Yang-Nan; Zhao, Guo-Qi

    2017-01-01

    The aim of this study was to establish bovine intestinal epithelial cell (BIEC) line and provide a novel clone cell method. Although various strategies of bovine cell culture and clone techniques have been reported, these methods remain not established. Here, we culture successfully primary BIECs and establish a novel clone cell method. Our result showed that BIECs could be successfully cultured and passaged about generation 5. These cellular aggregates and clusters were adherent loosely at day 2 of culture. Cell aggregates and clusters start to proliferate after approximately 4 d. The BIECs showed positive reaction against cytokeratin 18, E-cadherin, and characteristics of epithelial-like morphology. In addition, the fatty acid-binding proteins (FABPs), villin, and intestinal peptidase (IP) band were positive in BIECs. Our results suggest that the establishment of culturing and clone BIEC methods will apply to isolate and clone other primary cells. These BIECs could therefore contribute to the study of bovine intestinal nutrient absorption and regulation, immune regulation, and the pathogenesis of the bovine intestinal disease, which will provide intestinal cell model in vitro.

  20. Specific accumulation of 18F-deoxyglucose in three-dimensional long-term cultures of human and rodent brain tissue

    International Nuclear Information System (INIS)

    Hocke, C.; Prante, O.; Kuwert, T.; Bluemcke, I.; Jeske, I.; Romstoeck, J.; Stefan, H.

    2007-01-01

    Aim: Organotypic slice cultures (OSC) of human brain specimens represent an intriguing experimental model for translational studies addressing, e.g., stem cell transplantation in neurodegenerative diseases or targeting invasion by malignant glioma ex vivo. However, long-term viability and phenomena of structural reorganization of human OSC remain to be further characterized. Here, we report the use of 18 F-deoxyglucose (FDG) for evaluating the viability of brain slice preparations obtained either from postnatal rats or human hippocampal specimens. Methods: Anatomically well preserved human hippocampi obtained from epilepsy surgery and rat hippocampus slice cultures obtained from six day old Wistar rats were dissected into horizontal slices. The slices were incubated with FDG in phosphate buffered saline up to 1 h, either with or without supplementation of glucose at a concentration of 2.5 mg/ml. Radioactivity within the medium or slice cultures was measured using a gamma-counter. In addition, distribution of radioactivity was autoradiographically visualized and quantified as counts per mm 2 . Results: In rat hippocampal slices, FDG accumulated with 1 300 000 ± 68 000 counts/mm 2 , whereas the incorporation of the radioactive label in human slices was in the order of 1 500 000 ± 370 000 counts/mm 2 . The elevation of glucose concentration within the medium led to a significant three-fold decrease of FDG accumulation in rat slices and to a 2.4-fold decrease in human specimens. Conclusions: FDG accumulated in organotypic brain cultures of human or rodent origin. FDG is thus suited to investigate the viability of OSC. Furthermore, these preparations open new ways to study the factors governing cerebral FDG uptake in brain tissue ex vivo. (orig.)

  1. Comparison of the lysis centrifugation method with the conventional blood culture method in cases of sepsis in a tertiary care hospital.

    Science.gov (United States)

    Parikh, Harshal R; De, Anuradha S; Baveja, Sujata M

    2012-07-01

    Physicians and microbiologists have long recognized that the presence of living microorganisms in the blood of a patient carries with it considerable morbidity and mortality. Hence, blood cultures have become critically important and frequently performed test in clinical microbiology laboratories for diagnosis of sepsis. To compare the conventional blood culture method with the lysis centrifugation method in cases of sepsis. Two hundred nonduplicate blood cultures from cases of sepsis were analyzed using two blood culture methods concurrently for recovery of bacteria from patients diagnosed clinically with sepsis - the conventional blood culture method using trypticase soy broth and the lysis centrifugation method using saponin by centrifuging at 3000 g for 30 minutes. Overall bacteria recovered from 200 blood cultures were 17.5%. The conventional blood culture method had a higher yield of organisms, especially Gram positive cocci. The lysis centrifugation method was comparable with the former method with respect to Gram negative bacilli. The sensitivity of lysis centrifugation method in comparison to conventional blood culture method was 49.75% in this study, specificity was 98.21% and diagnostic accuracy was 89.5%. In almost every instance, the time required for detection of the growth was earlier by lysis centrifugation method, which was statistically significant. Contamination by lysis centrifugation was minimal, while that by conventional method was high. Time to growth by the lysis centrifugation method was highly significant (P value 0.000) as compared to time to growth by the conventional blood culture method. For the diagnosis of sepsis, combination of the lysis centrifugation method and the conventional blood culture method with trypticase soy broth or biphasic media is advocable, in order to achieve faster recovery and a better yield of microorganisms.

  2. Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods.

    Science.gov (United States)

    Edagawa, Akiko; Kimura, Akio; Kawabuchi-Kurata, Takako; Adachi, Shinichi; Furuhata, Katsunori; Miyamoto, Hiroshi

    2015-10-19

    We investigated Legionella contamination in bath water samples, collected from 68 bathing facilities in Japan, by culture, culture with amoebic co-culture, real-time quantitative PCR (qPCR), and real-time qPCR with amoebic co-culture. Using the conventional culture method, Legionella pneumophila was detected in 11 samples (11/68, 16.2%). Contrary to our expectation, the culture method with the amoebic co-culture technique did not increase the detection rate of Legionella (4/68, 5.9%). In contrast, a combination of the amoebic co-culture technique followed by qPCR successfully increased the detection rate (57/68, 83.8%) compared with real-time qPCR alone (46/68, 67.6%). Using real-time qPCR after culture with amoebic co-culture, more than 10-fold higher bacterial numbers were observed in 30 samples (30/68, 44.1%) compared with the same samples without co-culture. On the other hand, higher bacterial numbers were not observed after propagation by amoebae in 32 samples (32/68, 47.1%). Legionella was not detected in the remaining six samples (6/68, 8.8%), irrespective of the method. These results suggest that application of the amoebic co-culture technique prior to real-time qPCR may be useful for the sensitive detection of Legionella from bath water samples. Furthermore, a combination of amoebic co-culture and real-time qPCR might be useful to detect viable and virulent Legionella because their ability to invade and multiply within free-living amoebae is considered to correlate with their pathogenicity for humans. This is the first report evaluating the efficacy of the amoebic co-culture technique for detecting Legionella in bath water samples.

  3. The comparison of two methods to obtain human oral keratinocytes in primary culture

    International Nuclear Information System (INIS)

    Klingbeil, Maria Fatima Guarizo

    2006-01-01

    The therapeutic procedures frequently used in oral treatments for the pathological diseases are surgical, resulting in failures of the mucosal continuity.The possibility to obtain transplantable oral epithelia from an in vitro cell culture opens new utilization perspectives not only to where it comes from, but also as a reconstructive material for other parts of the human body, such as: urethra, epithelia corneo-limbal, cornea, ocular surface. Many researchers still use controversial methods for obtaining cells. It was therefore evaluated and compared the efficiency in both methods: enzymatic and direct explant to obtain oral keratinocytes from human oral mucosa. Fragments of intra oral epithelial tissues from healthy human subjects, undergoing dental surgeries, were donated to the research project. The keratinocytes were cultivated over a feeder-layer from a previously irradiated 3T3 Swiss albino fibroblasts. In this study it was compared the time needed in the cell obtention, the best cell amount between both methods, the life-span, the cell capacity to form an in vitro epithelia and its morphologic structure. The results in the assessment of both methods have shown the possibility to obtain keratinocytes from a small oral fragment, but at the same time we may verify the advantages and peculiar restrictions for each one of both analyzed methods. (author)

  4. Dentine microhardness after different methods for detection and removal of carious dentine tissue

    Directory of Open Access Journals (Sweden)

    Fernanda Brandão Mollica

    2012-08-01

    Full Text Available There are several methods for identifying carious dentinal tissue aiming to avoid removal of healthy dentinal tissue. OBJECTIVES: The purpose of this study was to test different methods for the detection of carious dentinal tissue regarding the amount of carious tissue removed and the remaining dentin microhardness after caries removal. MATERIAL AND METHODS: The dentin surfaces of 20 bovine teeth were exposed and half of the surface was protected with nail polish. Cariogenic challenge was performed by immersion in a demineralizing solution for 14 days. After transverse cross-section of the crown, the specimens were divided into four groups (n=10, according to the method used to identify and remove the carious tissue: "Papacárie", Caries-detector dye, DIAGNOdent and Tactile method. After caries removal, the cross-sectional surface was included in acrylic resin and polished. In a microhardness tester, the removed dentin thickness and the Vickers microhardness of the following regions were evaluated: remaining dentin after caries removal and superficial and deep healthy dentin. RESULTS: ANOVA and Tukey's test (α=0.05 were performed, except for DIAGNOdent, which did not detect the presence of caries. Results for removed dentin thickness were: "Papacárie" (424.7±105.0; a, Caries-detector dye (370.5±78.3; ab, Tactile method (322.8±51.5; bc. Results for the remaining dentin microhardness were: "Papacárie" (42.2±10.5; bc, Caries-detector dye (44.6±11.8; abc, Tactile method (24.3±9.0; d. CONCLUSIONS: DIAGNOdent did not detect the presence of carious tissue; Tactile method and "Papacárie" resulted in the least and the most dentinal thickness removal, respectively; Tactile method differed significantly from "Papacárie" and Caries-detector dye in terms of the remaining dentin microhardness, and Tactile method was the one which presented the lowest microhardness values.

  5. Coumarins and alkaloids in shoot culture of Ruta graveolens L.

    Directory of Open Access Journals (Sweden)

    Halina Ekiert

    2014-01-01

    Full Text Available A shoot culture of Ruta graveolens L. (Rutaceae was maintained in the stationary liquid phase. From the cultured shoots seven compounds were isolated and identified as psoralen, bergapten, xanthotoxin, isopimpinellin (linear furanocoumarins, rutamarin (linear dihydrofuranocoumarin, kokusaginine and skimmianine (furanoquinoline alkaloids by spectral methods. The compounds are known as secondary metabolites of the intact plant, as well as its cell and tissue cultures.

  6. Engineering systems for the generation of patterned co-cultures for controlling cell-cell interactions.

    Science.gov (United States)

    Kaji, Hirokazu; Camci-Unal, Gulden; Langer, Robert; Khademhosseini, Ali

    2011-03-01

    Inside the body, cells lie in direct contact or in close proximity to other cell types in a tightly controlled architecture that often regulates the resulting tissue function. Therefore, tissue engineering constructs that aim to reproduce the architecture and the geometry of tissues will benefit from methods of controlling cell-cell interactions with microscale resolution. We discuss the use of microfabrication technologies for generating patterned co-cultures. In addition, we categorize patterned co-culture systems by cell type and discuss the implications of regulating cell-cell interactions in the resulting biological function of the tissues. Patterned co-cultures are a useful tool for fabricating tissue engineered constructs and for studying cell-cell interactions in vitro, because they can be used to control the degree of homotypic and heterotypic cell-cell contact. In addition, this approach can be manipulated to elucidate important factors involved in cell-matrix interactions. Patterned co-culture strategies hold significant potential to develop biomimetic structures for tissue engineering. It is expected that they would create opportunities to develop artificial tissues in the future. This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine. 2010 Elsevier B.V. All rights reserved.

  7. Cloning crops in a CELSS via tissue culture: Prospects and problems

    Science.gov (United States)

    Carman, John G.; Hess, J. Richard

    1990-01-01

    Micropropagation is currently used to clone fruits, nuts, and vegetables and involves controlling the outgrowth in vitro of basal, axillary, or adventitious buds. Following clonal multiplication, shoots are divided and rooted. This process has greatly reduced space and energy requirements in greenhouses and field nurseries and has increased multiplication rates by greater than 20 fold for some vegetatively propagated crops and breeding lines. Cereal and legume crops can also be cloned by tissue culture through somatic embryogenesis. Somatic embryos can be used to produce 'synthetic seed', which can tolerate desiccation and germinate upon rehydration. Synthetic seed of hybrid wheat, rice, soybean and other crops could be produced in a controlled ecological life support system. Thus, yield advantages of hybreds over inbreds (10 to 20 percent) could be exploited without having to provide additional facilities and energy for parental-line and hybrid seed nurseries.

  8. Fabrication and characterization of PCL/gelatin composite nanofibrous scaffold for tissue engineering applications by electrospinning method

    International Nuclear Information System (INIS)

    Gautam, Sneh; Dinda, Amit Kumar; Mishra, Narayan Chandra

    2013-01-01

    In the present study, composite nanofibrous tissue engineering-scaffold consisting of polycaprolactone and gelatin, was fabricated by electrospinning method, using a new cost-effective solvent mixture: chloroform/methanol for polycaprolactone (PCL) and acetic acid for gelatin. The morphology of the nanofibrous scaffold was investigated by using field emission scanning electron microscopy (FE-SEM) which clearly indicates that the morphology of nanofibers was influenced by the weight ratio of PCL to gelatin in the solution. Uniform fibers were produced only when the weight ratio of PCL/gelatin is sufficiently high (10:1). The scaffold was further characterized by Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric (TG) analysis, and X-ray diffraction (XRD). FT-IR and TG analysis indicated some interactions between PCL and gelatin molecules within the scaffold, while XRD results demonstrated crystalline nature of PCL/gelatin composite scaffold. Cytotoxicity effect of scaffold on L929 mouse fibroblast cells was evaluated by MTT assay and cell proliferation on the scaffold was confirmed by DNA quantification. Positive results of MTT assay and DNA quantification L929 mouse fibroblast cells indicated that the scaffold made from the combination of natural polymer (gelatin) and synthetic polymer (PCL) may serve as a good candidate for tissue engineering applications. - Highlights: ► PCL/Gelatin scaffold was successfully fabricated by electrospinning method. ► PCL in CHCl 3 /CH 3 OH and gelatin in acetic acid: a novel polymer-solvent system. ► The morphology of nanofibers was influenced by the weight ratio of PCL/gelatin. ► Chemical interactions between PCL and gelatin molecules enhanced cell growth. ► Cell culture studies indicate the suitability of scaffold for tissue regeneration

  9. Towards modular bone tissue engineering using Ti-Co-doped phosphate glass microspheres: cytocompatibility and dynamic culture studies.

    Science.gov (United States)

    Peticone, Carlotta; De Silva Thompson, David; Owens, Gareth J; Kim, Hae-Won; Micheletti, Martina; Knowles, Jonathan C; Wall, Ivan

    2017-09-01

    The production of large quantities of functional vascularized bone tissue ex vivo still represent an unmet clinical challenge. Microcarriers offer a potential solution to scalable manufacture of bone tissue due to their high surface area-to-volume ratio and the capacity to be assembled using a modular approach. Microcarriers made of phosphate bioactive glass doped with titanium dioxide have been previously shown to enhance proliferation of osteoblast progenitors and maturation towards functional osteoblasts. Furthemore, doping with cobalt appears to mimic hypoxic conditions that have a key role in promoting angiogenesis. This characteristic could be exploited to meet the clinical requirement of producing vascularized units of bone tissue. In the current study, the human osteosarcoma cell line MG-63 was cultured on phosphate glass microspheres doped with 5% mol titanium dioxide and different concentrations of cobalt oxide (0%, 2% and 5% mol), under static and dynamic conditions (150 and 300 rpm on an orbital shaker). Cell proliferation and the formation of aggregates of cells and microspheres were observed over a period of two weeks in all glass compositions, thus confirming the biocompatibility of the substrate and the suitability of this system for the formation of compact micro-units of tissue. At the concentrations tested, cobalt was not found to be cytotoxic and did not alter cell metabolism. On the other hand, the dynamic environment played a key role, with moderate agitation having a positive effect on cell proliferation while higher agitation resulting in impaired cell growth. Finally, in static culture assays, the capacity of cobalt doping to induce vascular endothelial growth factor (VEGF) upregulation by osteoblastic cells was observed, but was not found to increase linearly with cobalt oxide content. In conclusion, Ti-Co phosphate glasses were found to support osteoblastic cell growth and aggregate formation that is a necessary precursor to tissue

  10. Method for increasing nuclear magnetic resonance signals in living biological tissue

    International Nuclear Information System (INIS)

    Krongrad, A.

    1995-01-01

    A method of enhancing a magnetic resonance comprising the steps of administering a quantity of a selected magnetic isotope to a living biological tissue at a concentration greater than the naturally occurring concentration of such isotope and detecting magnetic resonance signal from the administered magnetic isotope in the living biological tissue. (author)

  11. Qualitatively Monitoring Binding and Expression of the Transcription Factors Sp1 and NFI as a Useful Tool to Evaluate the Quality of Primary Cultured Epithelial Stem Cells in Tissue Reconstruction.

    Science.gov (United States)

    Le-Bel, Gaëtan; Ghio, Sergio Cortez; Larouche, Danielle; Germain, Lucie; Guérin, Sylvain L

    2018-05-27

    Electrophoretic mobility shift assays and Western blots are simple, efficient, and rapid methods to study DNA-protein interactions and protein expression, respectively. Primary cultures and subcultures of epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. The preservation of stem cells through the culture process is essential to produce high quality substitutes. However, the increase in the number of cell passages is associated with a decrease in their ability to proliferate until senescence is reached. This process is likely to be mediated by the altered expression of nuclear-located transcription factors such as Sp1 and NFI, whose expression has been documented to be required for cell adhesion, migration, and differentiation. In some of our recent studies, we observed a correlation between reconstructed tissues exhibiting poor histological and structural characteristics and a low expression of Sp1 in their constituting epithelial cells. Therefore, monitoring both the expression and DNA binding of these transcription factors in human skin and corneal epithelial cells is a useful tool for characterizing the quality of primary cultured epithelial cells.

  12. An ovine tracheal explant culture model for allergic airway inflammation

    Directory of Open Access Journals (Sweden)

    Abeynaike Latasha

    2010-08-01

    Full Text Available Abstract Background The airway epithelium is thought to play an important role in the pathogenesis of asthmatic disease. However, much of our understanding of airway epithelial cell function in asthma has been derived from in vitro studies that may not accurately reflect the interactive cellular and molecular pathways active between different tissue constituents in vivo. Methods Using a sheep model of allergic asthma, tracheal explants from normal sheep and allergic sheep exposed to house dust mite (HDM allergen were established to investigate airway mucosal responses ex vivo. Explants were cultured for up to 48 h and tissues were stained to identify apoptotic cells, goblet cells, mast cells and eosinophils. The release of cytokines (IL-1α, IL-6 and TNF-α by cultured tracheal explants, was assessed by ELISA. Results The general morphology and epithelial structure of the tracheal explants was well maintained in culture although evidence of advanced apoptosis within the mucosal layer was noted after culture for 48 h. The number of alcian blue/PAS positive mucus-secreting cells within the epithelial layer was reduced in all cultured explants compared with pre-cultured (0 h explants, but the loss of staining was most evident in allergic tissues. Mast cell and eosinophil numbers were elevated in the allergic tracheal tissues compared to naïve controls, and in the allergic tissues there was a significant decline in mast cells after 24 h culture in the presence or absence of HDM allergen. IL-6 was released by allergic tracheal explants in culture but was undetected in cultured control explants. Conclusions Sheep tracheal explants maintain characteristics of the airway mucosa that may not be replicated when studying isolated cell populations in vitro. There were key differences identified in explants from allergic compared to control airways and in their responses in culture for 24 h. Importantly, this study establishes the potential for the

  13. A new cell primo-culture method for freshwater benthic diatom communities

    OpenAIRE

    Debenest, Timothée; Silvestre, Jérôme; Coste, Michel; Delmas, François; Pinelli, Eric

    2009-01-01

    A new cell primo-culture method was developed for the benthic diatom community isolated from biofilm sampled in rivers. The approach comprised three steps: (1) scraping biofilm from river pebbles, (2) diatom isolation from biofilm, and (3) diatom community culture. With a view to designing a method able to stimulate the growth of diatoms, to limit the development of other microorganisms, and to maintain in culture a community similar to the original natural one, different factors were test...

  14. The effect of three culture methods on intensive culture system of pacific white shrimp ( Litopenaeus vannamei)

    Science.gov (United States)

    Ma, Zhen; Wan, Rong; Song, Xiefa; Gao, Lei

    2013-09-01

    Different culture methods may affect the intensive culture system of Pacific white shrimp ( Litopenaeus vannamei) regarding water quality and growth and economic performance. This study evaluated the potential effects of three culture methods through cultivation of juvenile shrimps under consistent tank management conditions for 84 d. The three methods involved shrimp cultivation in different tanks, i.e., outdoor tanks with cement bottom (mode-C), greenhouse tanks with cement bottom (mode-G) and outdoor tanks with mud-substrate (mode-M). Results showed that water temperature was significantly higher in mode-G than that in mode-C ( P shrimps. In the mid-late period, the average concentrations of TAN, NO2-N, DIP and COD were significantly lower in mode-M and mode-G compared with those in mode-C ( P shrimp weight among different treatments ( P > 0.05), mode-M had significantly higher shrimp yield, survival rate and feed conversion rate ( P < 0.05) than other modes. There were significant differences in revenue and net return among different treatments ( P < 0.05). These demonstrated that the treatments of mode-G and mode-M were conductive to the intensive culture system of L. vannamei.

  15. Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods

    Directory of Open Access Journals (Sweden)

    Akiko Edagawa

    2015-10-01

    Full Text Available We investigated Legionella contamination in bath water samples, collected from 68 bathing facilities in Japan, by culture, culture with amoebic co-culture, real-time quantitative PCR (qPCR, and real-time qPCR with amoebic co-culture. Using the conventional culture method, Legionella pneumophila was detected in 11 samples (11/68, 16.2%. Contrary to our expectation, the culture method with the amoebic co-culture technique did not increase the detection rate of Legionella (4/68, 5.9%. In contrast, a combination of the amoebic co-culture technique followed by qPCR successfully increased the detection rate (57/68, 83.8% compared with real-time qPCR alone (46/68, 67.6%. Using real-time qPCR after culture with amoebic co-culture, more than 10-fold higher bacterial numbers were observed in 30 samples (30/68, 44.1% compared with the same samples without co-culture. On the other hand, higher bacterial numbers were not observed after propagation by amoebae in 32 samples (32/68, 47.1%. Legionella was not detected in the remaining six samples (6/68, 8.8%, irrespective of the method. These results suggest that application of the amoebic co-culture technique prior to real-time qPCR may be useful for the sensitive detection of Legionella from bath water samples. Furthermore, a combination of amoebic co-culture and real-time qPCR might be useful to detect viable and virulent Legionella because their ability to invade and multiply within free-living amoebae is considered to correlate with their pathogenicity for humans. This is the first report evaluating the efficacy of the amoebic co-culture technique for detecting Legionella in bath water samples.

  16. Can we trust intraoperative culture results in nonunions?

    Science.gov (United States)

    Palmer, Michael P; Altman, Daniel T; Altman, Gregory T; Sewecke, Jeffrey J; Ehrlich, Garth D; Hu, Fen Z; Nistico, Laura; Melton-Kreft, Rachel; Gause, Trent M; Costerton, John W

    2014-07-01

    To identify the presence of bacterial biofilms in nonunions comparing molecular techniques (multiplex polymerase chain reaction and mass spectrometry, fluorescent in situ hybridization) with routine intraoperative cultures. Thirty-four patients with nonunions were scheduled for surgery and enrolled in this ongoing prospective study. Intraoperative specimens were collected from removed implants, surrounding tissue membrane, and local soft tissue followed by standard culture analysis, Ibis's second generation molecular diagnostics (Ibis Biosystems), and bacterial 16S rRNA-based fluorescence in situ hybridization (FISH). Confocal microscopy was used to visualize the tissue specimens reacted with the FISH probes, which were chosen based on the Ibis analysis. Thirty-four patient encounters were analyzed. Eight were diagnosed as infected nonunions by positive intraoperative culture results. Ibis confirmed the presence of bacteria in all 8 samples. Ibis identified bacteria in a total of 30 of 34 encounters, and these data were confirmed by FISH. Twenty-two of 30 Ibis-positive samples were culture-negative. Four samples were negative by all methods of analysis. No samples were positive by culture, but negative by molecular techniques. Our preliminary data indicate that molecular diagnostics are more sensitive for identifying bacteria than cultures in cases of bony nonunion. This is likely because of the inability of cultures to detect biofilms and bacteria previously exposed to antibiotic therapy. Diagnostic Level I. See Instructions for Authors for a complete description of levels of evidence.

  17. Developing 3D microstructures for tissue engineering

    DEFF Research Database (Denmark)

    Mohanty, Soumyaranjan

    casting process to generate various large scale tissue engineering constructs with single pore geometry with the desired mechanical stiffness and porosity. In addition, a new technique was developed to fa bricate dual-pore scaffolds for various tissue-engineering applications where 3D printing...... materials have been developed and tested for enhancing the differentiation of hiPSC-derived hepatocytes and fabricating biodegradable scaffolds for in-vivo tissue engineering applications. Along with various scaffolds fabrication methods we finally presented an optimized study of hepatic differentiation...... of hiPSC-derived DE cells cultured for 25 days in a 3D perfusion bioreactor system with an array of 16 small-scale tissue-bioreactors with integrated dual-pore pore scaffolds and flow rates. Hepatic differentiation and functionality of hiPSC-derived hepatocytes were successfully assessed and compared...

  18. Comparison of multiple assays for detecting human antibodies directed against antigens on normal and malignant tissue culture cells

    International Nuclear Information System (INIS)

    Rosenberg, S.A.; Schwarz, S.; Anding, H.; Hyatt, C.; Williams, G.M.; Johns Hopkins Univ., Baltimore, Md.

    1977-01-01

    Four separate assays of human antibody reactivity to four separate normal and malignant human tissue culture cells lines from two patients have been evaluated using a single highly-reactive allogeneic serum. The visual end-point cytolysis assay and the chromium-51 release assay were equally sensitive in measuring complement mediated antibody cytotoxicity and both were far more sensitive than a trypan blue dye exclusion assay. The assay of antibody reactivity by hemadsorption technique was about 10 times more sensitive than any of the cytotoxicity assays. This latter assay measures only IgG antibody however. These assays showed that cell lines from different patients may differ greatly in 'reactivity' to an allogeneic serum and emphasized the importance of utilizing tumor and normal cells from the same patient when using tissue culture cells to search for tumor specific reactivity. These observations emphasize the importance of utilizing multiple assays against paired normal and malignant cells from the same patient to be certain of the specificity and magnitude of the measured antibody

  19. Characteristics of ovulation method acceptors: a cross-cultural assessment.

    Science.gov (United States)

    Klaus, H; Labbok, M; Barker, D

    1988-01-01

    Five programs of instruction in the ovulation method (OM) in diverse geographic and cultural settings are described, and characteristics of approximately 200 consecutive OM acceptors in each program are examined. Major findings include: the religious background and family size of acceptors are variable, as is the level of previous contraceptive use. Acceptors are drawn from a wide range of socioeconomic and religious backgrounds; however, family planning intention was similarly distributed in all five countries. In sum, the ovulation method is accepted by persons from a variety of backgrounds within and between cultural setting.

  20. Improvement of potato tolerance to salinity using tissue culture techniques and irradiation with in vitro selection

    International Nuclear Information System (INIS)

    Al-Safadi, B.; Arabi, M. I. E.

    2006-01-01

    A mutation breeding program was conducted to improve potato (Solanum tuberosum) tolerance to salinity. In vitro cultured explants from potato cvs. Draga, Diamant, Spunta were irradiated with gamma doses 25, 30, and 35 Gy. Mutants were isolated to get rid of chimeral tissues and subsequently propagated for in vitro and pot selection pressure. Cultivar Sponta produced the highest number of tolerant plants (4) and only one plant was obtained from Diamant. (authors)

  1. Variation in tissue outcome of ovine and human engineered heart valve constructs : relevance for tissue engineering

    NARCIS (Netherlands)

    Geemen, van D.; Driessen - Mol, A.; Grootzwagers, L.G.M.; Soekhradj - Soechit, R.S.; Riem Vis, P.W.; Baaijens, F.P.T.; Bouten, C.V.C.

    AIM: Clinical application of tissue engineered heart valves requires precise control of the tissue culture process to predict tissue composition and mechanical properties prior to implantation, and to understand the variation in tissue outcome. To this end we investigated cellular phenotype and

  2. Methods for quantifying adipose tissue insulin resistance in overweight/obese humans.

    Science.gov (United States)

    Ter Horst, K W; van Galen, K A; Gilijamse, P W; Hartstra, A V; de Groot, P F; van der Valk, F M; Ackermans, M T; Nieuwdorp, M; Romijn, J A; Serlie, M J

    2017-08-01

    Insulin resistance of adipose tissue is an important feature of obesity-related metabolic disease. However, assessment of lipolysis in humans requires labor-intensive and expensive methods, and there is limited validation of simplified measurement methods. We aimed to validate simplified methods for the quantification of adipose tissue insulin resistance against the assessment of insulin sensitivity of lipolysis suppression during hyperinsulinemic-euglycemic clamp studies. We assessed the insulin-mediated suppression of lipolysis by tracer-dilution of [1,1,2,3,3- 2 H 5 ]glycerol during hyperinsulinemic-euglycemic clamp studies in 125 overweight or obese adults (85 men, 40 women; age 50±11 years; body mass index 38±7 kg m -2 ). Seven indices of adipose tissue insulin resistance were validated against the reference measurement method. Low-dose insulin infusion resulted in suppression of the glycerol rate of appearance ranging from 4% (most resistant) to 85% (most sensitive), indicating a good range of adipose tissue insulin sensitivity in the study population. The reference method correlated with (1) insulin-mediated suppression of plasma glycerol concentrations (r=0.960, PInsulin Resistance (Adipo-IR) index (fasting plasma insulin-NEFA product; r=-0.526, Pinsulin-glycerol product (r=-0.467, PInsulin Resistance Index (fasting plasma insulin-basal lipolysis product; r=0.460, PInsulin Sensitivity Check Index (QUICKI)-NEFA index (r=0.621, Pinsulin resistance (area under the curve ⩾0.801, Pinsulin sensitivity (that is, the antilipolytic action of insulin) can be reliably quantified in overweight and obese humans by simplified index methods. The sensitivity and specificity of the Adipo-IR index and the fasting plasma insulin-glycerol product, combined with their simplicity and acceptable agreement, suggest that these may be most useful in clinical practice.

  3. Studies on the use of gamma irradiation and tissue culture in improving brassica napus

    International Nuclear Information System (INIS)

    Khedr, E.K.A.

    2012-01-01

    The objectives of this study were to:1- Studying the effect of different doses of gamma rays on some growth and yield component traits of three Brassica napus cultivars (Serow6, Serow4 and Pactol) during four consecutive generations aiming to create new genotypes characterized with high yielding traits. 2- Studying the effect of different doses of gamma rays on in vitro biotechnology technique (tissue culture) used in improving Brassica napus. Seeds of three Brassica napus cultivars were irradiated with different gamma ray doses then sown for four consecutive seasons. Data were collected and recorded to clarify the effect gamma irradiation on some yield component traits which were days to flowering , plant height, number of main branches per plant, number of secondary branches per plant, number of pods per plant, number of seeds per pod, weight of 1000-seed, weight of grain yield/plant and oil content of seeds). Results showed that high doses of gamma radiation had enhanced all of the studied traits for each of the three tested cultivars (except the plant height trait for Serow6 and Pactol cultivars). Seven new mutant lines were selected for their superiority in one or more of the studied yield component traits. Regarding the effect of gamma rays on tissue culture techniques, the applied gamma radiation doses did not affect the percentage of seed germination of the three studied cultivars, whereas the percentage of callus induction decreased by increasing the dose of gamma rays for each of the three cultivars and in both types of explants (hypocotyl and cotyledons) used in this experiment.

  4. Research on fuzzy comprehensive assessment method of nuclear power plant safety culture

    International Nuclear Information System (INIS)

    Xiang Yuanyuan; Chen Xukun; Xu Rongbin

    2012-01-01

    Considering the traits of safety culture in nuclear plant, 38 safety culture assessment indexes are established from 4 aspects such as safety values, safety institution, safety behavior and safety sub- stances. Based on it, a comprehensive assessment method for nuclear power plant safety culture is constructed by using AHP (Analytic Hierarchy Process) approach and fuzzy mathematics. The comprehensive assessment method has the quality of high precision and high operability, which can support the decision making of safety culture development. (authors)

  5. Influence of ionizing radiation on synthesis and molecular heterogeneity of catalase in tissue culture of Rauwolfia serpentina

    International Nuclear Information System (INIS)

    Komov, V.P.; Bespalova, E.V.; Strelkova, M.A.

    1998-01-01

    Changes in activity and molecular heterogeneity of catalase in tissue culture of Rauwolfia serpentina following irradiation in early growth period at the doses of 8 and 50 Gy has been studied. Ionizing radiation accelerate the synthesis and degradation rates of catalase and total protein. A comparative study of changes in enzyme and protein turnover during growth on irradiated and non-irradiated medium has been made [ru

  6. Cell culture density affects the stemness gene expression of adipose tissue-derived mesenchymal stem cells.

    Science.gov (United States)

    Kim, Dae Seong; Lee, Myoung Woo; Lee, Tae-Hee; Sung, Ki Woong; Koo, Hong Hoe; Yoo, Keon Hee

    2017-03-01

    The results of clinical trials using mesenchymal stem cells (MSCs) are controversial due to the heterogeneity of human MSCs and differences in culture conditions. In this regard, it is important to identify gene expression patterns according to culture conditions, and to determine how the cells are expanded and when they should be clinically used. In the current study, stemness gene expression was investigated in adipose tissue-derived MSCs (AT-MSCs) harvested following culture at different densities. AT-MSCs were plated at a density of 200 or 5,000 cells/cm 2 . After 7 days of culture, stemness gene expression was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. The proliferation rate of AT-MSCs harvested at a low density (~50% confluent) was higher than that of AT-MSCs harvested at a high density (~90% confluent). Although there were differences in the expression levels of stemness gene, such as octamer-binding transcription factor 4, nanog homeobox ( Nanog ), SRY-box 2, Kruppel like factor 4, v-myc avian myelocytomatosis viral oncogene homolog ( c-Myc ), and lin-28 homolog A, in the AT-MSCs obtained from different donors, RT-qPCR analysis demonstrated differential gene expression patterns according to the cell culture density. Expression levels of stemness genes, particularly Nanog and c-Myc , were upregulated in AT-MSCs harvested at a low density (~50% confluent) in comparison to AT-MSCs from the same donor harvested at a high density (~90% confluent). These results imply that culture conditions, such as the cell density at harvesting, modulate the stemness gene expression and proliferation of MSCs.

  7. OBT analysis method using polyethylene beads for limited quantities of animal tissue

    International Nuclear Information System (INIS)

    Kim, S.B.; Stuart, M.

    2015-01-01

    This study presents a polyethylene beads method for OBT determination in animal tissues and animal products for cases where the amount of water recovered by combustion is limited by sample size or quantity. In the method, the amount of water recovered after combustion is enhanced by adding tritium-free polyethylene beads to the sample prior to combustion in an oxygen bomb. The method reduces process time by allowing the combustion water to be easily collected with a pipette. Sufficient water recovery was achieved using the polyethylene beads method when 2 g of dry animal tissue or animal product were combusted with 2 g of polyethylene beads. Correction factors, which account for the dilution due to the combustion water of the beads, are provided for beef, chicken, pork, fish and clams, as well as egg, milk and cheese. The method was tested by comparing its OBT results with those of the conventional method using animal samples collected on the Chalk River Laboratories (CRL) site. The results determined that the polyethylene beads method added no more than 25% uncertainty when appropriate correction factors are used. - Highlights: • Polyethylene beads method for OBT determination in animal tissues and animal products were determined. • The method reduces process time. • The polyethylene beads method added no more than 25% uncertainty when appropriate correction factors are used

  8. Molecular identification of Mucorales in human tissues: contribution of PCR electrospray-ionization mass spectrometry.

    Science.gov (United States)

    Alanio, A; Garcia-Hermoso, D; Mercier-Delarue, S; Lanternier, F; Gits-Muselli, M; Menotti, J; Denis, B; Bergeron, A; Legrand, M; Lortholary, O; Bretagne, S

    2015-06-01

    Molecular methods are crucial for mucormycosis diagnosis because cultures are frequently negative, even if microscopy suggests the presence of hyphae in tissues. We assessed PCR/electrospray-ionization mass spectrometry (PCR/ESI-MS) for Mucorales identification in 19 unfixed tissue samples from 13 patients with proven or probable mucormycosis and compared the results with culture, quantitative real-time PCR, 16S-23S rRNA gene internal transcribed spacer region (ITS PCR) and 18S PCR sequencing. Concordance with culture identification to both genus and species levels was higher for PCR/ESI-MS than for the other techniques. Thus, PCR/ESI-MS is suitable for Mucorales identification, within 6 hours, for tissue samples for which microscopy results suggest the presence of hyphae. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  9. Appendix A: The components of the culture media.

    Science.gov (United States)

    Loyola-Vargas, Víctor M

    2012-01-01

    The success in the technology and application of plant tissue culture is greatly influenced by the nature of the culture medium used. A better understanding of the nutritional requirements of cultured cells and tissues can help to choose the most appropriate culture medium for the explant used. It is also important to pay attention to a number of inaccuracies and errors which have appeared in several widely used plant tissue culture basal medium formulations.

  10. Optical clearing of tissues and blood using the immersion method

    International Nuclear Information System (INIS)

    Tuchin, Valery V

    2005-01-01

    This paper aims to review recent results on the optical clearing of the naturally turbid biological tissues and blood using the optical immersion technique, which is well known in physical science and is applied for the reduction of light scattering and undesirable reflections in the optical system. Basic principles of the technique, its advantages, limitations and future are discussed. The refractive index matching concept for enhancement of in-depth light penetration into tissues and blood is presented on the basis of in vitro and in vivo studies using optical spectroscopy, polarization and coherence-domain techniques. The index matching of scatterers and ground matter by means of administration of clearing agents is under discussion. The optical properties of tissues with basic multiple scattering, which are transformed to a low scattering mode, are analysed. It is shown that light reflection, transmission, scattering and polarization can be effectively controlled. The possibilities of using the optical immersion method for diagnostic purposes based on contrasting of abnormalities, on in-depth profiling of tissue and blood and on monitoring of endogenous and exogenous matter diffusion within tissue are demonstrated

  11. Comparing rapid and culture indicator bacteria methods at inland lake beaches

    Science.gov (United States)

    Francy, Donna S.; Bushon, Rebecca N.; Brady, Amie M.G.; Kephart, Christopher M.

    2013-01-01

    A rapid method, quantitative polymerase chain reaction (qPCR), for quantifying indicator bacteria in recreational waters is desirable for public health protection. We report that replacing current Escherichia coli standards with new US Environmental Protection Agency beach action values (BAVs) for enterococci by culture or qPCR may result in more advisories being posted at inland recreational lakes. In this study, concentrations of E. coli and enterococci by culture methods were compared to concentrations of Enterococcus spp. by qPCR at 3 inland lake beaches in Ohio. The E. coli and enterococci culture results were significantly related at all beaches; however, the relations between culture results and Enterococcus spp. qPCR results were not always significant and differed among beaches. All the qPCR results exceeded the new BAV for Enterococcus spp. by qPCR, whereas only 23.7% of culture results for E. coli and 79% of culture results for enterococci exceeded the current standard for E. coli or BAV for enterococci.

  12. Study on human chondrocyte culture viability for autologous transplantation in clinical application

    Directory of Open Access Journals (Sweden)

    Christiane Lombello

    2003-06-01

    Full Text Available Objective: The limited regenerative capacity of the cartilage tissuemakes the treatment of chondral lesions difficult. The techniquescurrently available to treat cartilage lesions may relieve symptoms,but do not regenerate the injured tissue. Autologous chondrocytetransplantation uses cell biology and cell culture techniques toregenerate the hyaline cartilage. Methods: In this study, we analyzechondrocyte biopsy collection and culture for autologoustransplantation. Ultrastructural analyses of hyaline cartilage biopsieswere performed 0, 6, 24 and 48 hours after collection. The tissue evenafter 48 hours. Eleven cell culture assays were performed to evaluateisolation, viability, morphology, proliferation and absence ofcontaminants. Results: The cell culture techniques used allowedchondrocyte proliferation. Rates on cell viability were maintained abovethe acceptable patterns (above 90. Control of cell culture laboratoryconditions showed absence of contaminants, assuring safety of theprocess. The chondrocytes obtained presented the morphology typicalof cultured cell monolayers. Conclusion: The results indicate viabilityof chondrocyte culture technique for clinical application in autologoustransplantation.

  13. Triangulation and the importance of establishing valid methods for food safety culture evaluation.

    Science.gov (United States)

    Jespersen, Lone; Wallace, Carol A

    2017-10-01

    The research evaluates maturity of food safety culture in five multi-national food companies using method triangulation, specifically self-assessment scale, performance documents, and semi-structured interviews. Weaknesses associated with each individual method are known but there are few studies in food safety where a method triangulation approach is used for both data collection and data analysis. Significantly, this research shows that individual results taken in isolation can lead to wrong conclusions, resulting in potentially failing tactics and wasted investments. However, by applying method triangulation and reviewing results from a range of culture measurement tools it is possible to better direct investments and interventions. The findings add to the food safety culture paradigm beyond a single evaluation of food safety culture using generic culture surveys. Copyright © 2017. Published by Elsevier Ltd.

  14. Human breast cancer histoid: an in vitro 3-dimensional co-culture model that mimics breast cancer tissue.

    Science.gov (United States)

    Kaur, Pavinder; Ward, Brenda; Saha, Baisakhi; Young, Lillian; Groshen, Susan; Techy, Geza; Lu, Yani; Atkinson, Roscoe; Taylor, Clive R; Ingram, Marylou; Imam, S Ashraf

    2011-12-01

    Progress in our understanding of heterotypic cellular interaction in the tumor microenvironment, which is recognized to play major roles in cancer progression, has been hampered due to unavailability of an appropriate in vitro co-culture model. The aim of this study was to generate an in vitro 3-dimensional human breast cancer model, which consists of cancer cells and fibroblasts. Breast cancer cells (UACC-893) and fibroblasts at various densities were co-cultured in a rotating suspension culture system to establish co-culture parameters. Subsequently, UACC-893, BT.20, or MDA.MB.453 were co-cultured with fibroblasts for 9 days. Co-cultures resulted in the generation of breast cancer histoid (BCH) with cancer cells showing the invasion of fibroblast spheroids, which were visualized by immunohistochemical (IHC) staining of sections (4 µm thick) of BCH. A reproducible quantitative expression of C-erbB.2 was detected in UACC-893 cancer cells in BCH sections by IHC staining and the Automated Cellular Imaging System. BCH sections also consistently exhibited qualitative expression of pancytokeratins, p53, Ki-67, or E-cadherin in cancer cells and that of vimentin or GSTPi in fibroblasts, fibronectin in the basement membrane and collagen IV in the extracellular matrix. The expression of the protein analytes and cellular architecture of BCH were markedly similar to those of breast cancer tissue.

  15. Comparison of the developmental potential of 2-week-old preantral follicles derived from vitrified ovarian tissue slices, vitrified whole ovaries and vitrified/transplanted newborn mouse ovaries using the metal surface method

    Directory of Open Access Journals (Sweden)

    Hsu Kung-Hao

    2008-04-01

    Full Text Available Abstract Background Cryopreservation of preantral follicles or ovarian tissues would enable the storage of large numbers of primordial follicles or preantral follicles and preserves the structural integrity of somatic and reproductive cells. In the present study, we compared the developmental potential of cryopreserved two-week-old mouse preantral follicles, ovarian tissue slices, two-week-old mouse ovaries and newborn mouse ovaries using a metal plate with a high cooling rate for cooling the droplet of vitrification solution. Methods Groups of 2 to 4 samples (including of 14-day old preantral follicles, ovarian tissue slices, whole ovaries, and whole newborn ovaries were exposed to 4% ethylene glycol (EG in DPBS + 10% FBS for 15 min and then rinsed in a vitrification solution composed of 6 M ethylene glycol and 0.4 M trehalose in DPBS + 10% FBS. Equilibration in room temperature was performed for 20–30 seconds for preantral follicle and 5 min equilibration was performed in an ice bath for ovaries. The samples were dropped onto the surface of metal plate around -180°C in the volume of 2 μl and 6 μl. After thawing, the ovarian tissue was mechanically isolated for collecting the preantral follicles. The thawed newborn ovaries were transplanted under the renal capsule of recipient male mice for 14 days. Preantral follicles collected from each groups were cultured individually in 20-μl droplets of α-MEM culture medium in culture dish for 12 days. On the day 12 of culture, the cumulus-oocyte complexes (COCs were collected for IVM and IVF. Fertilization and embryo cleavage were scored. Results After the vitrification of 14-day-old preantral follicles using 2 μl or 6 μl droplet onto surface of metal plate, the results indicated that no significant difference in survival rate, antral-like cavity formation, COCs collected, 2 cell embryo cleavage and blastocyst development was found in vitrification of the 2 μl and 6 μl droplet groups. As

  16. Evaluation of small intestine grafts decellularization methods for corneal tissue engineering.

    Directory of Open Access Journals (Sweden)

    Ana Celeste Oliveira

    Full Text Available Advances in the development of cornea substitutes by tissue engineering techniques have focused on the use of decellularized tissue scaffolds. In this work, we evaluated different chemical and physical decellularization methods on small intestine tissues to determine the most appropriate decellularization protocols for corneal applications. Our results revealed that the most efficient decellularization agents were the SDS and triton X-100 detergents, which were able to efficiently remove most cell nuclei and residual DNA. Histological and histochemical analyses revealed that collagen fibers were preserved upon decellularization with triton X-100, NaCl and sonication, whereas reticular fibers were properly preserved by decellularization with UV exposure. Extracellular matrix glycoproteins were preserved after decellularization with SDS, triton X-100 and sonication, whereas proteoglycans were not affected by any of the decellularization protocols. Tissue transparency was significantly higher than control non-decellularized tissues for all protocols, although the best light transmittance results were found in tissues decellularized with SDS and triton X-100. In conclusion, our results suggest that decellularized intestinal grafts could be used as biological scaffolds for cornea tissue engineering. Decellularization with triton X-100 was able to efficiently remove all cells from the tissues while preserving tissue structure and most fibrillar and non-fibrillar extracellular matrix components, suggesting that this specific decellularization agent could be safely used for efficient decellularization of SI tissues for cornea TE applications.

  17. Immunological methods for the detection and determination of connective tissue proteoglycans

    DEFF Research Database (Denmark)

    Caterson, B; Baker, J R; Christner, J E

    1982-01-01

    In this paper we report the use of immunological methods for specifically detecting and determining proteoglycan in cartilage and other connective tissues. Antibodies (polyclonal and monoclonal) have been raised against specific components of cartilage proteoglycan aggregates (i.e., proteoglycan...... surrounding invaginating hair follicles. These immunological procedures are currently being used to complement conventional biochemical analyses of proteoglycans found in different connective tissue matrices....

  18. Investigation of antibiotic residues in edible tissues of slaughtered broilers in Maragheh abattoir using FPT method (short comunication

    Directory of Open Access Journals (Sweden)

    Masumeh Abasi

    2016-05-01

    Full Text Available Antibiotic residues in food stuff and their transmission to the consumers have some consequences such as bacterial resistance, allergic reactions, intoxication, carcinogenic effects and disturbing of intestine natural flora. Among microbiologic methods, four plate test (FPT is used to detect antibiotic residues in food stuff, which performs in four culture media with different pH values and test bacteria. The aim of this study was investigation of antibiotic residues in edible tissues of slaughtered broilers in Maragheh abattoir using FPT method. For this reason, 40 slaughtered broilers carcasses in Maragheh abattoir (from 10 different poultry farms were sampled. The sampling was conducted randomly from breast and leg muscles, gizzard as well as liver of each carcass. According to results of current study, 60% of liver samples, 55% of leg samples, 52.5% of breast samples and 52.5% of gizzard samples contained antibiotic residues. Moreover, the amount of antibiotic residues among different samples did not show statistical significance (p>0.05. The highest occurrence of antibiotic residue was found in two flocks (100% and the lowest occurrence was recorded for another two flocks (0%. According to the health hazard of antibiotic residues in foods, continuous monitoring is recommended for edible tissues of broilers.

  19. Developing a Plant Culture Medium Composed of Vinasse Originating from Haematococcus Pluvialis Culture

    International Nuclear Information System (INIS)

    Gollo, A. L.; Silva, A. L. L. D.; Lima, K. K. D. D.; Camara, M. C.; Rodrigues, C.; Vandenberghe, L. P. D. S.; Soccol, V. T.; Soccol, C. R.; Biasi, L. A.

    2016-01-01

    The mineral nutrients in vinasse provide support for algal and plant growth. Algal culture releases organic compounds into its liquid culture medium. These organic and inorganic substances can be useful for formulating a plant tissue culture medium, because tissue culture medium is composed of organic and inorganic components. Therefore, the aims of this study were to develop a plant culture medium by using the vinasse that is employed for Haematococcus pluvialis culture (algal filtrate); to investigate the possible beneficial effects of the biocompounds in the micropropagation of Nidularium procerum (Bromeliaceae), to evaluate quercetin content, total phenolics content in vinasse and to evaluate the cytotoxicity of the media by performing a bioassay with Artemia salina. The vinasse that originated from H. pluvialis culture can be used to formulate plant tissue culture at a 3% dilution, and its mineral nutrients can support In vitro plant growth, but some nutrients must be supplemented to enhance its efficiency. An efficient micropropagation protocol was developed for N. procerum. The micropropagated plants were suitable for transfer to the field (they were acclimatized). This culture medium provides a way to reuse wastewater, gives a rational alternative to vinasse disposal and adds value to what is currently considered to be an undesirable residue. Moreover, this process can reduce the production costs of clonal seedlings and/or bioactive compounds in biofactories. There was no apparent biostimulatory effect by the algal filtrate on morphogenesis; however, it did increase quercetin production. The H. pluvialis culture that was grown in the vinasse decreased the cytotoxicity and phenolic compound contents, which prevented explant tissue necrosis and represented a treatment for this residue for safer disposal in the environment. (author)

  20. Automatic method for selective enhancement of different tissue densities at digital chest radiography

    International Nuclear Information System (INIS)

    McNitt-Gray, M.F.; Taira, R.K.; Eldredge, S.L.; Razavi, M.

    1991-01-01

    This paper reports that digital chest radiographs often are too bright and/or lack contrast when viewed on a video display. The authors have developed a method that can automatically provide a series of look-up tables that selectively enhance the radiographically soft or dense tissues on a digital chest radiograph. This reduces viewer interaction and improves displayed image quality. On the basis of a histogram analysis, gray-level ranges are approximated for the patient background, radiographically soft tissues, and radiographically dense tissues. A series of look-up tables is automatically created by varying the contrast in each range to achieve a level of enhancement for a selected tissue range. This is repeated for differing amounts of enhancement and for each tissue range. This allows the viewer to interactively select a tissue density range and degree of enhancement at the time of display via precalculated look-up tables. Preclinical trials in pediatric radiology using computed radiography images show that this method reduces viewer interaction and improves or maintains the displayed image quality

  1. Quantitative and informatics tools for studying the effect of low dose radiation on tissue and cell culture

    International Nuclear Information System (INIS)

    Parvin, B.; Yang, Q.; Fontenay, G.; Barcellos-Hoff, M.H.

    2003-01-01

    Full text: The challenge of the post-genomic era is functional genomics, i.e., understanding how the genome is expressed to produce myriad cell phenotypes. To use genomic information to understand the biology of complex organisms, one must understand the dynamics of phenotype generation and maintenance. A phenotype is the result of selective expression of the genome. In order to define cell 'phenomes,' one would track the kinetics and quantities of multiple constituent proteins, their cellular context and morphological features in large populations. Our aim is to extend the development of the BioSig imaging bioinformatics system for understanding how ionizing radiation alters tissue homeostasis and responses in cell culture experiments. Given several thousand antibodies and reagents for differentiating cell-specific protein components, biological heterogeneity, and other variables that affect cellular responses, there is a clear requirements for managing images and information about these images. Our focus is on the development of (1) quantitative methods for protein expression either in tissue or cell culture studies, (2) a adequate data model that couples quantitative results with the experimental variables, and (3) browsing and visualization tools that enable exploration of large scale image data in feature space in the context of biological heterogeneity. The framework provides the basis for studying the effect of low-dose radiation on the cellular microenvironment, inter-cell communication, and the underlying mechanisms. In turn, this information can then be used to more accurately predict more complex multicellular biological responses following exposure to different types of inhibitors. The BioSig informatics approach to microscopy and quantitative image analysis has been used to build a more detailed picture of the signaling that occurs between cells, as a result of an exogenous stimulus such as radiation, or as a consequence of endogenous programs leading

  2. A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection.

    Science.gov (United States)

    Braian, Clara; Svensson, Mattias; Brighenti, Susanna; Lerm, Maria; Parasa, Venkata R

    2015-10-05

    Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

  3. Method of tissue repair using a composite material

    Energy Technology Data Exchange (ETDEWEB)

    Hutchens, Stacy A.; Woodward, Jonathan; Evans, Barbara R.; O' Neill, Hugh M.

    2016-03-01

    A composite biocompatible hydrogel material includes a porous polymer matrix, the polymer matrix including a plurality of pores and providing a Young's modulus of at least 10 GPa. A calcium comprising salt is disposed in at least some of the pores. The porous polymer matrix can comprise cellulose, including bacterial cellulose. The composite can be used as a bone graft material. A method of tissue repair within the body of animals includes the steps of providing a composite biocompatible hydrogel material including a porous polymer matrix, the polymer matrix including a plurality of pores and providing a Young's modulus of at least 10 GPa, and inserting the hydrogel material into cartilage or bone tissue of an animal, wherein the hydrogel material supports cell colonization in vitro for autologous cell seeding.

  4. Method of tissue repair using a composite material

    Science.gov (United States)

    Hutchens, Stacy A; Woodward, Jonathan; Evans, Barbara R; O'Neill, Hugh M

    2014-03-18

    A composite biocompatible hydrogel material includes a porous polymer matrix, the polymer matrix including a plurality of pores and providing a Young's modulus of at least 10 GPa. A calcium comprising salt is disposed in at least some of the pores. The porous polymer matrix can comprise cellulose, including bacterial cellulose. The composite can be used as a bone graft material. A method of tissue repair within the body of animals includes the steps of providing a composite biocompatible hydrogel material including a porous polymer matrix, the polymer matrix including a plurality of pores and providing a Young's modulus of at least 10 GPa, and inserting the hydrogel material into cartilage or bone tissue of an animal, wherein the hydrogel material supports cell colonization in vitro for autologous cell seeding.

  5. Cell structure and proliferative activity of organ cultures of normal embryonic lung tissue of mice resistant (C57BL) and predisposed (A) to lung tumors

    International Nuclear Information System (INIS)

    Kolesnichenko, T.S.; Gor'kova, T.G.

    1985-01-01

    Local factors such as proliferative activity and the numerical ratio between epithelial and mesenchymal cells, and also the character of interaction between the tissue components in ontogeny may play an important role in the realization of sensitivity of mice of a particular line to the development of lung tumors. These characteristics of lung tissue in mice of lines A and C57BL are investigated under normal conditions and during induced carcinogenesis. Results are given of a comparative study of the relative numbers of epithelial and mesenchymal cells in organ cultures of embryonic lungs. 3 H-thymidine was added to the cultures on the 14th day of the experiment in a concentration of 1 microCi/m1 medium. An autoradiographic study of the cultures was performed

  6. Reduction of /sup 51/Cr-permeability of tissue culture cells by infection with herpes simplex virus type 1

    Energy Technology Data Exchange (ETDEWEB)

    Schlehofer, J.R.; Habermehl, K.O.; Diefenthal, W.; Hampl, H.

    1979-01-01

    Infection of different strains of tissue culture cells with herpes simplex virus type 1(HSV-1) resulted in a reduced /sup 51/Cr-permeability. A stability of the cellular membrane to Triton X-100, toxic sera and HSV-specific complement-mediated immune-cytolysis could be observed simultaneously. The results differed with respect to the cell strain used in the experiments.

  7. Evaluation of a Method for Quantifying Eugenol Concentrations in the Fillet Tissue from Freshwater Fish Species.

    Science.gov (United States)

    Meinertz, Jeffery R; Schreier, Theresa M; Porcher, Scott T; Smerud, Justin R

    2016-01-01

    AQUI-S 20E(®) (active ingredient, eugenol; AQUI-S New Zealand Ltd, Lower Hutt, New Zealand) is being pursued for approval as an immediate-release sedative in the United States. A validated method to quantify the primary residue (the marker residue) in fillet tissue from AQUI-S 20E-exposed fish was needed. A method was evaluated for determining concentrations of the AQUI-S 20E marker residue, eugenol, in freshwater fish fillet tissue. Method accuracies from fillet tissue fortified at nominal concentrations of 0.15, 1, and 60 μg/g from six fish species ranged from 88-102%. Within-day and between-day method precisions (% CV) from the fortified tissue were ≤8.4% CV. There were no coextracted compounds from the control fillet tissue of seven fish species that interfered with eugenol analyses. Six compounds used as aquaculture drugs did not interfere with eugenol analyses. The lower limit of quantitation (LLOQ) was 0.012 μg/g. The method was robust, i.e., in most cases, minor changes to the method did not impact method performance. Eugenol was stable in acetonitrile-water (3 + 7, v/v) for at least 14 days, in fillet tissue extracts for 4 days, and in fillet tissue stored at ~ -80°C for at least 84 days.

  8. Establishment and long-term culture of the cell lines derived from gonad tissues of Siberian sturgeon (Acipenser baerii

    Directory of Open Access Journals (Sweden)

    Jun Hyung Ryu

    2016-06-01

    Full Text Available Abstract To culture germline stem cells in vitro, establishment of the cell lines that can be used as the feeder cells is a prerequisite. In this study, we tried to establish gonad-derived cell lines in Siberian sturgeon (Acipenser baerii. Five 1-year-old A. baerii were used as a donor of gonad tissues, and gonad-dissociated cells were cultured in vitro. Subsequently, determination of growth conditions, long-term culture, characterization, and cryopreservation of the cell lines were also conducted. Five gonad-derived cell lines were stably established and cultured continuously over at least the 73th passage and 402 culture days under the media containing 20 % fetal bovine serum at 28 °C. All cell lines consisted of two main cell types based on morphology even if the ratio of the two cell types was different depending on cell lines. Despite long-term culture, all cell lines maintained diploid DNA contents and expression of several genes that are known to express in the A. baerii gonad. After freezing and thawing of the cell lines, post-thaw cell viabilities between 57.6 and 92.9 % depending on cell lines were indentified, suggesting that stable cryopreservation is possible. The results and the cell lines established in this study will contribute to the development of an in vitro system for A. baerii germline stem cell culture.

  9. Effect of x-ray irradiation on maize inbred line B73 tissue cultures and regenerated plants

    International Nuclear Information System (INIS)

    Wang, A.S.; Cheng, D.S.K.; Milcic, J.B.; Yang, T.C.

    1988-01-01

    In order to enhance variation induced by the tissue culture process and to obtain agronomically desirable mutants, friable embryogenic tissue cultures of maize (Zea mays L.) inbred line B73 were x-ray irradiated with 11 doses [0-8.4 kilorads (kR)]. Reductions in callus growth rate and embryogenic callus formation occurred with increasing x-ray doses 20 d and 3 months after irradiation. Callus irradiated with 0.8 kR showed a significant increase in growth rate and a 20% increase in embryogenic callus 9 months after irradiation. A total of 230 R 0 plants were regenerated for evaluation. Pollen fertility and seed set of R 0 plants decreased with increasing x-ray dosage. Days to anthesis and plant height of R 0 plants varied among x-ray treatments but were generally reduced with higher dosages. The number of chromosomal aberrations increased with x-ray dosage. The R 1 seeds taken from R 0 plants were also grown and tested for mutant segregation. Plants regenerated from irradiated calli had a two- to 10-fold increase in mutations over plants regenerated from unirradiated control callus. Germination frequency of seeds from R 0 plants decreased with increasing x-ray dosage. Although chlorophyll mutants were most frequently observed, a number of vigorous plants with earlier anthesis date were also recovered

  10. Three-dimensional spheroid cell culture of umbilical cord tissue-derived mesenchymal stromal cells leads to enhanced paracrine induction of wound healing.

    Science.gov (United States)

    Santos, Jorge M; Camões, Sérgio P; Filipe, Elysse; Cipriano, Madalena; Barcia, Rita N; Filipe, Mariana; Teixeira, Mariana; Simões, Sandra; Gaspar, Manuela; Mosqueira, Diogo; Nascimento, Diana S; Pinto-do-Ó, Perpétua; Cruz, Pedro; Cruz, Helder; Castro, Matilde; Miranda, Joana P

    2015-05-09

    The secretion of trophic factors by mesenchymal stromal cells has gained increased interest given the benefits it may bring to the treatment of a variety of traumatic injuries such as skin wounds. Herein, we report on a three-dimensional culture-based method to improve the paracrine activity of a specific population of umbilical cord tissue-derived mesenchymal stromal cells (UCX®) towards the application of conditioned medium for the treatment of cutaneous wounds. A UCX® three-dimensional culture model was developed and characterized with respect to spheroid formation, cell phenotype and cell viability. The secretion by UCX® spheroids of extracellular matrix proteins and trophic factors involved in the wound-healing process was analysed. The skin regenerative potential of UCX® three-dimensional culture-derived conditioned medium (CM3D) was also assessed in vitro and in vivo against UCX® two-dimensional culture-derived conditioned medium (CM2D) using scratch and tubulogenesis assays and a rat wound splinting model, respectively. UCX® spheroids kept in our three-dimensional system remained viable and multipotent and secreted considerable amounts of vascular endothelial growth factor A, which was undetected in two-dimensional cultures, and higher amounts of matrix metalloproteinase-2, matrix metalloproteinase-9, hepatocyte growth factor, transforming growth factor β1, granulocyte-colony stimulating factor, fibroblast growth factor 2 and interleukin-6, when compared to CM2D. Furthermore, CM3D significantly enhanced elastin production and migration of keratinocytes and fibroblasts in vitro. In turn, tubulogenesis assays revealed increased capillary maturation in the presence of CM3D, as seen by a significant increase in capillary thickness and length when compared to CM2D, and increased branching points and capillary number when compared to basal medium. Finally, CM3D-treated wounds presented signs of faster and better resolution when compared to untreated and CM

  11. Enhancement of contractile force generation of artificial skeletal muscle tissues by mild and transient heat treatment.

    Science.gov (United States)

    Sato, Masanori; Ikeda, Kazushi; Kanno, Shota; Ito, Akira; Kawabe, Yoshinori; Kamihira, Masamichi

    2014-01-01

    Artificial skeletal muscle tissues composed of cells are expected to be used for applications of regenerative medicine and drug screening. Generally, however, the physical forces generated by tissue-engineered skeletal muscle are lower than those of skeletal muscle tissues found in the body. Local hyperthermia is used for many diseases including muscle injuries. It was recently reported that mild heat treatment improved skeletal muscle functions. In this study, we investigated the effects of mild heat treatment on the tissue-engineered skeletal muscle tissues in vitro. We used magnetite cationic liposomes to label C2C12 myoblast cells magnetically, and constructed densely packed artificial skeletal muscle tissues by using magnetic force. Cell culture at 39°C promoted the differentiation of myoblast cells into myotubes. Moreover, the mild and transient heat treatment improved the contractile properties of artificial skeletal muscle tissue constructs. These findings indicate that the culture method using heat treatment is a useful approach to enhance functions of artificial skeletal muscle tissue.

  12. Cell Culturing of Cytoskeleton

    Science.gov (United States)

    2004-01-01

    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc., has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc., is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

  13. Standard Operating Procedure (SOP) for Rapid and Efficient Production of Stevia Tissue Culture Seedlings

    International Nuclear Information System (INIS)

    Norazlina Noordin; Peng, C.S.; Rusli Ibrahim

    2015-01-01

    Stevia rebaudiana Bertoni is a non-caloric natural sweetener which is 300 times sweeter than cane sugar. Extracts from stevia leaves has vast application in food and beverages based industries, can be added to tea and coffee, cooked or baked goods, processed foods and confectionary goods. Recently, stevia attained awareness owing to its natural, non-caloric sweetness by diet/ health conscious and diabetic persons (Arpita et al., 2011). This natural sweetener has high commercial value in global market, it was estimated that global market value for stevia is be around USD11 billion by year 2015. Although stevia is being largely popularized in Malaysia and other countries but large-scale propagation procedures for the continuous supply of planting materials in commercial plantation has yet to be established, optimized and standardized. Furthermore, propagation through stevia seeds is often very difficult due to self-incompatibility which results in sterile seeds (Sakaguchi et al., 1982). Tissue culture is the only rapid process for the mass propagation of stevia and there have been few reports of in vitro growth of stevia (Miyagaya et al., 1986) and in vitro micropropagation from shoot tip and leaf (Uddin et al., 2006). Hence, study was carried out to establish a suitable protocol for in vitro propagation of S. rebaudiana Bertoni that can be further up-scaled for mass propagation of stevia seedlings. The established Standard Operating Procedure (SOP) will ensure rapid and efficient production of stevia tissue culture seedlings for continuous supply of planting materials for commercial stevia plantations in Malaysia. Preparation of growth medium, multiplication of shoots, rooting of plant lets and hardening of ex-vitro rooted plant lets is discussed in this paper. (author)

  14. Inhibition of collagen production in scleroderma fibroblast cultures by a connective tissue glycoprotein extracted from normal dermis

    International Nuclear Information System (INIS)

    Maquart, F.X.; Bellon, G.; Cornillet-Stoupy, J.; Randoux, A.; Triller, R.; Kalis, B.; Borel, J.P.

    1985-01-01

    It was shown in a previous paper that a connective tissue glycoprotein (CTGP) extracted from normal rabbit dermis was able to inhibit total protein and collagen syntheses by normal dermis fibroblast cultures. In the present study, the effects of CTGP on scleroderma fibroblasts were investigated. [ 14 C]Proline incorporation into total proteins of the supernatant was not significantly different from that found in controls. By contrast, the amount of collagen, expressed as percentage of total secreted protein, was far higher in scleroderma cultures than in normal ones (14.4% +/- 6.0% vs 4.6% +/- 0.9%). Addition of CTGP to the medium induced a concentration-dependent inhibition of [ 14 C]proline incorporation into proteins from both control and scleroderma cells. In control cultures, no significant decrease of the percentage of collagen was observed, but over 60 micrograms/ml, both cytotoxic effects and inhibition of protein synthesis occurred. In scleroderma cultures, the inhibition was twice as effective on collagen as on noncollagen protein synthesis. The inhibition of collagen secretion was not related either to changes in collagen hydroxylation or to the intracellular catabolism of newly synthesized procollagen

  15. Does Polymerase Chain Reaction of Tissue Specimens Aid in the Diagnosis of Tuberculosis?

    Directory of Open Access Journals (Sweden)

    Yoo Jin Lee

    2016-11-01

    Full Text Available Background Mycobacterial culture is the gold standard test for diagnosing tuberculosis (TB, but it is time-consuming. Polymerase chain reaction (PCR is a highly sensitive and specific method that can reduce the time required for diagnosis. The diagnostic efficacy of PCR differs, so this study determined the actual sensitivity of TB-PCR in tissue specimens. Methods We retrospectively reviewed 574 cases. The results of the nested PCR of the IS6110 gene, mycobacterial culture, TB-specific antigen-induced interferon-γ release assay (IGRA, acid-fast bacilli (AFB staining, and histological findings were evaluated. Results The positivity rates were 17.6% for PCR, 3.3% for the AFB stain, 22.2% for mycobacterial culture, and 55.4% for IGRA. PCR had a low sensitivity (51.1% and a high specificity (86.3% based on the culture results of other studies. The sensitivity was higher (65.5% in cases with necrotizing granuloma but showed the highest sensitivity (66.7% in those with necrosis only. The concordance rate between the methods indicated that PCR was the best method compared to mycobacterial culture, and the concordance rate increased for the methods using positive result for PCR or histologic features. Conclusions PCR of tissue specimens is a good alternative to detect tuberculosis, but it may not be as sensitive as previously suggested. Its reliability may also be influenced by some histological features. Our data showed a higher sensitivity when specimens contained necrosis, which indicated that only specimens with necrosis should be used for PCR to detect tuberculosis.

  16. Cytogenetic studies on stevia rebaudiana produced by tissue culture and affected by gamma rays and drought

    International Nuclear Information System (INIS)

    Awad, A.S.A

    2009-01-01

    The present investigation was under taken to carry out in the laboratories of the Natural Products Department, National Center for Radiation Research and Technology, Atomic Energy authority, Nasr city, Cairo, Egypt, to study the effect of gamma radiation doses, osmostress and the combined effects between them on tissue culture, some biochemical analysis and molecular genetic marker in stevia rebaudiana bertoni. The results obtained were: Tissue culture 1- micropropagation media: stevia rebaudiana plantlets cultured on MS medium hormones free for micropropagation.Hormones such as BAP and NAA with different concentrations induced callus formation and give slight growth.Study the effect of gamma radiation, osmostress and the combined effects between them : 1)The effect of gamma radiation on buds survival: Gamma radiation doses (10, 20 and 30 Gy) induced decreasing in bud survival percentage with increasing radiation dose in stevia rebaudiana. The dose 30 Gy was induced 60% mortality.2) Study the effect of gamma radiation on some biochemical analysis: Gamma radiation doses induced increase in the total carbohydrate with doses (20 and 30 Gy) but decreased with dose 10 Gy. Proline contents increased in plantlets with increasing doses . The total protein was increased with doses (10 and 20 Gy), but the dose 30 Gy induced decrease in total protein. Gamma radiation doses induced decreasing in total DNA while, the nucleic acid RNA increased.3) The effect of osmostress on buds survival: The concentrations (40000,50000,60000,70000 and 80000 ppm) from sucrose or sorbitol decreased the bud survival and shoot length in stevia plantlets with increasing sucrose or sorbitol levels. 4) The effect of osmostress on some biochemical analysis: Sucrose and sorbitol concentrations (40000,50000,60000,70000 and 80000 ppm) caused decrease in total carbohydrate.

  17. A new LC-ESI-MS/MS method to measure long-chain acylcarnitine levels in cultured cells

    International Nuclear Information System (INIS)

    Jauregui, Olga; Sierra, Adriana Y.; Carrasco, Patricia; Gratacos, Esther; Hegardt, Fausto G.; Casals, Nuria

    2007-01-01

    The quantitative evaluation of long-chain acylcarnitines in lipid extracts from cultured cells or tissues is a prerequisite to study carnitine palmitoyltransferase (CPT) activity. There is thus a need for the accurate measurement of the concentration of long-chain acylcarnitines at the lowest concentration present in lipid extracts. Here we report a fast and reliable quantitative method based on the use of weak acid extraction and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to quantify acylcarnitines through hydrophilic interaction chromatography. The method was validated using isotopic dilution and the results allow the analysis of a large number of samples at low concentration levels (down to 0.35 nmol L -1 for palmitoylcarnitine) with good inter- and intra-day precision. The method was used for the quantitative study of changes in concentration of palmitoylcarnitine and other acylcarnitines in PC-12 cells over-expressing CPT1a gene. It was also used to measure CPT1 activity in mitochondria isolated from transfected cells, giving similar results to the more common radiometric method, but with higher sensitivity

  18. A new LC-ESI-MS/MS method to measure long-chain acylcarnitine levels in cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Jauregui, Olga [Scientific and Technical Services, University of Barcelona (Spain); Sierra, Adriana Y.; Carrasco, Patricia; Gratacos, Esther [Molecular and Cellular Unit, School of Health Sciences, International University of Catalonia (Spain); Hegardt, Fausto G. [Department of Biochemistry and Molecular Biology, University of Barcelona, School of Pharmacy (Spain); Casals, Nuria [Molecular and Cellular Unit, School of Health Sciences, International University of Catalonia (Spain)], E-mail: ncasals@csc.uic.es

    2007-09-05

    The quantitative evaluation of long-chain acylcarnitines in lipid extracts from cultured cells or tissues is a prerequisite to study carnitine palmitoyltransferase (CPT) activity. There is thus a need for the accurate measurement of the concentration of long-chain acylcarnitines at the lowest concentration present in lipid extracts. Here we report a fast and reliable quantitative method based on the use of weak acid extraction and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to quantify acylcarnitines through hydrophilic interaction chromatography. The method was validated using isotopic dilution and the results allow the analysis of a large number of samples at low concentration levels (down to 0.35 nmol L{sup -1} for palmitoylcarnitine) with good inter- and intra-day precision. The method was used for the quantitative study of changes in concentration of palmitoylcarnitine and other acylcarnitines in PC-12 cells over-expressing CPT1a gene. It was also used to measure CPT1 activity in mitochondria isolated from transfected cells, giving similar results to the more common radiometric method, but with higher sensitivity.

  19. Microbial Biofilms and Breast Tissue Expanders

    Directory of Open Access Journals (Sweden)

    Melissa J. Karau

    2013-01-01

    Full Text Available We previously developed and validated a vortexing-sonication technique for detection of biofilm bacteria on the surface of explanted prosthetic joints. Herein, we evaluated this technique for diagnosis of infected breast tissue expanders and used it to assess colonization of breast tissue expanders. From April 2008 to December 2011, we studied 328 breast tissue expanders at Mayo Clinic, Rochester, MN, USA. Of seven clinically infected breast tissue expanders, six (85.7% had positive cultures, one of which grew Propionibacterium species. Fifty-two of 321 breast tissue expanders (16.2%, 95% CI, 12.3–20.7% without clinical evidence of infection also had positive cultures, 45 growing Propionibacterium species and ten coagulase-negative staphylococci. While vortexing-sonication can detect clinically infected breast tissue expanders, 16 percent of breast tissue expanders appear to be asymptomatically colonized with normal skin flora, most commonly, Propionibacterium species.

  20. Utility of PCR, Culture, and Antigen Detection Methods for Diagnosis of Legionellosis.

    Science.gov (United States)

    Chen, Derrick J; Procop, Gary W; Vogel, Sherilynn; Yen-Lieberman, Belinda; Richter, Sandra S

    2015-11-01

    The goal of this retrospective study was to evaluate the performance of different diagnostic tests for Legionnaires' disease in a clinical setting where Legionella pneumophila PCR had been introduced. Electronic medical records at the Cleveland Clinic were searched for Legionella urinary antigen (UAG), culture, and PCR tests ordered from March 2010 through December 2013. For cases where two or more test methods were performed and at least one was positive, the medical record was reviewed for relevant clinical and epidemiologic factors. Excluding repeat testing on a given patient, 19,912 tests were ordered (12,569 UAG, 3,747 cultures, and 3,596 PCR) with 378 positive results. The positivity rate for each method was 0.4% for culture, 0.8% for PCR, and 2.7% for UAG. For 37 patients, at least two test methods were performed with at least one positive result: 10 (27%) cases were positive by all three methods, 16 (43%) were positive by two methods, and 11 (30%) were positive by one method only. For the 32 patients with medical records available, clinical presentation was consistent with proven or probable Legionella infection in 84% of the cases. For those cases, the sensitivities of culture, PCR, and UAG were 50%, 92%, and 96%, respectively. The specificities were 100% for culture and 99.9% for PCR and UAG. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  1. Electrospun gelatin biopapers as substrate for in vitro bilayer models of blood-brain barrier tissue.

    Science.gov (United States)

    Bischel, Lauren L; Coneski, Peter N; Lundin, Jeffrey G; Wu, Peter K; Giller, Carl B; Wynne, James; Ringeisen, Brad R; Pirlo, Russell K

    2016-04-01

    Gaining a greater understanding of the blood-brain barrier (BBB) is critical for improvement in drug delivery, understanding pathologies that compromise the BBB, and developing therapies to protect the BBB. In vitro human tissue models are valuable tools for studying these issues. The standard in vitro BBB models use commercially available cell culture inserts to generate bilayer co-cultures of astrocytes and endothelial cells (EC). Electrospinning can be used to produce customized cell culture substrates with optimized material composition and mechanical properties with advantages over off-the-shelf materials. Electrospun gelatin is an ideal cell culture substrate because it is a natural polymer that can aid cell attachment and be modified and degraded by cells. Here, we have developed a method to produce cell culture inserts with electrospun gelatin "biopaper" membranes. The electrospun fiber diameter and cross-linking method were optimized for the growth of primary human endothelial cell and primary human astrocyte bilayer co-cultures to model human BBB tissue. BBB co-cultures on biopaper were characterized via cell morphology, trans-endothelial electrical resistance (TEER), and permeability to FITC-labeled dextran and compared to BBB co-cultures on standard cell culture inserts. Over longer culture periods (up to 21 days), cultures on the optimized electrospun gelatin biopapers were found to have improved TEER, decreased permeability, and permitted a smaller separation between co-cultured cells when compared to standard PET inserts. © 2016 Wiley Periodicals, Inc.

  2. Methods and Techniques of Sampling, Culturing and Identifying of Subsurface Bacteria

    International Nuclear Information System (INIS)

    Lee, Seung Yeop; Baik, Min Hoon

    2010-11-01

    This report described sampling, culturing and identifying of KURT underground bacteria, which existed as iron-, manganese-, and sulfate-reducing bacteria. The methods of culturing and media preparation were different by bacteria species affecting bacteria growth-rates. It will be possible for the cultured bacteria to be used for various applied experiments and researches in the future

  3. Influence of epidermal growth factor (EGF) and hydrocortisone on the co-culture of mature adipocytes and endothelial cells for vascularized adipose tissue engineering.

    Science.gov (United States)

    Huber, Birgit; Czaja, Alina Maria; Kluger, Petra Juliane

    2016-05-01

    The composition of vascularized adipose tissue is still an ongoing challenge as no culture medium is available to supply adipocytes and endothelial cells appropriately. Endothelial cell medium is typically supplemented with epidermal growth factor (EGF) as well as hydrocortisone (HC). The effect of EGF on adipocytes is discussed controversially. Some studies say it inhibits adipocyte differentiation while others reported of improved adipocyte lipogenesis. HC is known to have lipolytic activities, which might result in mature adipocyte dedifferentiation. In this study, we evaluated the influence of EGF and HC on the co-culture of endothelial cells and mature adipocytes regarding their cell morphology and functionality. We showed in mono-culture that high levels of HC promoted dedifferentiation and proliferation of mature adipocytes, whereas EGF seemed to have no negative influence. Endothelial cells kept their typical cobblestone morphology and showed a proliferation rate comparable to the control independent of EGF and HC concentration. In co-culture, HC promoted dedifferentiation of mature adipocytes, which was shown by a higher glycerol release. EGF had no negative impact on adipocyte morphology. No negative impact on endothelial cell morphology and functionality could be seen with reduced EGF and HC supplementation in co-culture with mature adipocytes. Taken together, our results demonstrate that reduced levels of HC are needed for co-culturing mature adipocytes and endothelial cells. In co-culture, EGF had no influence on mature adipocytes. Therefore, for the composition of vascularized adipose tissue constructs, the media with low levels of HC and high or low levels of EGF can be used. © 2016 International Federation for Cell Biology.

  4. Use of the Culture Care Theory and ethnonursing method to discover how nursing faculty teach culture care.

    Science.gov (United States)

    Mixer, Sandra J

    2008-04-01

    As the world becomes increasingly multicultural, transcultural nursing education is critical to ensuring a culturally competent workforce. This paper presents a comprehensive review of literature and results of an ethnonursing pilot study using the Culture Care Theory (CCT) to discover how nursing faculty teach culture care. The literature revealed that despite 50 years of transcultural nursing knowledge development through theory, research and practice, there remains a lack of formal, integrated culture education in nursing. The importance of faculty providing generic and professional care to nursing students and using an organising framework to teach culture care was discovered. Additionally, care was essential for faculty health and well-being to enable faculty to teach culture care. This unique use of the theory and method demonstrates its usefulness in discovering and describing the complex nature of teaching culture care. Larger scale studies are predicted to further substantiate the CCT, building the discipline of nursing.

  5. A Stereological Method for the Quantitative Evaluation of Cartilage Repair Tissue

    Science.gov (United States)

    Nyengaard, Jens Randel; Lind, Martin; Spector, Myron

    2015-01-01

    Objective To implement stereological principles to develop an easy applicable algorithm for unbiased and quantitative evaluation of cartilage repair. Design Design-unbiased sampling was performed by systematically sectioning the defect perpendicular to the joint surface in parallel planes providing 7 to 10 hematoxylin–eosin stained histological sections. Counting windows were systematically selected and converted into image files (40-50 per defect). The quantification was performed by two-step point counting: (1) calculation of defect volume and (2) quantitative analysis of tissue composition. Step 2 was performed by assigning each point to one of the following categories based on validated and easy distinguishable morphological characteristics: (1) hyaline cartilage (rounded cells in lacunae in hyaline matrix), (2) fibrocartilage (rounded cells in lacunae in fibrous matrix), (3) fibrous tissue (elongated cells in fibrous tissue), (4) bone, (5) scaffold material, and (6) others. The ability to discriminate between the tissue types was determined using conventional or polarized light microscopy, and the interobserver variability was evaluated. Results We describe the application of the stereological method. In the example, we assessed the defect repair tissue volume to be 4.4 mm3 (CE = 0.01). The tissue fractions were subsequently evaluated. Polarized light illumination of the slides improved discrimination between hyaline cartilage and fibrocartilage and increased the interobserver agreement compared with conventional transmitted light. Conclusion We have applied a design-unbiased method for quantitative evaluation of cartilage repair, and we propose this algorithm as a natural supplement to existing descriptive semiquantitative scoring systems. We also propose that polarized light is effective for discrimination between hyaline cartilage and fibrocartilage. PMID:26069715

  6. [Biocybernetic approach to the thermometric methods of blood supply measurements of periodontal tissues].

    Science.gov (United States)

    Pastusiak, J; Zakrzewski, J

    1988-11-01

    Specific biocybernetic approach to the problem of the blood supply determination of paradontium tissues by means of thermometric methods has been presented in the paper. The compartment models of the measuring procedure have been given. Dilutodynamic methology and classification has been applied. Such an approach enables to select appropriate biophysical parameters describing the state of blood supply of paradontium tissues and optimal design of transducers and measuring methods.

  7. Nuclear analytical methods for trace element studies in calcified tissues

    International Nuclear Information System (INIS)

    Chaudhry, M.A.; Chaudhry, M.N.

    2001-01-01

    Full text: Various nuclear analytical methods have been developed and applied to determine the elemental composition of calcified tissues (teeth and bones). Fluorine was determined by prompt gamma activation analysis through the 19 F(p,ag) 16 O reaction. Carbon was measured by activation analysis with He-3 ions, and the technique of Proton-Induced X-ray Emission (PIXE) was applied to simultaneously determine Ca, P, and trace elements in well-documented teeth. Dental hard tissues, enamel, dentine, cement, and their junctions, as well as different parts of the same tissue, were examined separately. Furthermore, using a Proton Microprobe, we measured the surface distribution of F and other elements on and around carious lesions on the enamel. The depth profiles of F, and other elements, were also measured right up to the amelodentin junction

  8. Effects of Tissue Culture and Mycorrhiza Applications in Organic Farming on Concentrations of Phytochemicals and Antioxidant Capacities in Ginger (Zingiber officinale Roscoe) Rhizomes and Leaves.

    Science.gov (United States)

    Min, Byungrok R; Marsh, Lurline E; Brathwaite, Keegan; Daramola, Adebola O

    2017-04-01

    Tissue culture and mycorrhiza applications can provide disease-free seedlings and enhanced nutrient absorption, respectively, for organic farming. Ginger (Zingiber officinale Roscoe) is rich in phytochemicals and has various health-protective potentials. This study was aimed at determining effects of tissue culture and mycorrhiza applications alone or in combinations in organic farming on phytochemical contents (total phenolics and flavonoids [TP and TF, respectively], gingerol and shogaol homologues, phenolic acids, and carotenoids) and antioxidant capacities (DPPH [2,2-diphenyl-1-picrylhydrazyl] radical scavenging, oxygen radical absorbance (ORAC), and iron-chelating capacities [ICC]) in solvent-extractable (Free) and cell-wall-matrix-bound (Bound) fractions of ginger rhizome and Free fraction of the leaves in comparison with non-organics. Concentrations of the phytochemicals and antioxidant capacities, except for carotenoids and ICC, were significantly higher in organic ginger rhizomes and leaves than in non-organics regardless of the fractions and treatments (P < 0.05). Mycorrhiza application in organic farming significantly increased levels of TP, TF, gingerols, and ORAC in the Free fraction of the rhizome (P < 0.05). Furthermore, the combined application of tissue culture and mycorrhiza significantly increased concentrations of TF and gingerols and ORAC in the Free fraction of the rhizome (P < 0.05), suggesting their synergistic effects. Considerable amounts of phenolics were found in the Bound fractions of the rhizomes. Six-gingerol, ferulic acid, and lutein were predominant ones among gingerols, phenolic acids, and carotenoids, respectively, in ginger rhizomes. The results suggest that organic farming with mycorrhiza and tissue culture applications can increase concentrations of phytochemicals and antioxidant capacities in ginger rhizomes and leaves and therefore improve their health-protective potentials. © 2017 Institute of Food Technologists®.

  9. Rapid fabrication of detachable three-dimensional tissues by layering of cell sheets with heating centrifuge.

    Science.gov (United States)

    Haraguchi, Yuji; Kagawa, Yuki; Hasegawa, Akiyuki; Kubo, Hirotsugu; Shimizu, Tatsuya

    2018-01-18

    Confluent cultured cells on a temperature-responsive culture dish can be harvested as an intact cell sheet by decreasing temperature below 32°C. A three-dimensional (3-D) tissue can be fabricated by the layering of cell sheets. A resulting 3-D multilayered cell sheet-tissue on a temperature-responsive culture dish can be also harvested without any damage by only temperature decreasing. For shortening the fabrication time of the 3-D multilayered constructs, we attempted to layer cell sheets on a temperature-responsive culture dish with centrifugation. However, when a cell sheet was attached to the culture surface with a conventional centrifuge at 22-23°C, the cell sheet hardly adhere to the surface due to its noncell adhesiveness. Therefore, in this study, we have developed a heating centrifuge. In centrifugation (55g) at 36-37°C, the cell sheet adhered tightly within 5 min to the dish without significant cell damage. Additionally, centrifugation accelerated the cell sheet-layering process. The heating centrifugation shortened the fabrication time by one-fifth compared to a multilayer tissue fabrication without centrifugation. Furthermore, the multilayered constructs were finally detached from the dishes by decreasing temperature. This rapid tissue-fabrication method will be used as a valuable tool in the field of tissue engineering and regenerative therapy. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018. © 2018 American Institute of Chemical Engineers.

  10. Identity and quantity of microorganisms in necrotising fasciitis determined by culture and molecular methods

    DEFF Research Database (Denmark)

    Rudkjøbing, Vibeke Børsholt; Thomsen, Trine Rolighed; Nielsen, Per Halkjær

    communities were more common by molecular methods than culture. Correspondence between findings by culture and molecular methods indicates that the latter may be an appropriate method. The advantages of using molecular methods are: 1) identification of the pathogen(s) even when antibiotics have been...... involved in the disease may add to the knowledge of NF pathogenesis and influence the administration of antibiotics, thereby potentially improving the outcome for the patients. In this study the aim was to investigate the applicability of different molecular methods as compared to standard culture......-based methods. We investigated the microbial communities in 21 samples obtained during debridement of NF patients (n=8). Samples were examined by standard bacteriological examination (culture and microscopy) at Rigshospitalet (Copenhagen, Denmark) and a range of molecular methods. The best DNA extraction...

  11. Different methods of dentin processing for application in bone tissue engineering: A systematic review.

    Science.gov (United States)

    Tabatabaei, Fahimeh Sadat; Tatari, Saeed; Samadi, Ramin; Moharamzadeh, Keyvan

    2016-10-01

    Dentin has become an interesting potential biomaterial for tissue engineering of oral hard tissues. It can be used as a scaffold or as a source of growth factors in bone tissue engineering. Different forms of dentin have been studied for their potential use as bone substitutes. Here, we systematically review different methods of dentin preparation and the efficacy of processed dentin in bone tissue engineering. An electronic search was carried out in PubMed and Scopus databases for articles published from 2000 to 2016. Studies on dentin preparation for application in bone tissue engineering were selected. The initial search yielded a total of 1045 articles, of which 37 were finally selected. Review of studies showed that demineralization was the most commonly used dentin preparation process for use in tissue engineering. Dentin extract, dentin particles (tooth ash), freeze-dried dentin, and denatured dentin are others method of dentin preparation. Based on our literature review, we can conclude that preparation procedure and the size and shape of dentin particles play an important role in its osteoinductive and osteoconductive properties. Standardization of these methods is important to draw a conclusion in this regard. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2616-2627, 2016. © 2016 Wiley Periodicals, Inc.

  12. Comparison of DNA extraction protocols to detect Mycobacterium bovis in bovine tissue by PCR

    Directory of Open Access Journals (Sweden)

    Cássia Yumi Ikuta

    2016-11-01

    Full Text Available The current scenario of international beef trading has increased the pressure for better and faster diagnosis of bovine tuberculosis. Although traditional culture remains the gold standard method to confirm Mycobacterium bovis infection, it is exceedingly time consuming, and demands viable mycobacteria. Molecular methods overcome the flaws of the bacteriological methods with faster detection and identification. However, mycobacterial features like a complex cell wall and pathogen–host interaction make the molecular detection a challenge. Three protocols for DNA extraction (A, B and C from bovine tissues were tested to verify the most suitable technique for routine diagnostic assessment of their specificity and sensitivity. Thirty culture-positive and thirty culture-negative granulomatous lesions were included in the trial. From each sample, three tissue suspensions at different dilutions (10-1, 10-2 and 10-3 were prepared and submitted to DNA extraction. PCR procedures targeting IS6110 were performed, employing two volumes of DNA: 5 µL of all three dilutions, and 2.5 µL of the 10-1 dilution. Protocol A was able to detect members of the M. tuberculosis complex in most samples. The sensitivity of the test decreased with increase in tissue-suspension dilution. Although Protocol A presented the highest sensitivity followed by C and B, it showed the lowest specificity, which can be due to a failure in primary isolation caused by the lack of viable organisms or incubation time. Regardless classical bacteriological methods are still recommended by OIE, after evaluating the sensitivity of DNA extraction protocols and PCR procedures, we conclude that the best strategy for M. bovis detection is to follow Protocol A on concentrated tissue suspensions.

  13. In-air micro-pixe analysis of tissue samples

    International Nuclear Information System (INIS)

    Tanaka, A.; Ishii, K.; Komori, Y.

    2002-01-01

    Micro-PIXE is capable of providing spatial distributions of elements in the micro-meter scale and its application to biology is useful to elucidate the cellular metabolism. Since, in this method, a sample target is usually irradiated with proton or α-particle beams in vacuum, beam heating results in evaporation of volatile elements an shrinking of the sample. In order to avoid these side effects, we previously developed a technique of in-air micro-PIXE analysis for samples of cultured cells. In addition to these, analysis of exposed tissue samples from living subjects is highly desirable in biological and medical research. Here, we describe a technique of in-air micro-PIXE analysis of such tissue samples. The target samples of exposed tissue slices from a Donryu rat, in which a tumor had been transplanted, were analyzed with proton micro-beams of 2.6 MeV. We report that the shape of cells and the distribution of volatile elements in the tissue sample remain uncharged when using a target preparation based on a freeze-drying method. (author)

  14. Determination of americium and plutonium in autopsy tissue: methods and problems

    International Nuclear Information System (INIS)

    Boyd, H.A.; Eutsler, B.C.; McInroy, J.F.

    1979-01-01

    The current methods used by the tissue analysis program at LASL for the determination of americium and plutonium in autopsy tissue are described. Problems affecting radiochemical yield are discussed. Included are problems associated with sample preparation, separation of plutonium from large amounts of bone ash, and reagent contamination. The average 242 Pu tracer yield for 1800 Pu determinations is 78 +- 12%. The average 242 Am tracer yield is 85 +- 7% for 40 determinations

  15. Cell patch seeding and functional analysis of cellularized scaffolds for tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, P R Anil [Division of Implant Biology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala 695012 (India); Varma, H K [Bioceramics Laboratory, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala 695012 (India); Kumary, T V [Division of Implant Biology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala 695012 (India)

    2007-03-01

    Cell seeding has a direct impact on the final structure and function of tissue constructs, especially for applications like tissue engineering and regeneration. In this study seeding cell patches retrieved from the thermoresponsive poly(N-isopropylacrylamide) surface were used to generate in vitro tissue constructs. Porous and dense bone substitute materials were cellularized using osteoblast cells by a patch transfer and a trypsin method. The function and proliferation of cells was analyzed after 7 days of culture. The relative cell growth rate was found to be higher in cellularized porous hydroxyapatite (PHA) than in dense hydroxyapatite. Live-dead staining confirmed viable cells inside the pores of PHA. Increased alkaline phosphatase activity of cells transferred by the cell patch over the trypsin method revealed the significance of cell patch seeding. This novel method of generating tissue constructs by cell patch seeding was successful in cellularizing scaffolds with intact cell function.

  16. Cell patch seeding and functional analysis of cellularized scaffolds for tissue engineering

    International Nuclear Information System (INIS)

    Kumar, P R Anil; Varma, H K; Kumary, T V

    2007-01-01

    Cell seeding has a direct impact on the final structure and function of tissue constructs, especially for applications like tissue engineering and regeneration. In this study seeding cell patches retrieved from the thermoresponsive poly(N-isopropylacrylamide) surface were used to generate in vitro tissue constructs. Porous and dense bone substitute materials were cellularized using osteoblast cells by a patch transfer and a trypsin method. The function and proliferation of cells was analyzed after 7 days of culture. The relative cell growth rate was found to be higher in cellularized porous hydroxyapatite (PHA) than in dense hydroxyapatite. Live-dead staining confirmed viable cells inside the pores of PHA. Increased alkaline phosphatase activity of cells transferred by the cell patch over the trypsin method revealed the significance of cell patch seeding. This novel method of generating tissue constructs by cell patch seeding was successful in cellularizing scaffolds with intact cell function

  17. Engineered Muscle Actuators: Cells and Tissues

    National Research Council Canada - National Science Library

    Dennis, Robert G; Herr, Hugh; Parker, Kevin K; Larkin, Lisa; Arruda, Ellen; Baar, Keith

    2007-01-01

    .... Our primary objectives were to engineer living skeletal muscle actuators in culture using integrated bioreactors to guide tissue development and to maintain tissue contractility, to achieve 50...

  18. Surface characterization of retinal tissues for the enhancement of vitreoretinal surgical methods

    Science.gov (United States)

    Valentin-Rodriguez, Celimar

    Diabetic retinopathy is the most common ophthalmic complication of diabetes and the leading cause of blindness among adults, ages 30 to 70. Surgery to remove scar tissue in the eye is the only corrective treatment once the retina is affected. Visual recovery is often hampered by retinal trauma during surgery and by low patient compliance. Our work in this project aimed to improve vitreoretinal surgical methods from information gathered by sensitive surface analysis of pre-retinal tissues found at the vitreoretinal interface. Atomic force microscopy characterization of human retinal tissues revealed that surgically excised inner limiting membrane (ILM) has a heterogeneous surface and is mainly composed of globular and fibrous structures. ILM tissues also show low adhesion for clean unmodified surfaces as opposed to those with functional groups attractive to those on the ILM surface, due to their charge. Based on these observations, layer-by-layer films with embedded gold nanoparticles with a positive outer charge were designed. These modifications increased the adhesion between surgical instruments and ILM by increasing the roughness and tuning the film surface charge. These films proved to be stable under physiological conditions. Finally, the effect of vital dyes on the topographical characteristics of ILMs was characterized and new imaging modes to further reveal ILM topography were utilized. Roughness and adhesion force data suggest that second generation dyes have no effect on the surface nanostructure of ILMs, but increase adhesion at the tip sample interface. This project clearly illustrates that physicochemical information from tissues can be used to rationally re-design surgical procedures, in this case for tissue removal purposes. This rational design method can be applied to other soft tissue excision procedures as is the case of cataract surgery or laparoscopic removal of endometrial tissue.

  19. Dopaminergic Immunofluorescence Studies in Kidney Tissue.

    Science.gov (United States)

    Gildea, J J; Van Sciver, R E; McGrath, H E; Kemp, B A; Jose, P A; Carey, R M; Felder, R A

    2017-01-01

    The kidney is a highly integrated system of specialized differentiated cells that are responsible for fluid and electrolyte balance in the body. While much of today's research focuses on isolated nephron segments or cells from nephron segments grown in tissue culture, an often overlooked technique that can provide a unique view of many cell types in the kidney is slice culture. Here, we describe techniques that use freshly excised kidney tissue from rats to perform a variety of experiments shortly after isolating the tissue. By slicing the rat kidney in a "bread loaf" format, multiple studies can be performed on slices from the same tissue in parallel. Cryosectioning and staining of the tissue allow for the evaluation of physiological or biochemical responses in a wide variety of specific nephron segments. The procedures described within this chapter can also be extended to human or mouse kidney tissue.

  20. Development of a vinasse culture medium for plant tissue culture

    International Nuclear Information System (INIS)

    Silva, A.L.L.D.; Gollo, L.

    2014-01-01

    Vinasse is the main pollutant (effluent) obtained from the distillation of sugarcane in the production of fuel alcohol. However, this residue is rich in nutrients that are required by plants. We developed a new culture medium using vinasse for the In vitro propagation of an orchid. The vinasse was treated (decanted and filtered), and the nutrients were determined and quantified. Different formulations using vinasse were tested for an In vitro culture. The vinasse dilutions demonstrated a good buffering effect. The ideal vinasse dilution for media formulation was 2.5%. The best KC formulations with vinasse were KCV1 and KCV5. Compared to KC medium, these formulations demonstrated similar results for In vitro multiplication, with the exception of protocorm-like body number, which was inferior in the vinasse formulations. Conversely, for In vitro elongation and rooting, these vinasse media were superior to KC medium. KC medium promotes a low rooting rate (8%) compared to 68 and 100% obtained by KCV1 and KCV5, respectively. Moreover, plantlets cultured on KC medium become protocorm-like body clusters, which impeded the acclimatization of these explants. Plantlets elongated and rooted on KCV1 and KCV5 were successfully acclimatized with a 91% survival rate for both KC vinasse formulations. This study shows the great potential of this technology as a rational alternative to vinasse disposal and adds value to what is currently considered a waste product. (author)