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Sample records for tissue explant culture

  1. 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

  2. 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

  3. 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.

  4. Glucocorticoids affect 24 h clock genes expression in human adipose tissue explant cultures.

    Directory of Open Access Journals (Sweden)

    Purificación Gómez-Abellán

    Full Text Available to examine firstly whether CLOCK exhibits a circadian expression in human visceral (V and subcutaneous (S adipose tissue (AT in vitro as compared with BMAL1 and PER2, and secondly to investigate the possible effect of the glucocorticoid analogue dexamethasone (DEX on positive and negative clock genes expression.VAT and SAT biopsies were obtained from morbid obese women (body mass index ≥ 40 kg/m(2 (n = 6. In order to investigate rhythmic expression pattern of clock genes and the effect of DEX on CLOCK, PER2 and BMAL1 expression, control AT (without DEX and AT explants treated with DEX (2 hours were cultured during 24 h and gene expression was analyzed at the following times: 10:00 h, 14:00 h, 18:00 h, 22:00 h, 02:00 h and 06:00 h, using qRT-PCR.CLOCK, BMAL1 and PER2 expression exhibited circadian patterns in both VAT and SAT explants that were adjusted to a typical 24 h sinusoidal curve. PER2 expression (negative element was in antiphase with respect to CLOCK and in phase with BMAL1 expression (both positive elements in the SAT (situation not present in VAT. A marked effect of DEX exposure on both positive and negative clock genes expression patterns was observed. Indeed, DEX treatment modified the rhythmicity pattern towards altered patterns with a period lower than 24 hours in all genes and in both tissues.24 h patterns in CLOCK and BMAL1 (positive clock elements and PER2 (negative element mRNA levels were observed in human adipose explants. These patterns were altered by dexamethasone exposure.

  5. Cell number, tissue thickness and protein content as measures for development and variability in cultured neocortex explants

    NARCIS (Netherlands)

    de Jong, B. M.; Ruijter, J. M.

    1989-01-01

    The development of neuronal number, explant thickness and amount of protein was studied in several series of rat neocortex explants, cultured up to 21 days in vitro (DIV). In contrast to the dimensions of the explant, which rapidly stabilized, the amount of protein showed a prolonged increase with

  6. Somatic embryogenesis and plant regeneration in Carica papaya L. tissue culture derived from root explants.

    Science.gov (United States)

    Chen, M H; Wang, P J; Maeda, E

    1987-10-01

    The regeneration potential of shoot tip, stem, leaf, cotyledon and root explants of two papaya cultivars (Carica papaya cv. 'Solo' and cv. 'Sunrise') were studed. Callus induction of these two cultivars of papaya showed that the shoot tips and stems are most suitable for forming callus, while leaves, cotyledons and roots are comparatively difficult to induce callus. Callus induction also varied with the varities. Somatic embryogenesis was obtained from 3-month-old root cultures. A medium containing half strength of MS inorganic salts, 160 mg/l adenine sulfate, 1.0 mg/1 NAA, 0.5 mg/1 kinetin and 1.0 mg/1 GA3 was optimal for embryogenesis. The callus maintained high regenerative capacity after two years of culture on this medium. Plants derived from somatic embryos were obtained under green-house conditions.

  7. 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.

  8. Characterisation of a bystander effect induced in human tissue explant cultures by low LET radiation

    International Nuclear Information System (INIS)

    Motersill, C.; O'Malley, K.; Seymour, C.B.

    2002-01-01

    The existence of a bystander effect following both alpha and gamma irradiation of many cell lines is not now in dispute. The significance of this effect for cancer risk assessment and radiotherapy treatment planning requires demonstration of its relevance in vivo. The problem in demonstrating the existence of the effect in vivo is that other systemic effects may mask or confound the effect being investigated and it is practically impossible to attribute an effect in a particular cell to a signal produced in another irradiated cell. To approach this problem, an assay has been developed where fragments of human tissue can be irradiated ex vivo and the media harvested and added to unirradiated, clonogenic cells which have a well characterised and stable response to the bystander signal. The variation in the production of a signal from patient to patient can thus be assessed. The results of a study using tissue from over 100 patients attending Beaumont and St Vincent's Hospitals in Dublin for investigation of urological disorders including follow-up after treatment for transitional cell carcinoma (TCC) and resection of suspect prostatic lesions, are now available. Blood samples from the prostate group were also obtained. The results show that there is variation in the effect of the signal produced by irradiated tissue from different patients. This holds for bladder, prostate and blood. Gender, smoking status and the existence of a malignancy influence the expression of the signal by normal tissue. Male gender, smoking and a pre-existing malignancy all reduce the amount or effect of the signal produced into medium when the tissue is exposed. The effects of exposure to medium containing the signal are transmitted to distant progeny of the exposed cell population. The results may be important not only for understanding radiation risk mechanisms for protection but also for radiotherapy treatment planning where they may open new avenues for development of drugs for combined

  9. 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

  10. Cytoarchitecture in cultured rat neocortex explants

    NARCIS (Netherlands)

    de Jong, B. M.; Ruijter, J. M.; Romijn, H. J.

    1988-01-01

    Neocortex explants obtained from 6-day-old rat pups and cultured in a serum-free medium from 5 hr to 13 days in vitro (DIV) show preservation of cytoarchitectural characteristics. Major changes in the size of the explants and their layers occur during the first 2 DIV. A radial arrangement of neurons

  11. The Effect of Plant Growth Regulators and Different Explants on the Response of Tissue Culture and Cell Suspension Cultures of German Chamomile (Matricaria chamomilla L.

    Directory of Open Access Journals (Sweden)

    L. Koohi,

    2014-07-01

    Full Text Available German chamomile (Matricaria chamomilla L. is one of the most important medicinal plants that its essential oils used in different medicinal industries. In this study which was carried out in 2013 growing season at the Faculty of Agricultural Sciences of the University of Mohaghegh Ardabili, the in vitro response of leaf and hypocotyl explants of German Chamomile in B5 medium supplemented with different levels of plant growth regulators including 2,4-D, naphthalene acetic acid (NAA, kinetin and 6-benzylaminopurine (BAP were investigated in a factorial experiment based on completely randomized design (CRD.In addition, cell suspension cultures were established and characterized. Hypocotyl and leaf explants exhibited cell proliferation and produced callus within 1-2 weeks. The highest fresh weight of the callus (264.1 mg was produced by leaf explants in the medium supplemented with 0.5 mg/l 2,4-D and 1 mg/l BAP. However, the leaf explants cultured on medium containing 1.5 mg/l 2,4-D showed the lowest cell proliferation and callus yield (40.42 mg. The highest percentage of root induction from leaf explants (58.73% was observed on the medium containing 4 mg/l 2,4-D and 1 mg/l Kin, and from hypocotyl explants (48.61% was observed on medium supplemented with 1.5 mg/l NAA. The 42.22% of calli derived from hypocotyl explants on B5 medium supplemented with 4 mg/l NAA and 3 mg/l BAP, were friable. Cell suspension cultures of German chamomile were established by transferring of hypocotyl-derived friable calli into the MS medium supplemented with 1.5 mg/l 2,4-D and 1 mg/l kinetin. The growth curve of cell proliferations started 4 days after culture and continued to grow until day 13th, where the cells entered stationary phase.

  12. Culturing bovine nucleus pulposus explants by balancing medium osmolarity

    NARCIS (Netherlands)

    Dijk, van B.G.M.; Potier, E.; Ito, K.

    2011-01-01

    Regenerative therapies are promising treatments for early intervertebral disc degeneration. To test their efficacy, an in vitro tissue-level model would be valuable. Nucleus pulposus (NP) explant culture may constitute such a model, as the earliest signs of degeneration are in the NP. However, in NP

  13. 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

  14. Stabilization of gene expression and cell morphology after explant recycling during fin explant culture in goldfish

    International Nuclear Information System (INIS)

    Chenais, Nathalie; Lareyre, Jean-Jacques; Le Bail, Pierre-Yves; Labbe, Catherine

    2015-01-01

    The development of fin primary cell cultures for in vitro cellular and physiological studies is hampered by slow cell outgrowth, low proliferation rate, poor viability, and sparse cell characterization. Here, we investigated whether the recycling of fresh explants after a first conventional culture could improve physiological stability and sustainability of the culture. The recycled explants were able to give a supplementary cell culture showing faster outgrowth, cleaner cell layers and higher net cell production. The cells exhibited a highly stabilized profile for marker gene expression including a low cytokeratin 49 (epithelial marker) and a high collagen 1a1 (mesenchymal marker) expression. Added to the cell spindle-shaped morphology, motility behavior, and actin organization, this suggests that the cells bore stable mesenchymal characteristics. This contrast with the time-evolving expression pattern observed in the control fresh explants during the first 2 weeks of culture: a sharp decrease in cytokeratin 49 expression was concomitant with a gradual increase in col1a1. We surmise that such loss of epithelial features for the benefit of mesenchymal ones was triggered by an epithelial to mesenchymal transition (EMT) process or by way of a progressive population replacement process. Overall, our findings provide a comprehensive characterization of this new primary culture model bearing mesenchymal features and whose stability over culture time makes those cells good candidates for cell reprogramming prior to nuclear transfer, in a context of fish genome preservation. - Highlights: • Recycled fin explants outgrow cells bearing stable mesenchymal traits. • Cell production and quality is enhanced in the recycled explant culture system. • Fresh fin primary culture is highly variable and loose epithelial traits over time

  15. Stabilization of gene expression and cell morphology after explant recycling during fin explant culture in goldfish

    Energy Technology Data Exchange (ETDEWEB)

    Chenais, Nathalie; Lareyre, Jean-Jacques; Le Bail, Pierre-Yves; Labbe, Catherine, E-mail: catherine.labbe@rennes.inra.fr

    2015-07-01

    The development of fin primary cell cultures for in vitro cellular and physiological studies is hampered by slow cell outgrowth, low proliferation rate, poor viability, and sparse cell characterization. Here, we investigated whether the recycling of fresh explants after a first conventional culture could improve physiological stability and sustainability of the culture. The recycled explants were able to give a supplementary cell culture showing faster outgrowth, cleaner cell layers and higher net cell production. The cells exhibited a highly stabilized profile for marker gene expression including a low cytokeratin 49 (epithelial marker) and a high collagen 1a1 (mesenchymal marker) expression. Added to the cell spindle-shaped morphology, motility behavior, and actin organization, this suggests that the cells bore stable mesenchymal characteristics. This contrast with the time-evolving expression pattern observed in the control fresh explants during the first 2 weeks of culture: a sharp decrease in cytokeratin 49 expression was concomitant with a gradual increase in col1a1. We surmise that such loss of epithelial features for the benefit of mesenchymal ones was triggered by an epithelial to mesenchymal transition (EMT) process or by way of a progressive population replacement process. Overall, our findings provide a comprehensive characterization of this new primary culture model bearing mesenchymal features and whose stability over culture time makes those cells good candidates for cell reprogramming prior to nuclear transfer, in a context of fish genome preservation. - Highlights: • Recycled fin explants outgrow cells bearing stable mesenchymal traits. • Cell production and quality is enhanced in the recycled explant culture system. • Fresh fin primary culture is highly variable and loose epithelial traits over time.

  16. An in vitro model for detecting skin irritants: methyl green-pyronine staining of human skin explant cultures

    NARCIS (Netherlands)

    Jacobs, J. J. L.; Lehé, C.; Cammans, K. D. A.; Das, P. K.; Elliott, G. R.

    2002-01-01

    We evaluated the potential of human organotypic skin explant cultures (hOSECs) for screening skin irritants. Test chemicals were applied to the epidermis of the skin explants which were incubated for 4, 24 or 48 h in tissue culture medium. A decrease in epidermal RNA staining, visualised in frozen

  17. Stabilization of gene expression and cell morphology after explant recycling during fin explant culture in goldfish.

    Science.gov (United States)

    Chenais, Nathalie; Lareyre, Jean-Jacques; Le Bail, Pierre-Yves; Labbe, Catherine

    2015-07-01

    The development of fin primary cell cultures for in vitro cellular and physiological studies is hampered by slow cell outgrowth, low proliferation rate, poor viability, and sparse cell characterization. Here, we investigated whether the recycling of fresh explants after a first conventional culture could improve physiological stability and sustainability of the culture. The recycled explants were able to give a supplementary cell culture showing faster outgrowth, cleaner cell layers and higher net cell production. The cells exhibited a highly stabilized profile for marker gene expression including a low cytokeratin 49 (epithelial marker) and a high collagen 1a1 (mesenchymal marker) expression. Added to the cell spindle-shaped morphology, motility behavior, and actin organization, this suggests that the cells bore stable mesenchymal characteristics. This contrast with the time-evolving expression pattern observed in the control fresh explants during the first 2 weeks of culture: a sharp decrease in cytokeratin 49 expression was concomitant with a gradual increase in col1a1. We surmise that such loss of epithelial features for the benefit of mesenchymal ones was triggered by an epithelial to mesenchymal transition (EMT) process or by way of a progressive population replacement process. Overall, our findings provide a comprehensive characterization of this new primary culture model bearing mesenchymal features and whose stability over culture time makes those cells good candidates for cell reprogramming prior to nuclear transfer, in a context of fish genome preservation. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Hypoxia preferentially destroys GABAergic neurons in developing rat neocortex explants in culture

    NARCIS (Netherlands)

    Romijn, H. J.; Ruijter, J. M.; Wolters, P. S.

    1988-01-01

    The hypothesis that hypoxic ischemia before or during the human birth process preferentially destroys GABAergic nerve cells, particularly in the neocortex, was tested in a tissue culture model system. To that end, rat neocortex explants dissected from 6-day-old rat pups and cultured to a

  19. Immunocytochemical characterization of explant cultures of human prostatic stromal cells

    NARCIS (Netherlands)

    A. Kooistra (Anko); A.M.J. Elissen (Arianne ); J.J. Konig (Josee); M. Vermey; Th.H. van der Kwast (Theo); J.C. Romijn (Johannes); F.H. Schröder (Fritz)

    1995-01-01

    textabstractThe study of stromal-epithelial interactions greatly depends on the ability to culture both cell types separately, in order to permit analysis of their interactions under defined conditions in reconstitution experiments. Here we report the establishment of explant cultures of human

  20. A novel method for coral explant culture and micropropagation.

    Science.gov (United States)

    Vizel, Maya; Loya, Yossi; Downs, Craig A; Kramarsky-Winter, Esti

    2011-06-01

    We describe here a method for the micropropagation of coral that creates progeny from tissue explants derived from a single polyp or colonial corals. Coral tissue explants of various sizes (0.5-2.5 mm in diameter) were manually microdissected from the solitary coral Fungia granulosa. Explants could be maintained in an undeveloped state or induced to develop into polyps by manipulating environmental parameters such as light and temperature regimes, as well as substrate type. Fully developed polyps were able to be maintained for a long-term in a closed sea water system. Further, we demonstrate that mature explants are also amenable to this technique with the micropropagation of second-generation explants and their development into mature polyps. We thereby experimentally have established coral clonal lines that maintain their ability to differentiate without the need for chemical induction or genetic manipulation. The versatility of this method is also demonstrated through its application to two other coral species, the colonial corals Oculina patigonica and Favia favus.

  1. Explant culture of human peripheral lung. I. Metabolism of benzo[alpha]pyrene

    DEFF Research Database (Denmark)

    Stoner, G.D.; Harris, C.C.; Autrup, Herman

    1978-01-01

    the predominant alveolar epithelial cell type. Lamellar inclusion bodies were released from the type 2 cells and accumulated in the alveolar spaces. The metabolism of benzo[alpha]pyrene (BP) in human lung explants cultured for up to 7 days was investigated. Human lung explants had measurable aryl hydrocarbon......Human lung explants have been maintained in vitro for a period of 25 days. Autoradiographic studies indicated that the broncholar epithelial cells, type 2 alveolar epithelial cells, and stromal fibroblasts incorporated 3H-thymidine during the culture. After 7 to 10 days, type 2 cells were...... hydroxylase activity and could metabolize BP into forms that were bound to cellular DNA and protein. Peripheral lung had significantly lower aryl hydrocarbon hydroxylase activity than cultured bronchus but both tissues had similar binding levels of BP to DNA. Radioautographic studies indicated that all cell...

  2. Ex Vivo Produced Oral Mucosa Equivalent by Using the Direct Explant Cell Culture Technique

    Directory of Open Access Journals (Sweden)

    Kamile Öztürk

    2012-09-01

    Full Text Available Objective: The aim of this study is the histological and immunohistochemical evaluation of ex vivo produced oral mucosal equivalents using keratinocytes cultured by direct explant technique.Material and Methods: Oral mucosa tissue samples were obtained from the keratinized gingival tissues of 14 healthy human subjects. Human oral mucosa keratinocytes from an oral mucosa biopsy specimen were dissociated by the explant technique. Once a sufficient population of keratinocytes was reached, they were seeded onto the type IV collagen coated “AlloDerm” and taken for histological and immunohistochemical examinations at 11 days postseeding of the keratinocytes on the cadaveric human dermal matrix.Results: Histopathologically and immunohistochemically, 12 out of 14 successful ex vivo produced oral mucosa equivalents (EVPOME that consisted of a stratified epidermis on a dermal matrix have been developed with keratinocytes cultured by the explant technique.Conclusion: The technical handling involved in the direct explant method at the beginning of the process has fewer steps than the enzymatic method and use of the direct explant technique protocol for culturing of human oral mucosa keratinocyte may be more adequate for EVPOME production.

  3. Induction of bulb organogenesis in in vitro cultures of tarda tulip (Tulipa tarda Stapf.) from seed-derived explants.

    Science.gov (United States)

    Maślanka, Małgorzata; Bach, Anna

    2014-01-01

    A protocol for obtaining bulbs via in vitro organogenesis was developed for tarda tulip ( Tulipa tarda Stapf). Scale explants were obtained from bulbs formed at the base of seedlings or from adventitious bulbs that developed from callus tissue forming on stolons or on germinating seeds. Some explants were subjected to chilling at 5°C for 12 wk. The culture media contained 3 or 6% sucrose and was supplemented with either no growth regulators, either 0.5 μM 6-benzyl-aminopurine (BAP) or 18.9 or 94.6 μM abscisic acid (ABA). Cultures were maintained in the dark at 20°C. Callus tissue developed mainly on media without growth regulators or with BAP. Callus was formed from up to 96% of explants derived from non-chilled adventitious bulbs that were treated with 3% sucrose and 0.5 μM BAP. Less callus was formed from chilled explants compared with non-chilled explants. Newly formed adventitious bulbs appeared on the explants via direct and indirect organogenesis. The media with BAP promoted the formation of adventitious bulbs at a rate of 56-92% from non-chilled explants, whereas a maximum rate of 36% was observed from chilled explants. ABA inhibited the induction of adventitious bulbs and callus. The adventitious bulbs obtained in these experiments contained a meristem, which was evidence that they had developed properly.

  4. Effect of storage media and time on fin explants culture in the ...

    African Journals Online (AJOL)

    The effect of storage media and time was investigated on fin explants culture in the goldfish (Carassius auratus). Fin explants under sterile conditions were able to produce cells at different storage media and time. On the outgrowth of cells, fin explants stored for seven days before culturing showed significantly higher growth ...

  5. Simple explant culture of the embryonic chicken retina with long-term preservation of photoreceptors.

    Science.gov (United States)

    Thangaraj, Gopenath; Greif, Alexander; Layer, Paul G

    2011-10-01

    Structurally stable in vitro-model systems are indispensible to analyse neural development during embryogenesis, follow cellular differentiation and evaluate neurotoxicological or growth factor effects. Here we describe a three-dimensional, long-term in vitro-culture system of the embryonic chick retina which supports photoreceptor development. Retinal tissue was isolated from E6 chick eye, and cultured as explants by continuous orbital rotation to allow free floatation without any supporting materials. Young stage (E6) immature retinas were cultured for various time periods in order to follow the differentiation of cell types and plexiform layers by immunocytochemical methods. These explants could be cultured for at least 2-3 weeks with remarkable retention of retinal architecture. Interestingly, photoreceptors developed in the absence of pigment epithelium. Electron microscopic studies revealed formation of structures resembling photoreceptor outer segments, a feature not reported previously. Thus, the verification of photoreceptors, Müller cells, inner retinal cells and the inner plexiform layer described in our study establishes this explant culture as a valuable in vivo-like model system. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

  6. Participation of intracellular cysteine proteinases, in particular cathepsin B, in degradation of collagen in periosteal tissue explants

    NARCIS (Netherlands)

    Creemers, L. B.; Hoeben, K. A.; Jansen, D. C.; Buttle, D. J.; Beertsen, W.; Everts, V.

    1998-01-01

    The involvement of cysteine proteinases in the degradation of soft connective tissue collagen was studied in cultured periosteal explants. Using cysteine proteinase inhibitors that were active intracellularly or extracellularly (Ep453 and Ep475, respectively), it was shown that over-all collagen

  7. Effects of glucosamine on proteoglycan loss by tendon, ligament and joint capsule explant cultures.

    Science.gov (United States)

    Ilic, M Z; Martinac, B; Samiric, T; Handley, C J

    2008-12-01

    To investigate the effect of glucosamine on the loss of newly synthesized radiolabeled large and small proteoglycans by bovine tendon, ligament and joint capsule. The kinetics of loss of (35)S-labeled large and small proteoglycans from explant cultures of tendon, ligament and joint capsule treated with 10mM glucosamine was investigated over a 10-day culture period. The kinetics of loss of (35)S-labeled small proteoglycans and the formation of free [(35)S]sulfate were determined for the last 10 days of a 15-day culture period. The proteoglycan core proteins were analyzed by gel electrophoresis followed by fluorography. The metabolism of tendon, ligament and joint capsule explants exposed to 10mM glucosamine was evaluated by incorporation of [(3)H]serine and [(35)S]sulfate into protein and glycosaminoglycans, respectively. Glucosamine at 10mM stimulated the loss of small proteoglycans from ligament explant cultures. This was due to the increased loss of both macromolecular and free [(35)S]sulfate to the medium indicating that glucosamine affected the release of small proteoglycans as well as their intracellular degradation. The degradation pattern of small proteoglycans in ligament was not affected by glucosamine. In contrast, glucosamine did not have an effect on the loss of large or small proteoglycans from tendon and joint capsule or large proteoglycans from ligament explant cultures. The metabolism of cells in tendon, ligament and joint capsule was not impaired by the presence of 10mM glucosamine. Glucosamine stimulated the loss of small proteoglycans from ligament but did not have an effect on small proteoglycan catabolism in joint capsule and tendon or large proteoglycan catabolism in ligament, tendon or synovial capsule. The consequences of glucosamine therapy at clinically relevant concentrations on proteoglycan catabolism in joint fibrous connective tissues need to be further assessed in an animal model.

  8. Effect of environmental and cultural conditions on medium pH and explant growth performance of Douglas-fir ( Pseudotsuga menziesii) shoot cultures

    OpenAIRE

    Chen, Chien-Chih; Bates, Rick; Carlson, John

    2015-01-01

    The medium pH level of plant tissue cultures has been shown to be essential to many aspects of explant development and growth. Sensitivity or tolerance of medium pH change in vitro varies according to specific requirements of individual species. The objectives of this study are to 1) determine medium pH change over time in storage conditions and with presence of explants, 2) evaluate the effects of medium pH change on explant growth performance and 3) assess the effects of adding a pH stabili...

  9. Blockade of Toll-like receptor 2 prevents spontaneous cytokine release from rheumatoid arthritis ex vivo synovial explant cultures

    LENUS (Irish Health Repository)

    Nic An Ultaigh, Sinead

    2011-02-23

    Abstract Introduction The aim of this study was to examine the effect of blocking Toll-like receptor 2 (TLR2) in rheumatoid arthritis (RA) synovial cells. Methods RA synovial tissue biopsies, obtained under direct visualization at arthroscopy, were established as synovial explant cultures ex vivo or snap frozen for immunohistology. Mononuclear cell cultures were isolated from peripheral blood and synovial fluid of RA patients. Cultures were incubated with the TLR1\\/2 ligand, Pam3CSK4 (200 ng, 1 and 10 μg\\/ml), an anti-TLR2 antibody (OPN301, 1 μg\\/ml) or an immunoglobulin G (IgG) (1 μg\\/ml) matched control. The comparative effect of OPN301 and adalimumab (anti-tumour necrosis factor alpha) on spontaneous release of proinflammatory cytokines from RA synovial explants was determined using quantitative cytokine MSD multiplex assays or ELISA. OPN301 penetration into RA synovial tissue explants cultures was assessed by immunohistology. Results Pam3CSK4 significantly upregulated interleukin (IL)-6 and IL-8 in RA peripheral blood mononuclear cells (PBMCs), RA synovial fluid mononuclear cells (SFMCs) and RA synovial explant cultures (P < 0.05). OPN301 significantly decreased Pam3CSK4-induced cytokine production of tumour necrosis factor alpha (TNF-α), IL-1β, IL-6, interferon (IFN)-γ and IL-8 compared to IgG control in RA PBMCs and SFMCs cultures (all P < 0.05). OPN301 penetration of RA synovial tissue cultures was detected in the lining layer and perivascular regions. OPN301 significantly decreased spontaneous cytokine production of TNF-α, IL-1β, IFN-γ and IL-8 from RA synovial tissue explant cultures (all P < 0.05). Importantly, the inhibitory effect of OPN on spontaneous cytokine secretion was comparable to inhibition by anti-TNFα monoclonal antibody adalimumab. Conclusions These findings further support targeting TLR2 as a potential therapeutic agent for the treatment of RA.

  10. Organotypic Culture of Breast Tumor Explants as a Multicellular System for the Screening of Natural Compounds with Antineoplastic Potential

    Directory of Open Access Journals (Sweden)

    Irma Edith Carranza-Torres

    2015-01-01

    Full Text Available Breast cancer is the leading cause of death in women worldwide. The search for novel compounds with antitumor activity, with less adverse effects and higher efficacy, and the development of methods to evaluate their toxicity is an area of ​​intense research. In this study we implemented the preparation and culture of breast tumor explants, which were obtained from precision-cut breast tumor slices. In order to validate the model we are proposing to screen antineoplastic effect of natural compounds, we selected caffeic acid, ursolic acid, and rosmarinic acid. Using the Krumdieck tissue slicer, precision-cut tissue slices were prepared from breast cancer samples; from these slices, 4 mm explants were obtained and incubated with the selected compounds. Viability was assessed by Alamar Blue assay, LDH release, and histopathological criteria. Results showed that the viability of the explants cultured in the presence of paclitaxel (positive control decreased significantly (P<0.05; however, tumor samples responded differently to each compound. When the explants were coincubated with paclitaxel and compounds, a synergic effect was observed. This study shows that ex vivo culture of breast cancer explants offers a suitable alternative model for evaluating natural or synthetic compounds with antitumor properties within the complex microenvironment of the tumor.

  11. Culturated rat cerebral cortex explants and their application in the study of SPECT scan radiopharaceuticals

    International Nuclear Information System (INIS)

    Jong, B.M. de.

    1989-01-01

    In this thesis mechanics that result in the distinct localization of radiopharmaceuticals within the brain have been investigated. In order to 'get more insight' in uptake and binding of radiopharmaceuticals bu brain tissue, use has been made of the tissue culture technique. Tissue culture privides the opportunity of doing experiments with brain tissue under stable conditions, in the absence of a blood-brain barrier, and without interference by cerebral blood flow. The present thesis is presented in two sections. The first part focusses on longterm culture of 'organotypic' cerebral neocortex tissue, obtained from neonatal rat brain and explanted into a chemically defined medium. Procedures were developed which enabled culturing of this tissue without the occurence of central necrosis and with the preservation of a characteristic histiotypic organization. Morphological characteristics of the cultures were described and measured at various ages in vitro. In the second part, the cultures were used to study mechanisms that might contribute to the tissue uptake of radiopharmaceuticals which are in clinical use for SPECT brain imaging. (author). 369 refs.; 50 figs.; 13 tabs

  12. Effect of explant density and medium culture volumes on cassava micropropagation in Temporal Immersion System

    Directory of Open Access Journals (Sweden)

    Milagros Basail

    2003-04-01

    Full Text Available Due to the need of producing high quality planting material available to cassava growers, it has been necessary to look for alternatives in order to increase the efficiancy of in vitro propagation methods and their automation, such as the use of the Temporal Immersion Systems (RITA®. This work was carried out to increase the multiplication coefficient for cassava mass propagation through out Temporal Immersion Systems. The clone ‘CMC-40’ was used. Different medium volumes per explant, and material density per unit at a given Immersion frequency were tested. The highest results were obtained in the 2.8 multiplication coefficient with 20 ml culture medium volume and 3.2 using a density of 40 explants/flask. When the Temporal Immersion System is used with these results, a more efficient method for cassava micropropagation is established and also higher quality vitroplants for the rooting stage and further acclimatization in field conditions are produced. Key Words: Tissue Culture, liquid culture medium, Manihot esculenta Crantz

  13. The metabolic dynamics of cartilage explants over a long-term culture period

    Directory of Open Access Journals (Sweden)

    E.K Moo

    2011-01-01

    Full Text Available INTRODUCTION: Although previous studies have been performed on cartilage explant cultures, the generalized dynamics of cartilage metabolism after extraction from the host are still poorly understood due to differences in the experimental setups across studies, which in turn prevent building a complete picture. METHODS: In this study, we investigated the response of cartilage to the trauma sustained during extraction and determined the time needed for the cartilage to stabilize. Explants were extracted aseptically from bovine metacarpal-phalangeal joints and cultured for up to 17 days. RESULTS: The cell viability, cell number, proteoglycan content, and collagen content of the harvested explants were analyzed at 0, 2, 10, and 17 days after explantation. A high percentage of the cartilage explants were found to be viable. The cell density initially increased significantly but stabilized after two days. The proteoglycan content decreased gradually over time, but it did not decrease to a significant level due to leakage through the distorted peripheral collagen network and into the bathing medium. The collagen content remained stable for most of the culture period until it dropped abruptly on day 17. CONCLUSION: Overall, the tested cartilage explants were sustainable over long-term culture. They were most stable from day 2 to day 10. The degradation of the collagen on day 17 did not reach diseased levels, but it indicated the potential of the cultures to develop into degenerated cartilage. These findings have implications for the application of cartilage explants in pathophysiological fields.

  14. Effect of environmental and cultural conditions on medium pH and explant growth performance of Douglas-fir (Pseudotsuga menziesii shoot cultures [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Chien-Chih Chen

    2015-05-01

    Full Text Available The medium pH level of plant tissue cultures has been shown to be essential to many aspects of explant development and growth. Sensitivity or tolerance of medium pH change in vitro varies according to specific requirements of individual species. The objectives of this study are to 1 determine medium pH change over time in storage conditions and with presence of explants, 2 evaluate the effects of medium pH change on explant growth performance and 3 assess the effects of adding a pH stabilizer, 2-(N-morpholinoethanesulfonic acid (MES that is commonly used in Douglas-fir micropropagation medium. Vegetative buds were collected in the spring before breaking dormancy from juvenile and mature donor trees for conducting these evaluations. Medium, with or without MES, was pre-adjusted to five pH levels before adding MES, agar and autoclaving. Medium pH changes and explant growth parameters were measured at eight different incubation times. Overall, MES provided a more stable medium pH, relative to starting pH values, under both light and dark storage conditions as well as with presence of explants. A general trend of decreasing medium pH over time was found comparing explants from juvenile and mature donor genotypes. Explant height and weight growth increased over time, but differ among explants from juvenile and mature donor genotypes. Our findings suggest that a 21-day subculture practice may best sustain medium freshness, medium pH level and desirable explant growth.

  15. Evaluation of novel biodegradable three-armed- and hyper-branched tissue adhesives in a meniscus explant model.

    Science.gov (United States)

    Bochyńska, A I; Hannink, G; Verhoeven, R; Grijpma, D W; Buma, P

    2017-05-01

    Current treatment methods to repair meniscal tears do not bring fully satisfactory results. Tissue adhesives are considered promising alternatives, since they are easy to apply and cause minimal tissue trauma. The first aim of this study was to analyze the adhesive properties of and tissue response to two recently developed biodegradable block copolymeric three-armed- and hyper-branched tissue adhesives. The second aim was to investigate if tissue surface modification with collagenase improves the attachment of the adhesives and increases the healing potential of the tissue. Cylindrical explants were harvested from bovine menisci. The central core of the explants was removed and glued back into the defect, with or without incubation in collagenase solution prior to gluing, using one of the novel glues, Dermabond® or fibrin glue. The repair constructs were cultured in vitro for 1 and 28 days. Adhesion tests and histology were performed to analyze the effects of the glue in combination with the additional treatment. The adhesive strength of the novel glues was 40-50 kPa, which was significantly higher than that of fibrin glue (15 kPa). Cells were present in direct contact with the glues, and the tissue remained vital during the whole culture period. Increased cellularity around the tear in the collagenase treated explants was observed after 1 day. The two newly developed tissue adhesives are attractive materials to be used for repair of meniscal tears. The beneficial influence of collagenase treatment in treating meniscal tears with glues still needs to be confirmed in more clinical relevant studies. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1405-1411, 2017. © 2017 Wiley Periodicals, Inc.

  16. Development of efficient plant regeneration and transformation system for impatiens using Agrobacterium tumefaciens and multiple bud cultures as explants.

    Science.gov (United States)

    Dan, Yinghui; Baxter, Aaron; Zhang, Song; Pantazis, Christopher J; Veilleux, Richard E

    2010-08-09

    Impatiens (Impatiens walleriana) is a top selling floriculture crop. The potential for genetic transformation of Impatiens to introduce novel flower colors or virus resistance has been limited by its general recalcitrance to tissue culture and transformation manipulations. We have established a regeneration and transformation system for Impatiens that provides new alternatives to genetic improvement of this crop. In a first step towards the development of transgenic INSV-resistant Impatiens, we developed an efficient plant regeneration system using hypocotyl segments containing cotyledonary nodes as explants. With this regeneration system, 80% of explants produced an average of 32.3 elongated shoots per initial explant plated, with up to 167 elongated shoots produced per explant. Rooting efficiency was high, and 100% of shoots produced roots within 12 days under optimal conditions, allowing plant regeneration within approximately 8 weeks. Using this regeneration system, we developed an efficient Agrobacterium-mediated Impatiens transformation method using in vitro multiple bud cultures as explants and a binary plasmid (pHB2892) bearing gfp and nptII genes. Transgenic Impatiens plants, with a frequency up to 58.9%, were obtained within 12 to 16 weeks from inoculation to transfer of transgenic plants to soil. Transgenic plants were confirmed by Southern blot, phenotypic assays and T1 segregation analysis. Transgene expression was observed in leaves, stems, roots, flowers, and fruit. The transgenic plants were fertile and phenotypically normal. We report the development of a simple and efficient Agrobacterium-mediated transformation system for Impatiens. To the best of our knowledge, there have been no reports of Agrobacterium-mediated transformation of Impatiens with experimental evidence of stable integration of T-DNA and of Agrobacterium-mediated transformation method for plants using in vitro maintained multiple bud cultures as explants. This transformation system

  17. Development of Efficient Plant Regeneration and Transformation System for Impatiens Using Agrobacterium tumefaciens and Multiple Bud Cultures as Explants

    Directory of Open Access Journals (Sweden)

    Dan Yinghui

    2010-08-01

    Full Text Available Abstract Background Impatiens (Impatiens walleriana is a top selling floriculture crop. The potential for genetic transformation of Impatiens to introduce novel flower colors or virus resistance has been limited by its general recalcitrance to tissue culture and transformation manipulations. We have established a regeneration and transformation system for Impatiens that provides new alternatives to genetic improvement of this crop. Results In a first step towards the development of transgenic INSV-resistant Impatiens, we developed an efficient plant regeneration system using hypocotyl segments containing cotyledonary nodes as explants. With this regeneration system, 80% of explants produced an average of 32.3 elongated shoots per initial explant plated, with up to 167 elongated shoots produced per explant. Rooting efficiency was high, and 100% of shoots produced roots within 12 days under optimal conditions, allowing plant regeneration within approximately 8 weeks. Using this regeneration system, we developed an efficient Agrobacterium-mediated Impatiens transformation method using in vitro multiple bud cultures as explants and a binary plasmid (pHB2892 bearing gfp and nptII genes. Transgenic Impatiens plants, with a frequency up to 58.9%, were obtained within 12 to 16 weeks from inoculation to transfer of transgenic plants to soil. Transgenic plants were confirmed by Southern blot, phenotypic assays and T1 segregation analysis. Transgene expression was observed in leaves, stems, roots, flowers, and fruit. The transgenic plants were fertile and phenotypically normal. Conclusion We report the development of a simple and efficient Agrobacterium-mediated transformation system for Impatiens. To the best of our knowledge, there have been no reports of Agrobacterium-mediated transformation of Impatiens with experimental evidence of stable integration of T-DNA and of Agrobacterium-mediated transformation method for plants using in vitro maintained

  18. Immunocytochemical indications for neuronal co-localization of GABA and aspartate in cultured neocortex explants

    NARCIS (Netherlands)

    de Jong, B. M.; Ruijter, J. M.; Buijs, R. M.

    1989-01-01

    The application of postembedding immunocytochemistry on serial semithin plastic sections, revealed the presence of gamma-aminobutyric acid (GABA)-positive and aspartate-positive neurons in cultured neocortex explants. GABA-positive neurons were found in all layers of the cultured cortex, whereas

  19. Resolving browning during the establishment of explant cultures in Vicia faba L. for genetic transformation

    Directory of Open Access Journals (Sweden)

    Helena Klenotičová

    2013-01-01

    Full Text Available Optimisation of in vitro regeneration systems of two explant types for low-tannine cultivars of faba bean based on culturing of shoot apices and cotyledonary nodes were provided by usage of various antioxidants - ascorbic acid, citric acid, glutathione and activated charcoal. In subsequent testing, the combined effects of antioxidants with transformation co-cultivation compounds acetosyringone and L-cysteine was studied. The application of antioxidants lead to decreased callogenesis, citric acids treatments (50 mg.l−1 dramatically decreased necrotic response of explants. However, citric acid, used together with ascorbic acid completely inhibited shoot growth in shoot apex cultures. Glutathion evoked hyperhydricity of explants. Activated charcoal induced rooting on media which are commonly used for shoot proliferation. Combination of acetosyringone with antioxidants influenced shoot proliferation, except of variant with ascorbic acid. Citric acid was the best and universal antioxidant in faba bean in vitro cultures and its use is recommended for faba bean genetic transformation experiments.

  20. A procedure for culturing rat neocortex explants in a serum-free nutrient medium

    NARCIS (Netherlands)

    Romijn, H. J.; de Jong, B. M.; Ruijter, J. M.

    1988-01-01

    A procedure is described for long-term culturing of rat neocortex explants in a serum-free growth medium. Slices spanning the entire cortical depth from pial to ventricular side are prepared from 6-day-old rat pups. After preincubation in Hanks' balanced salt solution with extra glucose, the

  1. In vitro maintenance of spermatogenesis in Xenopus laevis testis explants cultured in serum-free media

    International Nuclear Information System (INIS)

    Risley, M.S.; Miller, A.; Bumcrot, D.A.

    1987-01-01

    Spermatogenesis has been maintained for extended periods in Xenopus laevis testis explants cultured in serum-free media supplemented with bovine serum albumin, insulin, transferrin, follicle-stimulating hormone, dihydrotestosterone, testosterone, retinol, ascorbate, and tocopherol. The organization of the testis fragments was maintained for 28 days, and all stages of development were present throughout the culture period. 3 H-Thymidine-labeled secondary (Type B) spermatogonia developed in 28 days into spermatids at the acrosomal vesicle stage whereas labeled zygotene spermatocytes became mature spermatids in 28 days. Spermatogonial proliferation also continued in vitro for 28 days. Germ cell differentiation was not dependent upon exogenous testosterone, ascorbate, or tocopherol since 3 H-labeled spermatogonia became mature spermatids in testes cultured 35 days in media lacking these supplements. Autoradiography demonstrated that 55% of the luminal sperm present in explants cultured 10 days had differentiated in vitro. Sperm from testes cultured 10-35 days were similar to sperm from freshly dissected testes with regard to motility and fecundity, and eggs fertilized with sperm from explant cultures developed normally into swimming tadpoles. The results demonstrate the feasibility of maintaining vertebrate spermatogenesis in culture and suggest that in vitro analysis of Xenopus spermatogenesis using defined media may provide important insights into the evolution of regulatory mechanisms in spermatogenesis

  2. Candidate Microbicides Block HIV-1 Infection of Human Immature Langerhans Cells within Epithelial Tissue Explants

    Science.gov (United States)

    Kawamura, Tatsuyoshi; Cohen, Sandra S.; Borris, Debra L.; Aquilino, Elisabeth A.; Glushakova, Svetlana; Margolis, Leonid B.; Orenstein, Jan M.; Offord, Robin E.; Neurath, A. Robert; Blauvelt, Andrew

    2000-01-01

    Initial biologic events that underlie sexual transmission of HIV-1 are poorly understood. To model these events, we exposed human immature Langerhans cells (LCs) within epithelial tissue explants to two primary and two laboratory-adapted HIV-1 isolates. We detected HIV-1Ba-L infection in single LCs that spontaneously emigrated from explants by flow cytometry (median of infected LCs = 0.52%, range = 0.08–4.77%). HIV-1–infected LCs downregulated surface CD4 and CD83, whereas MHC class II, CD80, and CD86 were unchanged. For all HIV-1 strains tested, emigrated LCs were critical in establishing high levels of infection (0.1–1 μg HIV-1 p24 per milliliter) in cocultured autologous or allogeneic T cells. HIV-1Ba-L (an R5 HIV-1 strain) more efficiently infected LC–T cell cocultures when compared with HIV-1IIIB (an X4 HIV-1 strain). Interestingly, pretreatment of explants with either aminooxypentane-RANTES (regulated upon activation, normal T cell expressed and secreted) or cellulose acetate phthalate (potential microbicides) blocked HIV-1 infection of LCs and subsequent T cell infection in a dose-dependent manner. In summary, we document HIV-1 infection in single LCs after exposure to virus within epithelial tissue, demonstrate that relatively low numbers of these cells are capable of inducing high levels of infection in cocultured T cells, and provide a useful explant model for testing of agents designed to block sexual transmission of HIV-1. PMID:11085750

  3. Changes in the metabolic footprint of placental explant-conditioned medium cultured in different oxygen tensions from placentas of small for gestational age and normal pregnancies.

    LENUS (Irish Health Repository)

    Horgan, R P

    2012-01-31

    Being born small for gestational age (SGA) confers significantly increased risks of perinatal morbidity and mortality. Accumulating evidence suggests that an SGA fetus results from a poorly perfused and abnormally developed placenta. Some of the placental features seen in SGA, such as abnormal cell turnover and impaired nutrient transport, can be reproduced by culture of placental explants in hypoxic conditions. Metabolic footprinting offers a hypothesis-generating strategy to investigate factors absorbed by and released from this tissue in vitro. Previously, metabolic footprinting of the conditioned culture media has identified differences in placental explants cultured under normoxic and hypoxic conditions and between normal pregnancies and those complicated by pre-eclampsia. In this study we aimed to examine the differences in the metabolic footprint of placental villous explants cultured at different oxygen (O(2)) tensions between women who deliver an SGA baby (n = 9) and those from normal controls (n = 8). Placental villous explants from cases and controls were cultured for 96 h in 1% (hypoxic), 6% (normoxic) and 20% (hyperoxic) O(2). Metabolic footprints were analysed by Ultra Performance Liquid Chromatography coupled to an electrospray hybrid LTQ-Orbitrap Mass Spectrometry (UPLC-MS). 574 metabolite features showed significant difference between SGA and normal at one or more of the oxygen tensions. SGA explant media cultured under hypoxic conditions was observed, on a univariate level, to exhibit the same metabolic signature as controls cultured under normoxic conditions in 49% of the metabolites of interest, suggesting that SGA tissue is acclimatised to hypoxic conditions in vivo. No such behaviour was observed under hyperoxic culture conditions. Glycerophospholipid and tryptophan metabolism were highlighted as areas of particular interest.

  4. 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.

  5. 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.

  6. Fumonisin B₁ (FB₁) Induces Lamellar Separation and Alters Sphingolipid Metabolism of In Vitro Cultured Hoof Explants.

    Science.gov (United States)

    Reisinger, Nicole; Dohnal, Ilse; Nagl, Veronika; Schaumberger, Simone; Schatzmayr, Gerd; Mayer, Elisabeth

    2016-03-24

    One of the most important hoof diseases is laminitis. Yet, the pathology of laminitis is not fully understood. Different bacterial toxins, e.g. endotoxins or exotoxins, seem to play an important role. Additionally, ingestion of mycotoxins, toxic secondary metabolites of fungi, might contribute to the onset of laminitis. In this respect, fumonsins are of special interest since horses are regarded as species most susceptible to this group of mycotoxins. The aim of our study was to investigate the influence of fumonisin B₁ (FB₁) on primary isolated epidermal and dermal hoof cells, as well as on the lamellar tissue integrity and sphingolipid metabolism of hoof explants in vitro. There was no effect of FB₁ at any concentration on dermal or epidermal cells. However, FB₁ significantly reduced the separation force of explants after 24 h of incubation. The Sa/So ratio was significantly increased in supernatants of explants incubated with FB₁ (2.5-10 µg/mL) after 24 h. Observed effects on Sa/So ratio were linked to significantly increased sphinganine concentrations. Our study showed that FB₁ impairs the sphingolipid metabolism of explants and reduces lamellar integrity at non-cytotoxic concentrations. FB₁ might, therefore, affect hoof health. Further in vitro and in vivo studies are necessary to elucidate the effects of FB₁ on the equine hoof in more detail.

  7. The effect of plant growth regulators, explants and cultivars on ...

    African Journals Online (AJOL)

    To achieve the best explants and media for spinach tissue culture, the effects of two different plant growth regulators, two explants and cultivars on adventitious shoot regeneration were tested. The Analysis of Variance (ANOVA) showed that the effects of plant growth regulators on spinach tissue culture were significant; ...

  8. [Influence of genotype, explant type and component of culture medium on in vitro callus induction and shoot organogenesis of tomato (Solanum lycopersicum L.)].

    Science.gov (United States)

    Khaliluev, M R; Bogoutdinova, L R; Baranova, G B; Baranova, E N; Kharchenko, P N; Dolgov, S V

    2014-01-01

    The influence of explant type as well as of the type of growth regulators and concentration on callus induction processes and somatic organogenesis of shoots was studied in vitro on four tomato genotypes of Russian breeding. Cytological study of callus tissue was conducted. It was established that tomato varieties possess a substantially greater ability to indirect shoot organogenesis compared with the F1 hybrid. The highest frequency of somatic organogenesis of shoots, as well as their number per explant, was observed for most of the genotypes studied during the cultivation of cotyledons on Murashige-Skoog culture medium containing 2 mg/l of zeatin in combination with 0.1 mg/l of 3-indoleacetic acid. An effective protocol of indirect somatic organogenesis of shoots from different explants of tomato varieties with a frequency of more than 80% was developed.

  9. Characterization of TLR-induced inflammatory responses in COPD and control lung tissue explants

    Directory of Open Access Journals (Sweden)

    Pomerenke A

    2016-09-01

    Full Text Available Anna Pomerenke,1 Simon R Lea,1 Sarah Herrick,2 Mark A Lindsay,3 Dave Singh1 1Centre for Respiratory Medicine and Allergy, Institute of Inflammation and Repair, Manchester Academic Health Science Centre, The University of Manchester and University Hospital of South Manchester, NHS Foundation Trust, 2Institute of Inflammation and Repair, Manchester Academic Health Science Centre, University of Manchester, Manchester, 3Department of Pharmacy and Pharmacology, University of Bath, Bath, UK Purpose: Viruses are a common cause of exacerbations in chronic obstructive pulmonary disease (COPD. They activate toll-like receptors (TLRs 3, 7, and 8, leading to a pro-inflammatory response. We have characterized the responses of TLR3 and TLR7/8 in lung tissue explants from COPD patients and control smokers.Methods: We prepared lung whole tissue explants (WTEs from patients undergoing surgery for confirmed or suspected lung cancer. In order to mimic the conditions of viral infection, we used poly(I:C for TLR3 stimulation and R848 for TLR7/8 stimulation. These TLR ligands were used alone and in combination. The effects of tumor necrosis factor α (TNFα neutralization and dexamethasone on TLR responses were examined. Inflammatory cytokine release was measured by enzyme-linked immunosorbent assay and gene expression by quantitative real-time polymerase chain reaction.Results: WTEs from COPD patients released higher levels of pro-inflammatory cytokines compared with WTEs from smokers. Activation of multiple TLRs led to a greater than additive release of TNFα and CCL5. TNFα neutralization and dexamethasone treatment decreased cytokine release.Conclusion: This WTE model shows an enhanced response of COPD compared with controls, suggesting an increased response to viral infection. There was amplification of innate immune responses with multiple TLR stimulation. Keywords: COPD, poly(I:C, R848, cytokines, lung explant

  10. E2f1 mediates high glucose-induced neuronal death in cultured mouse retinal explants.

    Science.gov (United States)

    Wang, Yujiao; Zhou, Yi; Xiao, Lirong; Zheng, Shijie; Yan, Naihong; Chen, Danian

    2017-10-02

    Diabetic retinopathy (DR) is the most common complication of diabetes and remains one of the major causes of blindness in the world; infants born to diabetic mothers have higher risk of developing retinopathy of prematurity (ROP). While hyperglycemia is a major risk factor, the molecular and cellular mechanisms underlying DR and diabetic ROP are poorly understood. To explore the consequences of retinal cells under high glucose, we cultured wild type or E2f1 -/- mouse retinal explants from postnatal day 8 with normal glucose, high osmotic or high glucose media. Explants were also incubated with cobalt chloride (CoCl 2 ) to mimic the hypoxic condition. We showed that, at 7 days post exposure to high glucose, retinal explants displayed elevated cell death, ectopic cell division and intact retinal vascular plexus. Cell death mainly occurred in excitatory neurons, such as ganglion and bipolar cells, which were also ectopically dividing. Many Müller glial cells reentered the cell cycle; some had irregular morphology or migrated to other layers. High glucose inhibited the hyperoxia-induced blood vessel regression of retinal explants. Moreover, inactivation of E2f1 rescued high glucose-induced ectopic division and cell death of retinal neurons, but not ectopic cell division of Müller glial cells and vascular phenotypes. This suggests that high glucose has direct but distinct effects on retinal neurons, glial cells and blood vessels, and that E2f1 mediates its effects on retinal neurons. These findings shed new light onto mechanisms of DR and the fetal retinal abnormalities associated with maternal diabetes, and suggest possible new therapeutic strategies.

  11. 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.

  12. The effect of radiation in combination with carcinogens on the growth of normal urothelium in explant culture

    International Nuclear Information System (INIS)

    Mothersill, C.; O'Brien, A.

    1990-01-01

    Radiation is known to be carcinogenic to humans but attempts to demonstrate the process using human tissue culture models have met with little success. In the present study explants were established from urothelium and exposed to radiation and a range of chemical carcinogens, suspected promotor or metabolic agents. The resulting outgrowth was monitored for growth rate, proliferating epithelial fraction and development and differentiation of endothelial cells in culture. The results indicate that enhanced growth of epithelial cells can be seen when cultures are irradiated in the presence of various nitrosamines, benzo(a)pyrene or aniline. Radiation alone reduced the overall growth area measured but several proliferative foci developed on the resulting outgrowth. Their ultrastructural appearance reveals that they carry severe mitochondrial damage and exposure of treated cultures to metabolic inhibitors confirms that their respiration is defective. Endothelial cells proliferated over the surface of the epithelial monolayer and both the number and the degree of differentiation of the endothelial cells increased with increasing dose up to 10 Gy. While the cultures are not immortalised by the treatment, it appears that the epithelial cells have an extended lifespan (division capacity) and that a subpopulation has undergone a number of premalignant changes. Changes in endothelial cell proliferation also occur. (orig.)

  13. Establishing axenic cultures from mature pecan embryo explants on media with low water availability.

    Science.gov (United States)

    Obeidy, A A; Smith, M A

    1990-12-01

    Endophytic fungi associated with mature pecan (Carya illinoensis (Wangenh.) C. Koch) nuts prevented successful, contaminant-free in vitro culture of embryo expiants, even after rigorous surface disinfestation of the nuts and careful aseptic shelling. Disinfestation with sodium hypochlorite after shell removal was also unsuccessful, because even dilute concentrations which were ineffective against the fungal contaminants prevented subsequent growth from the embryo. Explanting media with low water availability which would not sustain growth of fungal contaminants, but supported growth from mature pecan embryos, were developed as an alternative disinfestation method. The explanting media were supplemented with 0.9-1.5% agar, and other media components were selectively omitted to test their influence on water availability and fungal growth. Disinfestation of up to 65% of the cultures was accomplished, depending on the medium formulation, compared to 100% loss to contamination on control medium (0.5% agar). A complete medium (containing sucrose, salts, vitamins, 18 μM BAP, and 5 μM IBA) with 1.5% agar provided control of contamination, and encouraged subsequent regeneration from the embryo expiants, which remained free of contaminant growth through subsequent subcultures.

  14. Selection of valine-resistance in callus culture of Arabidopsis thaliana (L. Heynh. derived from leaf explants

    Directory of Open Access Journals (Sweden)

    Małgorzata D. Gaj

    2014-01-01

    Full Text Available The selection of valine-resistant mutants was carried out in leaf explant cultures of three Arabidopsis thaliana (L. Heynh. ecotypes: C-24, RLD and Columbia. The valine concentration used for in vitro selection, lethal for seed-growing plants, has not affected callus formation and growth. However, strong inhibition of shoot regeneration ability of calli growing under selection pressure was noticed. In total, 1043 explants were cultured on valine medium and 18 shoots were regenerated with an average frequency of 1.7 shoots per 100 calli. Most R1 shoots were sterile and seeds were collected from 3 plants. The transmission of valine-resistance to the sexual progeny of these plants was scored and the increased level of valine-resistance was found in progeny of one line - 61 C. This line originated from the culture of Columbia leaf explant and displayed tetraploid chromosome number.

  15. Neuronal differentiation of hair-follicle-bulge-derived stem cells co-cultured with mouse cochlear modiolus explants.

    Directory of Open Access Journals (Sweden)

    Timo Schomann

    Full Text Available Stem-cell-based repair of auditory neurons may represent an attractive therapeutic option to restore sensorineural hearing loss. Hair-follicle-bulge-derived stem cells (HFBSCs are promising candidates for this type of therapy, because they (1 have migratory properties, enabling migration after transplantation, (2 can differentiate into sensory neurons and glial cells, and (3 can easily be harvested in relatively high numbers. However, HFBSCs have never been used for this purpose. We hypothesized that HFBSCs can be used for cell-based repair of the auditory nerve and we have examined their migration and incorporation into cochlear modiolus explants and their subsequent differentiation. Modiolus explants obtained from adult wild-type mice were cultured in the presence of EF1α-copGFP-transduced HFBSCs, constitutively expressing copepod green fluorescent protein (copGFP. Also, modiolus explants without hair cells were co-cultured with DCX-copGFP-transduced HFBSCs, which demonstrate copGFP upon doublecortin expression during neuronal differentiation. Velocity of HFBSC migration towards modiolus explants was calculated, and after two weeks, co-cultures were fixed and processed for immunohistochemical staining. EF1α-copGFP HFBSC migration velocity was fast: 80.5 ± 6.1 μm/h. After arrival in the explant, the cells formed a fascicular pattern and changed their phenotype into an ATOH1-positive neuronal cell type. DCX-copGFP HFBSCs became green-fluorescent after integration into the explants, confirming neuronal differentiation of the cells. These results show that HFBSC-derived neuronal progenitors are migratory and can integrate into cochlear modiolus explants, while adapting their phenotype depending on this micro-environment. Thus, HFBSCs show potential to be employed in cell-based therapies for auditory nerve repair.

  16. 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.

  17. Micropropagation from cultured nodal explants of rose (Rosa hybrida L. cv. ‘Perfume Delight’

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    Kamnoon Kanchanapoom

    2010-01-01

    Full Text Available A method for the micropropagation of rose (Rosa hybrida L. cv. ‘Perfume Delight’ was developed. First to fifth nodal explants from young healthy shoots were excised and cultured on basal medium of Murashige and Skoog (1962, MS containing several concentrations of BA and NAA. Multiple shoot formation of up to 3 shoots was obtained on MS medium supplemented with 3 mg/l BA and 0.003 mg/l NAA. Shoot readily rooted on ¼MS medium devoid of growth regulators.Rooted plantlets were hardened and established in pots at 100% survival. In vitro flowering was observed on rose plantscultured on MS medium containing 3 mg/l BA and 0.003 mg/l NAA.

  18. Efficient regeneration of plants from shoot tip explants of ...

    African Journals Online (AJOL)

    Dendrobium densiflorum Lindl. is one of the horticulturally important orchids of Nepal due to its beautiful yellowish flower and medicinal properties. The present study was carried out for plant regeneration from shoot tip explants of D. densiflorum by tissue culture technique. The shoot tip explants of this species, obtained ...

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

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    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.

  20. Articular cartilage explant culture; an appropriate in vitro system to compare osteoarthritic and normal human cartilage

    NARCIS (Netherlands)

    Lafeber, F. P.; Vander Kraan, P. M.; van Roy, J. L.; Huber-Bruning, O.; Bijlsma, J. W.

    1993-01-01

    Proteoglycan metabolism of normal and histologically mild to moderate osteoarthritic cartilage explants were studied. Explants were obtained from the human knee of donors aged over 40 years. Proteoglycan content, synthesis and release were very similar in normal cartilage obtained from donors with

  1. 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

  2. In vitro plant regeneration of two cucumber (Cucumis sativum L. genotypes: Effects of explant types and culture medium

    Directory of Open Access Journals (Sweden)

    Grozeva Stanislava

    2014-01-01

    Full Text Available The effect of different phytohormone concentrations on callusogenesis and organogenesis in two cucumber genotypes were studied. It was established that the rate of plant regeneration depends on genotype, explant type and culture medium. Hypocotyls were found to be more responsive than cotyledons in morphogenesis. In vitro planlet-regenerants have been obtained in hypocotyls explants on culture medium with 1.0 and 2.0 mgL-1 BA for cultivar Gergana and in 1.0 and 3.0 mgL-1K-line 15B. Induction of regeneration in cotyledons were established only in cultivar Gergana on culture medium supplemented with 3.0 mgL-1 BA and in combination of 0.5 mgL-1IAA.

  3. Effects of donor plant age and explants on in vitro culture of Cedrela montana Moritz ex Turcz

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    Sofía Basto

    2012-12-01

    Full Text Available To evaluate the organogenic potential of Cedrela montana Moritz ex Turcz. Explants from mature (10-20 year-old and juvenile (7-18 month-old trees were collected. The first grouping included buds, leaves, and nodes derived from juvenile basal offshoots and rejuvenated shoots from cuttings. The second, included leaves, petioles, nodes, internodes and nodes of in vitro elongated shoots. The highest organogenic potential was observed in nodes from juvenile trees: 45.8% of explants presented axillary bud elongation, while 56.2% presented rooting in a growth regulator free culture medium. Fifty-one percent of elongated shoots produced adventitious shoots with 0.5 μM NAA and 0.5 μM BA; 30% with 0.5 μM NAA and 1 μM BA; and 30% with 1 μM BA. Twenty percent presented roots with 0.5 μM NAA. Root formation was stimulated in a medium supplemented with activated charcoal (5 gL-1. The acclimatization of eighty percent of plantlets regenerated from nodes, and of 72.5% in vitro generated shoots was successful. On the contrary, mature trees material presented low organogenic response. Axillary bud elongation was recorded just in 10.7% of explants from juvenile shoots and in 6.7% of explants from rejuvenated shoots. The age of donor plant and type of explant affect the organogenic potential of C. montana. This study contributes to the understanding of this species’ response under in vitro conditions.

  4. Nanoparticles containing siRNA to silence CD4 and CCR5 reduce expression of these receptors and inhibit HIV-1 infection in human female reproductive tract tissue explants

    Directory of Open Access Journals (Sweden)

    Susan K. Eszterhas

    2011-09-01

    Full Text Available Human Immunodeficiency Virus-type 1 (HIV- 1 binds to CD4 and CCR5 receptors on target cells in the human female reproductive tract. We sought to determine whether reducing levels of messenger RNA (mRNA transcripts that encode these receptors in female reproductive tract cells could protect mucosal tissue explants from HIV- 1 infection. Explants prepared from the endometrium, endocervix, and ectocervix of hysterectomy tissues from HIV-1 sero-negative women were exposed to nanoparticles containing CD4- and CCR5-specific short-interfering RNA (siRNA sequences. Explants were then exposed two days later to HIV-1, and HIV-1 reverse transcripts were measured five days post-infection. Explants treated with nanoparticles containing CD4- and CCR5-specific siRNA showed reduced levels of CD4 and CCR5 transcripts, and significantly lower levels of HIV-1 reverse transcripts compared to those treated with an irrelevant siRNA. In female reproductive tract explants and in peripheral blood cell cultures, siRNA transfection induced the secretion of IFN-alpha (IFN-α, a potent antiviral cytokine. In female mice, murine-specific Cd4-siRNA nanoparticles instilled within the uterus significantly reduced murine Cd4 transcripts by day 3. Our findings demonstrate that siRNA nanoparticles reduce expression of HIV-1 infectivity receptors in human female reproductive tract tissues and also inhibit HIV-1 infection. Murine studies demonstrate that nanoparticles can penetrate the reproductive tract tissues in vivo and silence gene expression. The induction of IFN-α after siRNA transfection can potentially contribute to the antiviral effect. These findings support the therapeutic development of nanoparticles to deliver siRNA molecules to silence host cell receptors in the female reproductive tract as a novel microbicide to inhibit mucosal HIV-1 transmission.

  5. Inflammatory Response of Human Gestational Membranes to Ureaplasma parvum Using a Novel Dual-Chamber Tissue Explant System.

    Science.gov (United States)

    Potts, Lauren C; Feng, Liping; Seed, Patrick C; Jayes, Friederike L; Kuchibhatla, Maragatha; Antczak, Brian; Nazzal, Matthew K; Murtha, Amy P

    2016-05-01

    Preterm premature rupture of membranes (PPROM) is often associated with intra-amniotic inflammation and infection. Current understanding of the pathogenesis of PPROM includes activation of pro-inflammatory cytokines and proteolytic enzymes leading to compromise of membrane integrity. The impact of exposure to bacterial pathogens, including Ureaplasma parvum, on gestational membranes is poorly understood. Our objective was to develop a dual-chamber system to characterize the inflammatory response of gestational membranes to U. parvum in a directional nature. Full-thickness human gestational membrane explants, with either choriodecidua or amnion oriented superiorly, were suspended between two washers in a cylindrical device, creating two distinct compartments. Brilliant green dye was introduced into the top chamber to assess the integrity of the system. Tissue viability was evaluated after 72 h using a colorimetric cell proliferation assay. Choriodecidua or amnion was exposed to three doses of U. parvum and incubated for 24 h. Following treatment, media from each compartment were used for quantification of U. parvum (quantitative PCR), interleukin (IL)-8 (enzyme-linked immunosorbent assay), and matrix metalloproteinase (MMP)-2 and MMP-9 activity (zymography). We observed that system integrity and explant viability were maintained over 72 h. Dose-dependent increases in recovered U. parvum, IL-8 concentration, and MMP-2 activity were detected in both compartments. Significant differences in IL-8 concentration and MMP-9 activity were found between the choriodecidua and amnion. This tissue explant system can be used to investigate the inflammatory consequences of directional bacterial exposure for gestational membranes and provides insight into the pathogenesis of PPROM and infectious complications of pregnancy. © 2016 by the Society for the Study of Reproduction, Inc.

  6. The effects of low-level ionising radiation on primary explant cultures of rainbow trout Pronephros

    International Nuclear Information System (INIS)

    Olwell, P.; Ni Shuilleabhain, S.; Mothersill, C.; Seymour, C.; Cottell, D.C.; Lyng, F.M.

    2004-01-01

    It has long been known that the haematopoietic tissue of mammals is one of the most radiosensitive tissues. In vitro studies on prawn have also shown that low doses of radiation has an extremely deleterious effect on cells cultured from this animal's blood forming tissues. This raises the question on the relative effects of radiation between animals from different species. One of the most important aquatic animals, from both an economic and ecologic point of view, is the fish. With this in mind, primary cultures of the blood forming tissues of rainbow trout were exposed to radiation followed by a morphological comparison between control and irradiated cultures. The cultured cells were characterised as macrophages following incubation with non-specific fluorescent beads and human apoptotic PMN. The cells demonstrated both specific and non-specific phagocytosis, by consuming the non-indigenous bodies, and were classified as phagocytic leucocytes. These cells were found in two morphological forms, stretched and rounded. It was shown that there was a commensurate increase in the number of stretched cells following application of radiation. Radiation was also shown to cause a dose-dependent increase in the amounts of apoptosis and necrosis in cells over time. The phagocytic efficacy of the irradiated leucocytes compared to controls was also investigated. (author)

  7. Label-Free Imaging of Umbilical Cord Tissue Morphology and Explant-Derived Cells

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    Raf Donders

    2016-01-01

    Full Text Available In situ detection of MSCs remains difficult and warrants additional methods to aid with their characterization in vivo. Two-photon confocal laser scanning microscopy (TPM and second harmonic generation (SHG could fill this gap. Both techniques enable the detection of cells and extracellular structures, based on intrinsic properties of the specific tissue and intracellular molecules under optical irradiation. TPM imaging and SHG imaging have been used for label-free monitoring of stem cells differentiation, assessment of their behavior in biocompatible scaffolds, and even cell tracking in vivo. In this study, we show that TPM and SHG can accurately depict the umbilical cord architecture and visualize individual cells both in situ and during culture initiation, without the use of exogenously applied labels. In combination with nuclear DNA staining, we observed a variance in fluorescent intensity in the vessel walls. In addition, antibody staining showed differences in Oct4, αSMA, vimentin, and ALDH1A1 expression in situ, indicating functional differences among the umbilical cord cell populations. In future research, marker-free imaging can be of great added value to the current antigen-based staining methods for describing tissue structures and for the identification of progenitor cells in their tissue of origin.

  8. Effect of Pre-culture Irradiation and Explant Types on Efficiency of Brassica napus Genetic Transformation

    International Nuclear Information System (INIS)

    Amer, I.M.; Moustafa, H.A.M.; Azzam, C.R.

    2008-01-01

    The irradiated seeds of canola cv. Drakkar ( Brassica napus l. ) were germinated under aspect conditions, cotyledonary petioles and hypocotyl of 6 days old seedlings were used for Agrobacterium-mediated transformation. Agrobacterium tumefaciens has construct with the selectable marker gene (NPT II) and the desirable gene (HPPD). Direct and indirect shoot organogenesis were obtained from the both explants. Cotyledonary petioles was higher responded than hypocotyl with respective 26% and 14% of the explants producing NPT II-positive shoots after the selection on 50mg/l kanamycin. Calli might develop on and not in the agar medium were un transformation. This explains the higher number of escapes detected in hypocotyl explants than in experiments with cotyledons. The frequency of transformation plants as a function of indirect organogenesis was more than direct shoot regeneration from explants. The pre- irradiation with 75 Gy of gamma rays enhanced the genetic transformation frequencies by about 10 % as compared to that of the un-irradiated material. The obtained shoots were rooted and regenerated mature plants

  9. Effects of plant growth regulators on in vitro cultured nodal explants ...

    African Journals Online (AJOL)

    SAM

    2014-07-09

    Jul 9, 2014 ... sterilized potting mix of forest soil, well-decomposed coffee husk, and red sand at the respective ratio of 1:1:2 by volume (JARC experience) and the stock plants were established in the main- tenance greenhouse of the biotechnology division at an average temperature of 25 ± 2°C. Explant sterilization and ...

  10. Effect of Macrophage Migration Inhibitory Factor (MIF) in Human Placental Explants Infected with Toxoplasma gondii Depends on Gestational Age

    Science.gov (United States)

    de Oliveira Gomes, Angelica; de Oliveira Silva, Deise Aparecida; Silva, Neide Maria; de Freitas Barbosa, Bellisa; Franco, Priscila Silva; Angeloni, Mariana Bodini; Fermino, Marise Lopes; Roque-Barreira, Maria Cristina; Bechi, Nicoletta; Paulesu, Luana Ricci; dos Santos, Maria Célia; Mineo, José Roberto; Ferro, Eloisa Amália Vieira

    2011-01-01

    Because macrophage migration inhibitory factor (MIF) is a key cytokine in pregnancy and has a role in inflammatory response and pathogen defense, the objective of the present study was to investigate the effects of MIF in first- and third-trimester human placental explants infected with Toxoplasma gondii. Explants were treated with recombinant MIF, IL-12, interferon-γ, transforming growth factor-β1, or IL-10, followed by infection with T. gondii RH strain tachyzoites. Supernatants of cultured explants were assessed for MIF production. Explants were processed for morphologic analysis, immunohistochemistry, and real-time PCR analysis. Comparison of infected and stimulated explants versus noninfected control explants demonstrated a significant increase in MIF release in first-trimester but not third-trimester explants. Tissue parasitism was higher in third- than in first-trimester explants. Moreover, T. gondii DNA content was lower in first-trimester explants treated with MIF compared with untreated explants. However, in third-trimester explants, MIF stimulus decreased T. gondii DNA content only at the highest concentration of the cytokine. In addition, high expression of MIF receptor was observed in first-trimester placental explants, whereas MIF receptor expression was low in third-trimester explants. In conclusion, MIF was up-regulated and demonstrated to be important for control of T. gondii infection in first-trimester explants, whereas lack of MIF up-regulation in third-trimester placentas may be involved in higher susceptibility to infection at this gestational age. PMID:21641401

  11. Establishment and Characterization of Primary Cultures from Iranian Oral Squamous Cell Carcinoma Patients by Enzymatic Method and Explant Culture

    Directory of Open Access Journals (Sweden)

    Meysam Ganjibakhsh

    2017-10-01

    Full Text Available Objectives: Oral Squamous Cell Carcinoma (OSCC is the most frequent oral cancer worldwide. It is known as the eighth most common cancer in men and as the fifth most common cancer in women. Cytogenetic and biochemical studies in recent decades have emphasized the necessity of providing an appropriate tool for such researches. Cancer cell culture is a useful tool for investigations on biochemical, genetic, molecular and immunological characteristics of different cancers, including oral cancer. Here, we explain the establishment process of five primary oral cancer cells derived from an Iranian population.Materials and Methods: The specimens were obtained from five oral cancer patients. Enzymatic, explant culture and magnetic-activated cell sorting (MACS methods were used for cell isolation. After quality control tests, characterization and authentication of primary oral cancer cells were performed by short tandem repeats (STR profiling, chromosome analysis, species identification, and monitoring the growth, morphology and the expression of CD326 and CD133 markers.Results: Five primary oral cancer cells were established from an Iranian population. The flow cytometry results showed that the isolated cells were positive for CD326 and CD133 markers. Furthermore, the cells were free from mycoplasma, bacterial and fungal contamination. No misidentified or cross-contaminated cells were detected by STR analysis.Conclusions: Human primary oral cancer cells provide an extremely useful platform for studying carcinogenesis pathways of oral cancer in Iranian population. They may be helpful in explaining the ethnic differences in cancer biology and the individuality in anticancer drug response in future studies.

  12. 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

  13. 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.

  14. 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

  15. Plantlet regeneration potential from seedling explants of vitegnus (Vitex agnus castus).

    Science.gov (United States)

    Chamandoosti, F

    2007-11-15

    In this research a simple and repeatable method for regeneration of a important medicinal plant (Vitex agnus castus) described. Different seedling explants such as hypocotyl, cotyledon, root and apical meristem were cultured in MS basal media with different kinds and concentrations of PGRs. Root and apical meristem explants were the only explants that have regeneration whole plantlets potential. It was interesting that regeneration whole plantlets from root and apical meristem explants have different developmental pathways. Whole plantlets from apical meristem explants regenerated by passing phase callusing whereas regeneration whole plantlets from root was direct and without phase callusing. This subject implies that we can have many manipulation possibilities in order to different objects of tissue culture by selecting different explants in vitegnus.

  16. 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

  17. 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.

  18. 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)

  19. The Effects of Polyphenol Oxidase and Cycloheximide on the Early Stage of Browning in Phalaenopsis Explants

    Directory of Open Access Journals (Sweden)

    Xu Chuanjun

    2015-11-01

    Full Text Available Explant browning is one of the major problems in the tissue culture process, and polyphenol oxidase (PPO, is the major proteases involved in plant tissue browning. We investigated the effects of polyphenol oxidase on the early stage of browning in explants of the orchid Phalaenopsis. Our results show that PPO activity was significantly higher in explants cultured for 3 d than in the 0 h control. The levels of PPO transcripts and PPO protein were significantly higher in explants cultured for 6 h compared to the 0 h control; these high expression levels were maintained over increasing cultivation time. Cycloheximide (CHX treatment reduced PPO transcript levels, PPO protein levels, and PPO enzyme activity. High levels of PPO mRNA and PPO protein were detected in the cytoplasm and vascular bundles of Phalaenopsis explants cultured for 6 h compared to explants cultured for 0 h, 24 h, and 3 d. CHX treatment did not significantly affect the distribution of PPO mRNA and PPO protein in explant tissues, but their levels were significantly lower than those of the untreated control.

  20. Morphogenic responses of three explants of Lupinus montanus (H.B.K. cultured in vitro

    Directory of Open Access Journals (Sweden)

    Gabriel Ramírez-González

    2015-01-01

    Full Text Available Resumen El necrosamiento y la respuesta morfogénica de explantes de cotiledón, epicótilo e hipocótilo de plántulas de Lupinus montanus (H.B.K. germinadas in vitro se evaluaron para establecer las condiciones básicas de micropropagación. El necrosamiento se evaluó en medio MS con 0.40 mg·litro-1 de tiamina, 100 mg·litro-1 de mioinositol, 3 % de sacarosa y 7 g·litro-1 de agar-agar y distintas dosis de carbón activado (CA y ácido cítrico. Los tratamientos con CA (50 mg·litro-1 y 100 mg·litro-1 fueron los mejores en el control del necrosamiento (P = 0.001; el cotiledón tuvo mayor nivel de necrosamiento ( 70 % a diferencia del cotiledón, cuya respuesta fue primordialmente callogénica (50 %. El epicótilo cultivado en medio MS con 3.0 μM de AIA y 1.0 μM de BA tuvo el mayor número de brotes (10 y altura (11.4 ± 2.6 cm.

  1. Long-term culture of sponge explants: conditions enhancing survival and growth, and assessment of bioactivity

    NARCIS (Netherlands)

    Caralt, de S.; Agell, G.; Uriz, M.J.

    2003-01-01

    Sponges are an important source of secondary metabolites with pharmaceutical interest. This is the main reason for the increasing interest of sponge culture recent years. The optimal culture system depends on the species to be cultured: while some species easily produce sponge aggregates after

  2. Factors Influencing the Tissue Culture and the Agrobacterium tumefaciens-Mediated Transformation of Hybrid Aspen and Poplar Clones.

    Science.gov (United States)

    De Block, M

    1990-07-01

    Tissue culture conditions and transformation have been established for both aspen and poplar. The use of previously described culture conditions resulted in shoot tip necrosis in the shoot cultures and necrosis of stem and leaf explants. Shoot tip necrosis could be overcome by buffering the medium with 2-(N-morpholino)ethanesulfonic acid and Ca-gluconate and by growing the shoots below 25 degrees C. Necrosis of the explants was probably due to an accumulation of ammonium in the explants and could be overcome by adapting the NO(3) (-)/NH(4) (+) ratio of the media. Stem explants of established shoot cultures of the aspen hybrid Populus alba x P. tremula and of the poplar hybrid Populus trichocarpa x P. deltoides were cocultivated with Agrobacterium strains having chimeric bar and neo genes on their disarmed tDNAs. Transformed aspen shoots were obtained from 30 to 40% of the explants, while transformed poplar shoots were obtained from 10% of the explants. Extracts from the transformed trees contained high phosphinotricin acetyltransferase and neomycin phosphotransferase activities, and the trees contained one to three copies of the chimeric genes. The transformed trees were completely resistant to the commercial preparations of the herbicide phosphinotricin (glufosinate), while control trees were not.

  3. 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

  4. 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 ...

  5. 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)

  6. Notochord manipulation does not impact oesophageal and tracheal formation from isolated foregut in 3D explant culture.

    Science.gov (United States)

    Mc Laughlin, Danielle; Murphy, Paula; Puri, Prem

    2016-01-01

    Tracheo-oesophageal malformations result from disturbed foregut separation during early development. The notochord, a specialised embryonic structure, forms immediately adjacent to the dividing foregut. In the Adriamycin mouse model of oesophageal atresia, foregut and notochord abnormalities co-exist, and the site and severity of foregut malformations closely correlate to the position and extent of the notochord defects. Notochord and foregut abnormalities also co-exist in the Noggin Knockout mouse as well in a small number of human cases. The notochord is a source of powerful molecular signals during early embryogenesis, being particularly important for neural crest development. The influence of notochord signaling on the adjacent foregut is not known. The purpose of this study was to examine the impact of notochord manipulation on foregut separation using a robust 3D explant method for culturing isolated foregut which permits oeosphageal and tracheal formation in vitro. Foregut was micro-dissected from embryonic day 9 mice (License B100/4447 Irish Medicines Board), embedded in collagen and cultured for 48 h with native notochord intact (n = 6), notochord removed (n = 10) or additional notochord transplanted from stage matched controls (n = 8). Specimens were analysed for foregut morphology and molecular patterning using immunohistochemistry for Hnf3b (an endoderm marker) and Sox2 (a notochord and oesophageal marker) on cryosections. Foregut separation into distinct oesophagus and trachea was observed in isolated foregut specimens with or without their native notochord. In specimens with additional notochord transplants, foregut morphology and molecular patterning were comparable to controls whether or not the native notochord was maintained. In particular foregut separation was not disrupted by the transplantation of additional notochord at the dorsal foregut endoderm. The relationship between the embryonic foregut and notochord is complex and ill

  7. Low-intensity pulsed ultrasound (LIPUS) and pulsed electromagnetic field (PEMF) treatments affect degeneration of cultured articular cartilage explants

    NARCIS (Netherlands)

    Tan, Lijun; Ren, Yijin; van Kooten, Theo G.; Grijpma, Dirk W.; Kuijer, Roelof

    PURPOSE: Articular cartilage has some capacity for self-repair. Clinically used low-intensity pulsed ultrasound (LIPUS) and pulsed electromagnetic field (PEMF) treatments were compared in their potency to prevent degeneration using an explant model of porcine cartilage. METHODS: Explants of porcine

  8. Low-intensity pulsed ultrasound (LIPUS) and pulsed electromagnetic field (PEMF) treatments affect degeneration of cultured articular cartilage explants

    NARCIS (Netherlands)

    Tan, Lijun; Tan, Lijun; Ren, Yijin; van Kooten, Theo G.; Grijpma, Dirk W.; Kuijer, Roel

    2015-01-01

    Purpose: Articular cartilage has some capacity for self-repair. Clinically used low-intensity pulsed ultrasound (LIPUS) and pulsed electromagnetic field (PEMF) treatments were compared in their potency to prevent degeneration using an explant model of porcine cartilage. Methods: Explants of porcine

  9. The Stimulatory Effect of Notochordal-Cell Conditioned Medium in a Nucleus Pulposus Explant Culture

    NARCIS (Netherlands)

    de Vries, Stefan; Doeselaar, Marina van; Meij, Björn; Tryfonidou, M; Ito, Keita

    2015-01-01

    OBJECTIVES: Notochordal cell-conditioned medium (NCCM) has previously shown to have a stimulatory effect on nucleus pulposus cells (NPCs) and bone marrow stromal cells (BMSCs) in alginate and pellet cultures. These culture methods provide a different environment than the nucleus pulposus (NP)

  10. The Stimulatory Effect of Notochordal Cell-Conditioned Medium in a Nucleus Pulposus Explant Culture

    NARCIS (Netherlands)

    de Vries, Stefan A H; van Doeselaar, Marina; Meij, Björn P; Tryfonidou, Marianna A; Ito, K

    2016-01-01

    Objectives: Notochordal cell-conditioned medium (NCCM) has previously shown to have a stimulatory effect on nucleus pulposus cells (NPCs) and bone marrow stromal cells (BMSCs) in alginate and pellet cultures. These culture methods provide a different environment than the nucleus pulposus (NP)

  11. 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)

  12. In vitro performances of hypocotyl and cotyledon explants of tomato ...

    African Journals Online (AJOL)

    A plant tissue culture technique is a good method for the evaluation and screening of plant genotypes for salt tolerance. In vitro evaluations of sodium chloride (NaCl) effects on two tomato cultivars (Pearl and Beril) were investigated with four NaCl levels (0, 25, 50 and 75 mM) using hypocotyl and cotyledon explants.

  13. The effect of plant growth regulators, explants and cultivars on ...

    African Journals Online (AJOL)

    ONOS

    2010-07-05

    Jul 5, 2010 ... The effect of plant growth regulators, explants and cultivars on spinach (Spinacia oleracea L.) tissue culture. Taha Roodbar Shojaei1*, Vahid Salari2, Darioush Ramazan3, Mahdi Ehyaei1, Javad. Gharechahi4 and Roya Motallebi Chaleshtori5. 1Department of Agronomy and Plant Breeding, College of ...

  14. 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.

  15. 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.

  16. 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)

  17. 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.

  18. The Influence of Explant Types and Orientation on in Vitro Culture

    Directory of Open Access Journals (Sweden)

    Kamnoon KANCHANAPOOM

    2011-08-01

    Full Text Available Inflorescence, apical and lateral buds of Musa balbisiana ‘Kluai Hin’ (BBB group were used to culture on MS medium supplemented with 22 μM BA and 15% (v/v coconut water. Comparison of bud orientation showed that the best response of in vitro shoot tip proliferation was obtained with abaxial surface of buds lying down i.e. one side touching the medium (tilt. Mass propagation of shoot tips was obtained when cultured buds on MS medium containing 44 μM BA. Rooting was achieved by transferring the isolated shoots to MS basal medium without growth regulators. Rooted plantlets were acclimatized and successfully established in soil.

  19. 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...

  20. Tumorigenic risk of human induced pluripotent stem cell explants cultured on mouse SNL76/7 feeder cells

    Energy Technology Data Exchange (ETDEWEB)

    Kamada, Mizuna; Mitsui, Youji, E-mail: y-mitsui8310@hb.tp1.jp; Kumazaki, Tsutomu; Kawahara, Yuta; Matsuo, Taira; Takahashi, Tomoko, E-mail: t-takahashi@kph.bunri-u.ac.jp

    2014-10-24

    Highlights: • hiPS cell explants formed malignant tumors when SNL76/7 feeder cells were used. • Multi type tumors developed by interaction of SNL76/7 feeder cells with hiPS cells. • Tumorigenic risk occurs by co-culture of hiPS cells with SNL76/7 feeder cells. - Abstract: The potential for tumor formation from transplanted human induced pluripotent stem cell (hiPSC) derivatives represents a high risk in their application to regenerative medicine. We examined the genetic origin and characteristics of tumors, that were formed when 13 hiPSC lines, established by ourselves, and 201B7 hiPSC from Kyoto University were transplanted into severe combined immune-deficient (SCID) mice. Though teratomas formed in 58% of mice, five angiosarcomas, one malignant solitary fibrous tumor and one undifferentiated pleomorphic sarcoma formed in the remaining mice. Three malignant cell lines were established from the tumors, which were derived from mitomycin C (MMC)-treated SNL76/7 (MMC-SNL) feeder cells, as tumor development from fusion cells between MMC-SNL and hiPSCs was negative by genetic analysis. While parent SNL76/7 cells produced malignant tumors, neither MMC-SNL nor MMC-treated mouse embryo fibroblast (MEF) produced malignant tumors. When MMC-SNL feeder cells were co-cultured with hiPSCs, growing cell lines were generated, that expressed genes similar to the parent SNL76/7 cells. Thus, hiPSCs grown on MMC-SNL feeder cells have a high risk of generating feeder-derived malignant tumors. The possible mechanism(s) of growth restoration and the formation of multiple tumor types are discussed with respect of the interactions between MMC-SNL and hiPSC.

  1. 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)

  2. Suppression of MMP activity in bovine cartilage explants cultures has little if any effect on the release of aggrecanase-derived aggrecan fragments

    DEFF Research Database (Denmark)

    Wang, Bijue; Chen, Pingping; Jensen, Anne-Christine Bay

    2009-01-01

    BACKGROUND: Progressive loss of articular cartilage is a central hallmark in many joint disease, however, the relative importance of individual proteolytic pathways leading to cartilage erosion is at present unknown. We therefore investigated the time-dependant release ex vivo of MMP- and aggreca......BACKGROUND: Progressive loss of articular cartilage is a central hallmark in many joint disease, however, the relative importance of individual proteolytic pathways leading to cartilage erosion is at present unknown. We therefore investigated the time-dependant release ex vivo of MMP......- and aggrecanase-derived fragments of aggrecan and type II collagen into the supernatant of bovine cartilage explants cultures using neo-epitope specific immunoassays, and to associate the release of these fragments with the activity of proteolytic enzymes using inhibitors. FINDINGS: Bovine cartilage explants were...... cultured in the presence or absence of the catabolic cytokines oncostatin M (OSM) and tumor necrosis factor alpha (TNFalpha). In parallel, explants were co-cultured with protease inhibitors such as GM6001, TIMP1, TIMP2 and TIMP3. Fragments released into the supernatant were determined using a range of neo...

  3. Streptococcus pneumoniae-induced ototoxicity in organ of Corti explant cultures.

    Science.gov (United States)

    Perny, Michael; Solyga, Magdalena; Grandgirard, Denis; Roccio, Marta; Leib, Stephen L; Senn, Pascal

    2017-07-01

    Hearing loss remains the most common long-term complication of pneumococcal meningitis (PM) reported in up to 30% of survivors. Streptococcus pneumoniae have been shown to possess different ototoxic properties. Here we present a novel ex vivo experimental setup to examine in detail the pattern of hair cell loss upon exposure to different S. pneumoniae strains, therefore recapitulating pathogen derived aspects of PM-induced hearing loss. Our results show a higher susceptibility towards S. pneumoniae-induced cochlear damage for outer hair cells (OHC) compared to inner hair cells (IHC), which is consistent with in vivo data. S. pneumoniae-induced hair cell loss was both time and dose-dependent. Moreover, we have found significant differences in the level of cell damage between tissue from the basal and the apical turns. This shows that the higher vulnerability of hair cells located at high frequency regions observed in vivo cannot be explained solely by the spatial organisation and bacterial infiltration from the basal portion of the cochlea. Using a wild type D39 strain and a mutant defective for the pneumolysin (PLY) gene, we also have shown that the toxin PLY is an important factor involved in ototoxic damages. The obtained results indicate that PLY can cause both IHC and OHC loss. Finally, we are reporting here for the first time a higher vulnerability of HC located at the basal and middle cochlear region to pneumolysin-induced damage. The detailed description of the susceptibility of hair cells to Streptococcus pneumoniae provided in this report can in the future determine the choice and the development of novel otoprotective therapies during pneumococcal meningitis. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  4. 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.

  5. Suppression of MMP activity in bovine cartilage explants cultures has little if any effect on the release of aggrecanase-derived aggrecan fragments

    Directory of Open Access Journals (Sweden)

    Sondergaard Bodil-Cecilie

    2009-12-01

    Full Text Available Abstract Background Progressive loss of articular cartilage is a central hallmark in many joint disease, however, the relative importance of individual proteolytic pathways leading to cartilage erosion is at present unknown. We therefore investigated the time-dependant release ex vivo of MMP- and aggrecanase-derived fragments of aggrecan and type II collagen into the supernatant of bovine cartilage explants cultures using neo-epitope specific immunoassays, and to associate the release of these fragments with the activity of proteolytic enzymes using inhibitors. Findings Bovine cartilage explants were cultured in the presence or absence of the catabolic cytokines oncostatin M (OSM and tumor necrosis factor alpha (TNFα. In parallel, explants were co-cultured with protease inhibitors such as GM6001, TIMP1, TIMP2 and TIMP3. Fragments released into the supernatant were determined using a range of neo-epitope specific immunoassays; (1 sandwich 342FFGVG-G2 ELISA, (2 competition NITEGE373ELISA (3 sandwich G1-NITEGE373 ELISA (4 competition 374ARGSV ELISA, and (5 sandwich 374ARGSV-G2 ELISA all detecting aggrecan fragments, and (6 sandwich CTX-II ELISA, detecting C-telopeptides of type II collagen. We found that (1 aggrecanase-derived aggrecan fragments are released in the early (day 2-7 and mid phase (day 9-14 into the supernatant from bovine explants cultures stimulated with catabolic cytokines, (2 the release of NITEGE373 neo-epitopes are delayed compared to the corresponding 374ARGSV fragments, (3 the MMP inhibitor GM6001 did not reduce the release of aggrecanase-derived fragment, but induced a further delay in the release of these fragments, and finally (4 the MMP-derived aggrecan and type II collagen fragments were released in the late phase (day 16-21 only. Conclusion Our data support the model, that aggrecanases and MMPs act independently in the processing of the aggrecan molecules, and furthermore that suppression of MMP-activity had little if

  6. Metabolism of 14C-aspartate during shoot bud formation in cultured cotyledon explants of radiata pine

    International Nuclear Information System (INIS)

    Konschuh, M.N.; Thorpe, T.A.

    1997-01-01

    Aspartate metabolism was investigated in excised cotyledons of radiata pine (Pinus radiate D. Don). These cotyledons were cultured under shoot-forming (plus N 6 -benzyladenine, SF), non-shoot-forming (minus N 6 -benzyladenine, NSF) and unresponsive (plus N 6 -benzyladenine, OLD) conditions, then incubated with [ 14 C]-aspartate for 3-h pulse treatments followed by 3-h chase treatments with cold aspartate. The majority of label was recovered in the CO 2 , amino acid, organic acid and pellet fractions. Uptake was greatest in all tissue types early in culture. Most (over 80%) of the [ 14 C 9-aspartate taken up by the tissues was converted to CO 2 at day 0 in SF and NSF tissues. CO 2 accounted for less than 50% of the total radioactivity in other tissues. Greater incorporation into fractions was observed in SF tissues during promeristemoid formation, while in NSF tissues the greatest incorporation was observed during a period of rapid elongation. Generally, less incorporation was observed in OLD cotyledons than in SF and NSF cotyledons. Analysis of the amino acid fraction showed that labelled aspartate was converted to other amino acids, mainly glutamate, glutamine, asparagine and 4-aminobutyric acid. (au)

  7. Application of tissue culture to cashew ( Anacardium occidentale L ...

    African Journals Online (AJOL)

    Summary of the previous works on the in vitro culture of cashew is highlighted with emphasis on the critical factors that influence the explants response and plantlet regeneration. The recalcitrant nature of cashew has been attributed to the limited success recorded so far in the in vitro culture of the crop and abnormal ...

  8. 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.

    2005-06-01

    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.Growing shoots were cut and re-cultured every 2 weeks until the 4th generation (MV 4 ) to make sure no chimeral tissues still existed in the mutant material. Plantlets were subsequently propagated to obtain enough explants for in vitro selection pressure. Around 3000 plantlets from the three cultivars were subjected to selection pressure. MV 4 explants were cultured on MS medium supplemented with the NaCl in varying concentrations ranging between 50 to 200 mM. Surviving plantlets were propagated and re-cultured on a similar medium to insure their tolerance to salinity. Tolerant plantlets were acclimatized and transferred to pots and grown under glasshouse conditions. Plants were later subjected to another selection pressure, by irrigating them using water containing NaCl in concentrations ranging between 50-250 mM in addition to controls irrigated with normal water. Cultivar Spunta produced the highest number of tolerant plants. Four plants of Spunta appeared to be tolerant to salinity whereas only one plant from Diamant and was tolerant and no plants from cultivar Draga were tolerant. Mutant plants varied in number of produced minitubers from 8 - 14. Also, weight of these minitubers varied from less than 1 to 31 grams. (author)

  9. Tissue explant coculture model of the hypothalamic-pituitary-gonadal-liver axis of the fathead minnow (Pimephales promelas) as a predictive tool for endocrine disruption.

    Science.gov (United States)

    Johnston, Theresa K; Perkins, Edward; Ferguson, Duncan C; Cropek, Donald M

    2016-10-01

    Endocrine-disrupting compounds (EDCs) can impact the reproductive system by interfering with the hypothalamic-pituitary-gonadal (HPG) axis. Although in vitro testing methods have been developed to screen chemicals for endocrine disruption, extrapolation of in vitro responses to in vivo action shows inconsistent accuracy. The authors describe a tissue coculture of the fathead minnow (Pimephales promelas) HPG axis and liver (HPG-L) as a tissue explant model that mimics in vivo results. Brain (hypothalamus), pituitary, gonad, and liver tissue explants from adult fish were examined for function both individually and in coculture to determine combinations and conditions that could replicate in vivo behavior. Only cocultures had the ability to respond to an EDC, trenbolone, similarly to in vivo studies, based on estradiol, testosterone, and vitellogenin production trends, where lower exposure doses suppressed hormone production but higher doses increased production, resulting in distinctive U-shaped curves. These data suggest that a coculture system with all components of the HPG-L axis can be used as a link between in vitro and in vivo studies to predict endocrine system disruption in whole organisms. This tissue-based HPG-L system acts as a flexible deconstructed version of the in vivo system for better control and examination of the minute changes in system operation and response on EDC exposure with options to isolate, interrogate, and recombine desired components. Environ Toxicol Chem 2016;35:2530-2541. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US Government work and, as such, is in the public domain in the United States of America. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US Government work and, as such, is in the public domain in the United States of America.

  10. Effects of Low Intensity Continuous Ultrasound (LICU on Mouse Pancreatic Tumor Explants

    Directory of Open Access Journals (Sweden)

    Despina Bazou

    2017-12-01

    Full Text Available This paper describes the effects of low intensity continuous ultrasound (LICU on the inflammatory response of mouse pancreatic tumor explants. While there are many reports focusing on the application of low-intensity pulsed ultrasound (LIPUS on cell cultures and tissues, the effects of continuous oscillations on biological tissues have never been investigated. Here we present an exploratory study of the effects induced by LICU on mouse pancreatic tumor explants. We show that LICU causes significant upregulation of IFN-γ, IL-1β, and TNF-α on tumor explants. No detectable effects were observed on tumor vasculature or collagen I deposition, while thermal and mechanical effects were not apparent. Tumor explants responded as a single unit to acoustic waves, with spatial pressure variations smaller than their size.

  11. 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.

  12. Comparison of spiculogenesis in in vitro ADCP-primmorph and explants culture of marine sponge Hymeniacidon perleve with 3-TMOSPU supplementation.

    Science.gov (United States)

    Cao, Xupeng; Yu, Xingju; Zhang, Wei

    2007-01-01

    This study aims to test the feasibility of introducing functional chemical groups into biogenic silica spicules by examining the effect of supplementing a silican coupler [3-(trimethoxysilyl)propyl]urea (3-TMOSPU) as silica source in the cultures of archaeocytes-dominant-cell-population (ADCP) primmorphs and explants of the marine sponge Hymeniacidon perleve. Analysis by Fourier Transform Infrared Spectroscopy (FT-IR) confirmed that the organic group in 3-TMOSPU was introduced into silica spicules. By comparing ADCP-primmorph cultures when supplemented with Na2SiO3, 3-TMOSPU supplementation showed no notable effect on the primmorphs development and cell locomotion behaviors. A decline in silicatein expression quantified by real-time RT-PCR was, however, observed during spiculogenesis. The decline was slower for the 3-TMOSPU group whereas significantly fewer spicules were formed. When sponge papillae explants were cultured, 3-TMOSPU supplementation had no negative effect on sponge growth but inhibited the growth biofouling of the diatom Nitzschia closterium. By monitoring the detectable Si concentration, it seemed that 3-TMOSPU was converted by the sponge and its conversion was related to spiculogenesis. Analysis of spicule dimensional changes indicated that the inhibition of spiculogenesis by 3-TMOSPU supplementation was less in ADCP-primmorphs culture due to lower 3-TMOSPU/detectable Si ratio in the media.

  13. 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

  14. The Role of Chronic Mesh Infection in Delayed-Onset Vaginal Mesh Complications or Recurrent Urinary Tract Infections: Results From Explanted Mesh Cultures.

    Science.gov (United States)

    Mellano, Erin M; Nakamura, Leah Y; Choi, Judy M; Kang, Diana C; Grisales, Tamara; Raz, Shlomo; Rodriguez, Larissa V

    2016-01-01

    Vaginal mesh complications necessitating excision are increasingly prevalent. We aim to study whether subclinical chronically infected mesh contributes to the development of delayed-onset mesh complications or recurrent urinary tract infections (UTIs). Women undergoing mesh removal from August 2013 through May 2014 were identified by surgical code for vaginal mesh removal. Only women undergoing removal of anti-incontinence mesh were included. Exclusion criteria included any women undergoing simultaneous prolapse mesh removal. We abstracted preoperative and postoperative information from the medical record and compared mesh culture results from patients with and without mesh extrusion, de novo recurrent UTIs, and delayed-onset pain. One hundred seven women with only anti-incontinence mesh removed were included in the analysis. Onset of complications after mesh placement was within the first 6 months in 70 (65%) of 107 and delayed (≥6 months) in 37 (35%) of 107. A positive culture from the explanted mesh was obtained from 82 (77%) of 107 patients, and 40 (37%) of 107 were positive with potential pathogens. There were no significant differences in culture results when comparing patients with delayed-onset versus immediate pain, extrusion with no extrusion, and de novo recurrent UTIs with no infections. In this large cohort of patients with mesh removed for a diverse array of complications, cultures of the explanted vaginal mesh demonstrate frequent low-density bacterial colonization. We found no differences in culture results from women with delayed-onset pain versus acute pain, vaginal mesh extrusions versus no extrusions, or recurrent UTIs using standard culture methods. Chronic prosthetic infections in other areas of medicine are associated with bacterial biofilms, which are resistant to typical culture techniques. Further studies using culture-independent methods are needed to investigate the potential role of chronic bacterial infections in delayed vaginal mesh

  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. The use of tissue culture techniques with irradiation to improve potato resistance to late blight

    International Nuclear Information System (INIS)

    Al-Safadi, B.; Arabi, M.I.E.

    2004-01-01

    A mutation breeding program was conducted to improve potato (Solanum tuberosum) resistance to late blight disease caused by Phytophthora infestans. In vitro cultured explants from potato cvs. Draga, Diamant, Spunta were irradiated with gamma ray doses 25, 30, and 35 Gy. Growing shoots were cut and re-cultured every 2 weeks until the 4 t h generation (MV 4 ) to make sure no chimeral tissues still existed in the mutant material. Plantlets were subsequently propagated to obtain enough explants for in vitro selection pressure. Around 3000 plantlets from the three cultivars were subjected to selection pressure using co-culture technique. MV 4 explants were incubated in jars, containing MS medium, with mycelia of P. infestans. Surviving plantlets were propagated and re-incubated with the pathogen for three consecutive generations. Resistant plantlets were acclimatized and transferred to pots and grown under glasshouse conditions. Plants were later inoculated, at the adult stage, with sporangial suspension. Cultivar Draga produced the highest number of resistant plants. Ten plants of Draga appeared to be resistant to late blight whereas only one plant from each of the other 2 cultivars was resistant. Mutant plants varied in number of produced minitubers from 13 to 70, Also, weight of these minitubers varied from less than 1 to 35 grams. Selected mutant lines will undergo further testing under field conditions for P. infestans resistance and other agronomic characteristics. (author)

  17. 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)

  18. 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)

  19. Multiplicação in vitro do porta-enxerto de macieira cv. Marubakaido: efeito da orientação do explante no meio de cultura In vitro multiplication of the apple rootstock cv. Marubakaido: effect of the orientation of explant in the medium of culture

    Directory of Open Access Journals (Sweden)

    ALAN CRISTIANO ERIG

    2002-08-01

    Full Text Available Objetivou-se avaliar o efeito da orientação do explante, vertical ou horizontal, no meio de cultura, na multiplicação in vitro, do porta-enxerto de macieira cv. Marubakaido. O meio de cultura utilizado foi o MS com N (nitrogênio reduzido a ¾ da concentração original, 100mg.L-1 de mio-inositol, 40g.L-1 de sacarose e 6g.L-1 de ágar, suplementado com 4,44mM de BAP (6-benzilaminopurina e 0,2ml.L-1 de PPM TM ("Plant Preservative Mixture". Segmentos caulinares com duas gemas e o ápice excisado foram utilizados como explantes. Após a inoculação, os frascos com os explantes foram incubados a 16 horas de fotoperíodo, à temperatura de 25±2ºC, com radiação de 25µmoles.m-2.s-1. O número de brotações, o número de gemas por explante, a taxa de multiplicação e a altura da brotação maior foram avaliados aos quarenta dias de cultivo. O maior número de brotações, o maior número de gemas e a maior taxa de multiplicação foram obtidos com o explante na orientação horizontal no meio de cultura. Não houve diferença significativa quanto à orientação vertical e horizontal do explante no meio de cultura para a altura da brotação maior.The aim of this study was evaluate the effect of the vertical and the horizontal orientation of the explant in the culture medium, in the in vitro multiplication, for the apple rootstock cv. Marubakaido. The culture medium used was the MS with N reduced to ¾ of the original concentration, myo-inositol (100mg.L-1, sucrose (40g.L-1 and agar (6g.L-1, suplemented with BAP (4.44mM and PPM TM (0.2ml.L-1. Stem segments with two buds and the apex excised were used as explants. After the inoculation, the flasks with the explants were incubated at 16 hour of photoperiod, 25±2ºC temperature, with irradiation of 25µmoles.m-2.s-1. The number of shoots and buds, the rate of multiplication and the height of the larger shoot were evaluated after 40 days of cultivation. The highests shoot number, number of buds

  20. 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

  1. 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.

  2. Effect of triiodothyronine and insulin on glucose metabolism in tissue explants and isolated adipocytes from lean and obese Zucker rats

    International Nuclear Information System (INIS)

    Bailey, J.W.

    1985-01-01

    Glucose metabolism in adipocytes from 6 week old lean and obese Zucker rats were sensitive to direct and chronic treatment with insulin and triidothyronine (T 3 ). Insulin had a large stimulatory effect on glucose metabolism in acutely isolated adipocytes. This effect was greater in the lean than in the obese. Fatty acid, CO 2 , and glycerol-glyceride formation from radiolabeled glucose was elevated in the obese over the leans. Pretreatment of isolated adipocytes with pharmacological concentrations of T 3 for 30 minutes prior to the measurement of glucose metabolism had a greater effect on lean than obese adipocytes. The presence of insulin was required to observe the acute effects of T 3 . A 2-hour exposure to physiological levels of T 3 in the presence of insulin in both lean and obese adipocytes decreased lipogenesis. In the absence of insulin, a 2 hour pretreatment with physiological levels of T 3 in tissue from a euthyroid animal produced increased lipogenesis

  3. 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.

  4. 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.

  5. Prevention of the disrupted enamel phenotype in Slc4a4-null mice using explant organ culture maintained in a living host kidney capsule.

    Directory of Open Access Journals (Sweden)

    Xin Wen

    Full Text Available Slc4a4-null mice are a model of proximal renal tubular acidosis (pRTA. Slc4a4 encodes the electrogenic sodium base transporter NBCe1 that is involved in transcellular base transport and pH regulation during amelogenesis. Patients with mutations in the SLC4A4 gene and Slc4a4-null mice present with dysplastic enamel, amongst other pathologies. Loss of NBCe1 function leads to local abnormalities in enamel matrix pH regulation. Loss of NBCe1 function also results in systemic acidemic blood pH. Whether local changes in enamel pH and/or a decrease in systemic pH are the cause of the abnormal enamel phenotype is currently unknown. In the present study we addressed this question by explanting fetal wild-type and Slc4a4-null mandibles into healthy host kidney capsules to study enamel formation in the absence of systemic acidemia. Mandibular E11.5 explants from NBCe1-/- mice, maintained in host kidney capsules for 70 days, resulted in teeth with enamel and dentin with morphological and mineralization properties similar to cultured NBCe1+/+ mandibles grown under identical conditions. Ameloblasts express a number of proteins involved in dynamic changes in H+/base transport during amelogenesis. Despite the capacity of ameloblasts to dynamically modulate the local pH of the enamel matrix, at least in the NBCe1-/- mice, the systemic pH also appears to contribute to the enamel phenotype. Extrapolating these data to humans, our findings suggest that in patients with NBCe1 mutations, correction of the systemic metabolic acidosis at a sufficiently early time point may lead to amelioration of enamel abnormalities.

  6. 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.

  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. 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...

  9. Effect of Hormones on Direct Shoot Regeneration in Hypocotyl Explants of Tomato

    Directory of Open Access Journals (Sweden)

    Rizwan RASHID

    2010-03-01

    Full Text Available This study was conducted for developing a high frequency regeneration system in two genotypes of tomato (Lycopersicon esculentum Mill., �Punjab Upma� and �IPA-3� for direct shoot regeneration from hypocotyl explants. The explants were excised from in vitro tomato seedlings and cultured on MS medium supplemented with different concentrations and combinations of hormones. Direct regeneration was significantly influenced by the genotype hormones combination and concentrations. The MS medium supplemented with (Kinetin 0.5 mg/l and (BAP 0.5 mg/l was found optimum for inducing direct shoot regeneration and number of shoots per explant from hypocotyl explants on this medium. Shoot regeneration per cent in �Punjab Upma� and �IPA-3� per cent was recorded to be highest i.e (86.02 and (82.57 respectively. Besides this, average number shoots per explant was also highest i.e (3.16 in case of �Punjab Upma� and (2.93 in case of �IPA-3�. A significant decline was observed in percent shoot regeneration and average number of shoots per explant with increase in the hormonal concentration. Shoots were obtained and transferred to the elongation medium (MS + BAP 0.3 mg/l. Hundred per cent rooting was induced in separated shoots upon culturing on MS and � MS basal media. Hardening on moist cotton showed maximum plantlet survival rate in case of both genotypes. After hardening, plants were transferred to soil. Thus, a tissue culture base line was established in tomato for obtaining direct regeneration using hypocotyl as explants.

  10. Effect of Hormones on Direct Shoot Regeneration in Hypocotyl Explants of Tomato

    Directory of Open Access Journals (Sweden)

    Rizwan RASHID

    2010-03-01

    Full Text Available This study was conducted for developing a high frequency regeneration system in two genotypes of tomato (Lycopersicon esculentum Mill., Punjab Upma and IPA-3 for direct shoot regeneration from hypocotyl explants. The explants were excised from in vitro tomato seedlings and cultured on MS medium supplemented with different concentrations and combinations of hormones. Direct regeneration was significantly influenced by the genotype hormones combination and concentrations. The MS medium supplemented with (Kinetin 0.5 mg/l and (BAP 0.5 mg/l was found optimum for inducing direct shoot regeneration and number of shoots per explant from hypocotyl explants on this medium. Shoot regeneration per cent in Punjab Upma and IPA-3 per cent was recorded to be highest i.e (86.02 and (82.57 respectively. Besides this, average number shoots per explant was also highest i.e (3.16 in case of Punjab Upma and (2.93 in case of IPA-3. A significant decline was observed in percent shoot regeneration and average number of shoots per explant with increase in the hormonal concentration. Shoots were obtained and transferred to the elongation medium (MS + BAP 0.3 mg/l. Hundred per cent rooting was induced in separated shoots upon culturing on MS and MS basal media. Hardening on moist cotton showed maximum plantlet survival rate in case of both genotypes. After hardening, plants were transferred to soil. Thus, a tissue culture base line was established in tomato for obtaining direct regeneration using hypocotyl as explants.

  11. Proteomic Analyses of the Acute Tissue Response for Explant Rabbit Corneas and Engineered Corneal Tissue Models Following In Vitro Exposure to 1540 nm Laser Light

    National Research Council Canada - National Science Library

    Eurell, T. E; Johnson, T. E; Roach, W. P

    2005-01-01

    Two-dimensional electrophoresis and histomorphometry were used to determine if equivalent protein changes occurred within native rabbit corneas and engineered corneal tissue models following in vitro...

  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. Application of stem-cell media to explant culture of human periosteum: An optimal approach for preparing osteogenic cell material.

    Science.gov (United States)

    Uematsu, Kohya; Nagata, Masaki; Kawase, Tomoyuki; Suzuki, Kenji; Takagi, Ritsuo

    2013-01-01

    As part of our clinical tests on bone regeneration using cultured periosteal sheets, here, we prepared cultured periosteal sheets in two types of stem-cell culture media, STK1 and STK3. Human periosteum was expanded either in 1% human serum-supplemented STK1 for 28 days, in 1% human serum-supplemented STK1 for 14 days followed by 1% human serum-supplemented STK3 for 14 days (1% human serum-supplemented STK1+3), or in 10% fetal bovine serum-supplemented Medium 199 for 28 days (control). Cultured periosteal sheet diameter and DNA content were significantly higher, and the multilayer structure was prominent in 1% human serum-supplemented STK1 and 1% human serum-supplemented STK1+3. The messenger RNA of osteoblastic markers was significantly upregulated in 1% human serum-supplemented STK1+3. Osteopontin-immunopositive staining and mineralization were evident across a wide area of the cultured periosteal sheet in 1% human serum-supplemented STK1+3. Subcutaneous implantation in nude mice following expansion in 1% human serum-supplemented STK1+3 produced the highest cultured periosteal sheet osteogenic activity. Expansion in 1% human serum-supplemented STK1+3 successfully induced cultured periosteal sheet growth while retaining osteogenic potential, and subsequent osteoblastic induction promoted the production of homogeneous cell material.

  14. Micropropagation of Dalbergia sissoo Roxb. through tissue culture technique.

    Science.gov (United States)

    Sahu, Jyoti; Khan, Shagufta; Sahu, Ram Kumar; Roy, Amit

    2014-04-01

    Multiple shoots of Dalbergia sissoo Roxb. (Sissoo) were incited from seeds through indirect somatic embryogenesis method. Seeds were inoculated in Murashige and Skoog's medium without any growth hormone. Than cotyledonary leaves were struck and used for callus induction on MS medium amplified with 2, 4-dichlorophenoxyacetic acid (0.5 to 4 mg mL(-1)). After 3 to 4 weeks the embryogenic callus clumps was transferred to medium supplemented with cytokinin (BAP 1 to 5 mg L(-1), kinetin 1-5.0 mg L(-1)) for embryo maturation and germination. The high-frequency shoot proliferation (82%) and maximum number of shoots per explants were recorded in MS medium containing NAA (0.5)+BAP (0.5). The findings of recent investigations have shown that, it is possible to induce indirect somatic embryogenesis in Dalbergia sissoo and plant regeneration from callus cultures derived from cotyledonary leaves as explants.

  15. Micropropagation of six Paulownia genotypes through tissue culture

    Directory of Open Access Journals (Sweden)

    Lydia Shtereva

    2014-12-01

    Full Text Available We investigated the effect of genotype and culture medium on the in vitro germination and development of plantlets from seeds of 6 different Paulownia genotypes (P. tomentosa, hybrid lines P. tomentosa P. fortunei (Mega, Ganter and Caroline, P. elongata and hybrid line P. elongata P. fortunei. Nodal and shoot tip explants were used for micropropagation of Paulownia genotypes by manipulating plant growth regulators. The highest germination percentage for all genotypes was obtained for seeds inoculated on medium supplemented with 50 mg*L GA3 (MSG2. On Thidiazuron containing media, the explants of hybrid line P. elongata P. fortunei exhibited the highest frequency of axillary shoot proliferation following by P. tomentosa P. fortunei. The results are discussed with the perspective of applying an improved protocol for in vitro seed germination and plantlet formation in several economically valuable Paulownia genotypes.

  16. 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 ...

  17. 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.

  18. 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

  19. Effect Of Explant Source And Different Medium Culture On Friable Embryogenic Callus Induction Of Four Cultivars Of Cassava Manihot Esculenta Crantz

    Directory of Open Access Journals (Sweden)

    Simplice Prosper Yandia

    2015-08-01

    Full Text Available In order to obtain Friable Embryogenic Callus FEC for protoplast isolation we have evaluated in this research the competance for Friable Embryogenic Callus FEC of four cassava cultivars M61033 Rendre Yalipe and Six-mois in media containing MS supplemented with 8mgl 24-D MS supplemented with 10 mgl BAP and GD supplemented with 12mgl picloram using apical bud AB and immature leaves lobes ILL as explants. In general in the medium GD12mgl picloram the highest efficiencies of FEC ranged from 58 to 87 and the highest score of FEC ranged from 4.2 to 5.4 with explants AB however we have observed with explants ILL the efficiencies of somatic embryos ranged form 41 to 75 and the score ranged from 4.1 to 4.4. The mediums MS28 mgl 24-D have induced with explants AB the efficiencies of FEC ranged from 43 to 57 and the score ranged from 3.1 to 3.8 however with ILL explants the efficiencies of FEC ranged from 39 to 49 and the score ranged from 2.9 to 3.7. The least FEC were observed in the medium MS210 mgl BAP with BA explants however the efficiencies ranged from 6 to 11 and the score ranged from 1.1 to 1.8. Whereas the efficiencies of FEC with ILL explants ranged from 4 to 7 and the score ranged from 0.5 to 0.8. All of four cultivars showed capability of producing FEC although their efficiency varied according to gonotype donors explants and medium taking into acount. Abbreviations GD Gressoff and Doy MS Murashige and Skoog 24-dichloro phenoxyacetic acid BAP Benzylamino-purin-Acid AB Apical Bud ILL Immature Leaves lobes

  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. 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...

  2. 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

  3. Adventitious shoot regeneration from leaf explants of the valuable ...

    African Journals Online (AJOL)

    The objective of this study was to develop an efficient protocol for adventitious shoot regeneration for Plectranthus barbatus Andrews using leaf explants. The explants were cultured on MS (Murashige and Skoog, 1962) medium containing various concentration of kinetin (KN), 6-benzylaminopurine (BAP) and thidiazuron ...

  4. 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.

  5. 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.

  6. 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.

  7. Cultura in vitro de embriões e de gemas de mudas de pau-rosa (Aniba rosaeodora Ducke Culture in vitro of rosewood (Aniba rosaeodora Ducke embryos´seeds and buds explants

    Directory of Open Access Journals (Sweden)

    Lucia Handa

    2005-01-01

    Full Text Available Este trabalho teve como objetivo o estabelecimento in vitro de embriões e de gemas de mudas de pau-rosa (Aniba rosaeodora Ducke livres de contaminações e de oxidação fenólica. As gemas foram obtidas da rebrota de mudas cultivadas em viveiro e os embriões a partir de sementes em diversos estágios de maturação. Para a assepsia dos explantes foram utilizados dois antibiótico (Ampicilina e Agrimicina, etanol (70% e hipoclorito de sódio, em concentrações e tempo de exposição variando em função do tratamento. Para o controle da oxidação foram utilizados imersão em ácido ascórbico (250 mg/l e PVP (Polivinilpirrolidona no meio Murashige & Skoog (MS. O delineamento estatístico empregado foi o inteiramente ao acaso com tratamentos e repetições em função do tipo de explante. Foi observado 71% de sobrevivência e 53% de germinação de embriões tratados com hipoclorito de sódio (50% e 2% de cloro ativo por 10 minutos e inoculados em meio MS contendo 20 mg/l de água de côco após 45 dias. As gemas das rebrotas de mudas tratadas com solução de Sulfato de Estreptomicina (Agrimicina na concentração de 500 mg/l (1h apresentaram 51% de sobrevivência. Quando submetidas ao pré-tratamento com o emprego de bomba a vácuo (180 mmHg contendo a Agrimicina (500 mg/l, apresentaram 25% de sobrevivência.This study deals with the establishment in vitro of Aniba rosaeodora Ducke explants, free from fungical and endogenous contaminations and phenolic oxidation. Bud explants and embryos' seeds from many maturation stages were used in this trial. The explants were disinfected with Ampicilin antibiotic, Streptomicine Sulphate (Agrimicina, etanol (70%, sodium hipoclorite in many concentrations and exposure time acording to the type of explant. For the phenolic oxidation control, the immersion on ascorbic acid and PVP (Polyvinilpirrolidone in culture medium were used. The explants were inoculated in MS medium. The statistical design was the

  8. A Novel 3D Skin Explant Model to Study Anaerobic Bacterial Infection

    Directory of Open Access Journals (Sweden)

    Grazieli Maboni

    2017-09-01

    Full Text Available Skin infection studies are often limited by financial and ethical constraints, and alternatives, such as monolayer cell culture, do not reflect many cellular processes limiting their application. For a more functional replacement, 3D skin culture models offer many advantages such as the maintenance of the tissue structure and the cell types present in the host environment. A 3D skin culture model can be set up using tissues acquired from surgical procedures or post slaughter, making it a cost effective and attractive alternative to animal experimentation. The majority of 3D culture models have been established for aerobic pathogens, but currently there are no models for anaerobic skin infections. Footrot is an anaerobic bacterial infection which affects the ovine interdigital skin causing a substantial animal welfare and financial impact worldwide. Dichelobacter nodosus is a Gram-negative anaerobic bacterium and the causative agent of footrot. The mechanism of infection and host immune response to D. nodosus is poorly understood. Here we present a novel 3D skin ex vivo model to study anaerobic bacterial infections using ovine skin explants infected with D. nodosus. Our results demonstrate that D. nodosus can invade the skin explant, and that altered expression of key inflammatory markers could be quantified in the culture media. The viability of explants was assessed by tissue integrity (histopathological features and cell death (DNA fragmentation over 76 h showing the model was stable for 28 h. D. nodosus was quantified in all infected skin explants by qPCR and the bacterium was visualized invading the epidermis by Fluorescent in situ Hybridization. Measurement of pro-inflammatory cytokines/chemokines in the culture media revealed that the explants released IL1β in response to bacteria. In contrast, levels of CXCL8 production were no different to mock-infected explants. The 3D skin model realistically simulates the interdigital skin and has

  9. Multiplicação e enraizamento in vitro de amoreira-preta 'Xavante': efeito da concentração de sais, do tipo de explante e de carvão ativado no meio de cultura Blackberry 'Xavante' in vitro multiplication and rooting: salt concentrations effect, explant type, and activated coal on culture medium

    Directory of Open Access Journals (Sweden)

    Luciane Nolasco Leitzke

    2009-01-01

    concentrations in culture medium for. The first experiment constituted of nodal segments with 1 cm, originated from of the in vitro cultivation, inoculated at four different MS medium concentrations, supplemented with 7.5 µM BAP, 30 g L-1 sucrose, 100 mg L-1 myo-inositol and 6 g L-1 agar, and with two types of explants, which totaled 8 treatments. The second experiment constituted of apex microcutting, having about 1 to 1.5 cm length, and two leaves were inoculated at three different concentrations of MS medium, added of 30 g L-1 sucrose, 100 mg L-1 myo-inositol and 6 g L-1 agar and supplemented with three different activated coal concentrations, totaling nine treatments. It can be inferred that T1, in other words, explants with leaves, were more efficient at 125% MS salt concentration on culture medium, thereby inducing larger number of leaves, buds, shoots and shoot length, and that the use of 75% salt concentration without activated coal is the most suitable one for the blackberry 'Xavante' in vitro rooting.

  10. Mucus glycoprotein secretion by tracheal explants: effects of pollutants

    International Nuclear Information System (INIS)

    Last, J.A.; Kaizu, T.

    1980-01-01

    Tracheal slices incubated with radioactive precursors in tissue culture medium secrete labeled mucus glycoproteins into the culture medium. We have used an in vivtro approach, a combined method utilizing exposure to pneumotoxins in vivo coupled with quantitation of mucus secretion rates in vitro, to study the effects of inhaled pollutants on mucus biosynthesis by rat airways. In addition, we have purified the mucus glycoproteins secreted by rat tracheal explants in order to determine putative structural changes that might by the basis for the observed augmented secretion rates after exposure of rats to H2SO4 aerosols in combination with high ambient levels of ozone. After digestion with papain, mucus glycoproteins secreted by tracheal explants may be separated into five fractions by ion-exchange chromatography, with recovery in high yield, on columns of DEAE-cellulose. Each of these five fractions, one neutral and four acidic, migrates as a single unique spot upon cellulose acetate electrophoresis at pH values of 8.6 and 1.2. The neutral fraction, which is labeled with [3H] glucosamine, does not contain radioactivity when Na2 35SO4 is used as the precursor. Acidic fractions I to IV are all labeled with either 3H-glucosamine or Na2 35SO4 as precursor. Acidic fraction II contains sialic acid as the terminal sugar on its oligosaccharide side chains, based upon its chromatographic behavior on columns of wheat-germ agglutinin-Agarose. Treatment of this fraction with neuraminidase shifts its elution position in the gradient to a lower salt concentration, coincident with acidic fraction I. After removal of terminal sialic acid residues with either neuraminidase or low pH treatment, the resultant terminal sugar on the oligosaccharide side chains is fucose. These results are identical with those observed with mucus glycoproteins secreted by cultured human tracheal explants and purified by these same techniques

  11. 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 ...

  12. 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

  13. Refining the application of direct embryogenesis in sugarcane: Effect of the developmental phase of leaf disc explants and the timing of DNA transfer on transformation efficiency.

    Science.gov (United States)

    Snyman, S J; Meyer, G M; Richards, J M; Haricharan, N; Ramgareeb, S; Huckett, B I

    2006-10-01

    A rapid in vitro protocol using direct somatic embryogenesis and microprojectile bombardment was investigated to establish the developmental phases most suitable for efficient sugarcane transformation. Immature leaf roll disc explants with and without pre-emergent inflorescence tissue were compared. It was shown that for effective transformation to occur, explants should be cultured for several days to allow initiation of embryo development prior to bombardment. Leaf roll discs with pre-emergent inflorescences showed a higher degree of embryogenic competence than non-flowering explants, and transformation efficiency was higher when explants containing floral initials were bombarded. Despite the occurrence of high numbers of phenotypically negative plants, combining the use of inflorescent leaf roll discs with direct embryogenic regeneration has the potential to improve the speed and efficiency of transgenesis in sugarcane.

  14. In vitro regeneration from internodal explants of bitter melon ...

    African Journals Online (AJOL)

    Thiru

    2012-04-24

    Apr 24, 2012 ... shoots per internodal explant after 80 days of culture. Key words: ... grown in the tropical regions of Asia, Amazon, east Africa and the ... Tamilnadu, India. .... expressed as the mean ± standard error (SE) of three experiments.

  15. 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.

  16. Studies on Callus Induction and Regeneration of Medicinal Plant Chicory (Cichorium intybus L. from Leaf and Petiole Explants

    Directory of Open Access Journals (Sweden)

    H. Hadizadeh

    2016-07-01

    Full Text Available Introduction: Chicory (Cichorium intybus L. belongs to Asteraceae family is commonly known as witloof chicory. The leaves and the roots of this medicinal plant are edible and commonly used as salad. Some varieties are also cultivated as coffee substitute after roasting the roots. All parts of the plant contain these volatile oils, with the majority of the toxic components concentrated in the plant's root. In folk medicine, the plant is used for the treatment of diarrhea, spleen enlargement, fever, and vomiting. Antihepatotoxic activity on damaged rat’s liver sections and anti-bacterial activity of this crop has been recently reported. In vitro regeneration from leaf explants with various hormonal combinations has been reported previously. Moreover, in vitro regeneration of Chicory from cotyledon explants using different combinations of plant growth regulators has been studied. Also, a protocol for the regeneration of plantlets from leaf and petiole explants of witloof chicory has been developed. The aim of the present investigation was optimization of callus induction and shoot regeneration from leaf and petiole tissues of Chicory (Esfahan genotype. Materials and Methods: In this investigation, Esfahan genotype was used for callus induction and direct shoot regeneration. Seeds were first washed with running tap water for 30 min then seeds were surface sterilized by dipping in 70% ethanol for 90 s and rinsed with sterile distilled water, followed by immersing in 5% sodium hypochlorite solution for 25 min and thereafter rinsed for 30 min with sterile distilled water. The basal medium used in this investigation was MS. For shoot regeneration, leaf and petiole explants (5 mm segments were excised from 4-week-old sterile seedlings and cultured on MS medium containing different combinations of NAA / BA and KIN / BA in two separate experiments. Experiments were performed factorial based on completely randomized design. Cultures were incubated at 25

  17. [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.

  18. Seasonal variations in the pattern of RNA metabolism of tuber tissue in response to excision and culture

    International Nuclear Information System (INIS)

    Macleod, A.J.; Mills, E.D.; Yeoman, M.M.

    1979-01-01

    Between December 1975 and June 1976 explants excised from Jerusalem artichoke tubers were cultured in the presence and in the absence of 2,4-D, the cells in the tissue dividing only in the presence of 2,4-D, in which the length of the first cell cycle increased nonlinearly from 18 hours to 40 hours as the tubers aged in storage at 4 0 C. Simultaneously the amount of RNA in the tissue declined linearly from 8 to 5 μg RNA per explant. Detailed examination of the RNA metabolism in dividing and in non-dividing cells during February and June 1976 revealed superimposed but independent responses to wounding during excision and to stimulation into growth by 2,4-D. The responses to wounding involved only a very low level of metabolic activity, were complete within a few hours of excision and changed very little with the storage of the tubers. Tissue treated with 2,4-D showed a much higher level of metabolic activity including the periodic accumulation of RNA coupled to its discontinuous synthesis. The features of these growth-related responses changed considerably during the investigation. (author)

  19. 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

  20. Exogenous transforming growth factor-β1 enhances smooth muscle differentiation in embryonic mouse jejunal explants.

    Science.gov (United States)

    Coletta, Riccardo; Roberts, Neil A; Randles, Michael J; Morabito, Antonino; Woolf, Adrian S

    2017-01-13

    An ex vivo experimental strategy that replicates in vivo intestinal development would in theory provide an accessible setting with which to study normal and dysmorphic gut biology. The current authors recently described a system in which mouse embryonic jejunal segments were explanted onto semipermeable platforms and fed with chemically defined serum-free media. Over 3 days in organ culture, explants formed villi and they began to undergo spontaneous peristalsis. As defined in the current study, the wall of the explanted gut failed to form a robust longitudinal smooth muscle (SM) layer as it would do in vivo over the same time period. Given the role of transforming growth factor β1 (TGFβ1) in SM differentiation in other organs, it was hypothesized that exogenous TGFβ1 would enhance SM differentiation in these explants. In vivo, TGFβ receptors I and II were both detected in embryonic longitudinal jejunal SM cells and, in organ culture, exogenous TGFβ1 induced robust differentiation of longitudinal SM. Microarray profiling showed that TGFβ1 increased SM specific transcripts in a dose dependent manner. TGFβ1 proteins were detected in amniotic fluid at a time when the intestine was physiologically herniated. By analogy with the requirement for exogenous TGFβ1 for SM differentiation in organ culture, the TGFβ1 protein that was demonstrated to be present in the amniotic fluid may enhance intestinal development when it is physiologically herniated in early gestation. Future studies of embryonic intestinal cultures should include TGFβ1 in the defined media to produce a more faithful model of in vivo muscle differentiation. Copyright © 2017 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd. Copyright © 2017 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd.

  1. Effect of induced mutagenesis in rice tissue culture

    International Nuclear Information System (INIS)

    Maddumage, R.

    1994-01-01

    The influence of chemical mutagens and ionising radiation on growth, regenerative capacity of rice callus culture and the effect o9f mutagens on frequency and spectrum of mutant regenerants, derived from calli and determination of approximate semi-lethal dose of each mutagen on rice calli was studied. Intact mature de-husked grains and pieces of primordial particles of four varieties were used as explants in the experiment. Organogenesis was induced using MS media supplemented with agar. After thirty days calluses were subjected to varying concentrations/dosage of mutagens. The effect of mutagens on growth of callus was stimulative in low concentration/doses at short exposure, but in higher concentration/doses at longer exposure it was oppressive. In x-radiation treatment all the studied doses showed only stimulative effect on growth. The effect of mutagenic treatment on regenerative capacity was negative. No specificity was found even between two chemical mutagens of their action on studied characters

  2. 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...

  3. Somatic Embryogenesis in Peach-Palm (Bactris gasipaes) Using Different Explant Sources.

    Science.gov (United States)

    Steinmacher, Douglas A; Heringer, Angelo Schuabb; Jiménez, Víctor M; Quoirin, Marguerite G G; Guerra, Miguel P

    2016-01-01

    Peach palm (Bactris gasipaes Kunth) is a member of the family Arecaceae and is a multipurpose but underutilized species. Nowadays, fruit production for subsistence and local markets, and heart-of-palm production for local, national, and international markets are the most important uses of this plant. Conventional breeding programs in peach palm are long-term efforts due to the prolonged generation time, large plant size, difficulties with controlled pollination and other factors. Although it is a caespitose palm, its propagation is currently based on seeds, as off-shoots are difficult to root. Hence, tissue culture techniques are considered to be the most likely strategy for efficient clonal plantlet regeneration of this species. Among various techniques, somatic embryogenesis offers the advantages of potential automated large-scale production and putative genetic stability of the regenerated plantlets. The induction of somatic embryogenesis in peach palm can be achieved by using different explant sources including zygotic embryos, immature inflorescences and thin cell layers from the young leaves and shoot meristems. The choice of a particular explant depends on whether clonal propagation is desired or not, as well as on the plant conditions and availability of explants. Protocols to induce and express somatic embryogenesis from different peach palm explants, up to acclimatization of plantlets, are described in this chapter.

  4. 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.

  5. 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)

  6. The effect of caspase-3 inhibition on interdigital tissue regression in explant cultures of developing mouse limbs

    Czech Academy of Sciences Publication Activity Database

    Kudělová, Judita; Tucker, A.S.; Dubská, L.; Chlastáková, Ivana; Doubek, J.; Matalová, Eva

    2012-01-01

    Roč. 16, č. 4 (2012), s. 295-301 ISSN 1976-8354 R&D Projects: GA AV ČR IAA600450904; GA ČR GA203/08/1680 Institutional research plan: CEZ:AV0Z50450515 Keywords : caspases * cell death * digitalization Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.639, year: 2012

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. Enhanced micropropagation and tiller formation in sugarcane through pretreatment of explants with thidiazuron (TDZ).

    Science.gov (United States)

    Kumari, Kavita; Lal, Madan; Saxena, Sangeeta

    2017-10-01

    An efficient, simple and commercially applicable protocol for rapid micropropagation of sugarcane has been designed using variety Co 05011. Pretreatment of shoot tip explants with thidiazuron (TDZ) induced high frequency regeneration of shoot cultures with improved multiplication ratio. The highest frequency (80%) of shoot initiation in explants pretreated with 10 mg/l of TDZ was obtained during the study. Maximum 65% shoot cultures could be established from the explants pretreated with TDZ as compared to minimum 40% establishment in explants without pretreatment. The explants pretreated with 10 mg/l of TDZ required minimum 40 days for the establishment of shoot cultures as compared to untreated explants which required 60 days. The highest average number of shoots per culture (19.1) could be obtained from the explants pretreated with 10 mg/l of TDZ, indicating the highest multiplication ratio (1:6). Highest rooting (over 94%) was obtained in shoots regenerated from pretreated explants on ½ strength MS medium containing 5.0 mg/l of NAA and 50 g/l of sucrose within 15 days. Higher number of tillers/clump (15.3) could be counted in plants regenerated from pretreated explants than untreated ones (10.9 tillers/clump) in field condition, three months after transplantation. Molecular analysis using RAPD and DAMD markers suggested that the pretreatment of explants with TDZ did not adversely affect the genetic stability of regenerated plants and maintained high clonal purity.

  12. 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)

  13. 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.

  14. 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

  15. A novel 3D skin explant model to study anaerobic bacterial infection

    DEFF Research Database (Denmark)

    Maboni, Grazieli; Davenport, Rebecca; Sessford, Kate

    2017-01-01

    of the tissue structure and the cell types present in the host environment. A 3D skin culture model can be set up using tissues acquired from surgical procedures or post slaughter, making it a cost effective and attractive alternative to animal experimentation. The majority of 3D culture models have been......Skin infection studies are often limited by financial and ethical constraints, and alternatives, such as monolayer cell culture, do not reflect many cellular processes limiting their application. For a more functional replacement, 3D skin culture models offer many advantages such as the maintenance...... bacterium and the causative agent of footrot. The mechanism of infection and host immune response to D. nodosus is poorly understood. Here we present a novel 3D skin ex vivo model to study anaerobic bacterial infections using ovine skin explants infected with D. nodosus. Our results demonstrate that D...

  16. 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.

  17. Co-culture in cartilage tissue engineering.

    NARCIS (Netherlands)

    Hendriks, J.A.A.; Riesle, J.U.; van Blitterswijk, Clemens

    2007-01-01

    For biotechnological research in vitro in general and tissue engineering specifically, it is essential to mimic the natural conditions of the cellular environment as much as possible. In choosing a model system for in vitro experiments, the investigator always has to balance between being able to

  18. Plant regeneration from cotyledonary explants of Eucalyptus camaldulensis Regeneração de plantas de Eucalyptus camaldulensis a partir das explantes cotiledonares

    Directory of Open Access Journals (Sweden)

    Roberson Dibax

    2005-08-01

    Full Text Available Breeding methods based on genetic transformation techniques need to be implemented for Eucalyptus camaldulensis to shorten the long breeding cycles and avoid manipulation of adult trees; that requires the development of plant regeneration protocols enabling development of plants from transformed tissues. The present work aimed to optimise the regeneration process already established for the species. Cotyledonary leaves of E. camaldulensis were cultured in MS medium supplemented with naphthaleneacetic acid (NAA and 6-benzylaminopurine (BAP combinations. The most efficient treatment for bud indirect regeneration (2.7 µmol L-1 NAA and 4.44 µmol L-1 BAP was used for further experiments. When explants were kept in the dark during the first 30 days, the percentage of explants forming calluses increased and explant necrosis was reduced in comparison with light-cultured explants. Mineral medium modifications were compared and half-strength MS mineral medium turned out to be as efficient as full-strength medium, producing 54% and 47% of explants with buds, respectively. For shoot elongation, MS medium with half-strength nitrate and ammonium salts, and 0.2% activated charcoal yielded rooted shoots 1 to 8 cm high after one month. The procedure is an efficient protocol for E. camadulensis plant regeneration, reducing the stages necessary for the obtention of complete plants.A implementação, para espécies florestais, de técnicas de melhoramento baseadas em métodos de transformação genética, permitirá reduzir os longos ciclos de melhoramento e evitar a manipulação de árvores adultas. Isto implica dispor de um protocolo de regeneração que permita o desenvolvimento de plantas a partir de tecidos transformados. Este trabalho teve como objetivo otimizar este protocolo de regeneração para Eucalyptus camaldulensis. Folhas cotiledonares foram cultivadas em meio de cultura MS suplementado com combinações de ácido naftalenoacético (ANA e 6

  19. 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

  20. Effect of medium composition and explant size on embryogenic calli formation of cassava (Manihot esculenta Crantz local genotypes

    Directory of Open Access Journals (Sweden)

    ENNY SUDARMONOWATI

    2006-07-01

    Full Text Available Cassava (Manihot esculenta Crantz is an important tropical crop species used for human consumption, feed and raw material for various industries. Genetic transformation through embryogenic tissues is known as an effective method for cassava genetic improvements. Objective of this study was to obtain a suitable medium and length of explants to induce embryogenic callus on friable embryogenic callus (FEC as a target for genetic transformation. Immature leaf lobes (1-3 mm, 3-5 mm and larger than 5 mm in length of local genotypes of cassava (Adira 4. Menti, Iding, Gebang, Rawi and Timtim-29 cultured in vitro were used as explants. The explants were incubated for 2 and 4 weeks on MS (Murashige-Skoog or GD (Greshooff & Doy semi solid medium containing 10 mg/L picloram, 6 mg/L NAA supplemented with 4% sucrose and 4 µM CuSO4. Results showed that the highest percentage (100% of embryogenic calli formation for 4 weeks obtained by culturing Iding of 3-5 mm length on GD semi solid medium, whereas the lowest (33% one obtained by incubation 5 mm leaf lobe of Timtim-29 on the same medium. The most suitable medium for callus induction was GD, whereas the optimum length of explants was 5 mm or larger. Further study needs to be done to obtain friable embryogenic calli (FEC by employing different concentration of picloram and varying other critical factors.

  1. 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.

  2. 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

  3. Clonal propagation of eucalyptus by tissue culture

    Energy Technology Data Exchange (ETDEWEB)

    Mehra-Palta, A.

    1982-07-01

    Multiple adventitious buds were induced on cotyledons, shoot tips and nodal stem segments of Eucalyptus species cultured on a defined nutrient medium supplemented with the cytokinin zeatin and the auxin indole-3-butyric acid (IBA). The adventitious buds could be recycled on cytokinin medium to produce more buds thus providing the possibility of producing large clones from selected genotypes. The adventitious shoots were rooted in auxin medium and some of the resulting propagules were outplanted in the field. These techniques have the potential for use in the genetic improvement of Eucalyptus. (Refs. 15).

  4. Callus induction from epicotyl and hypocotyl explants of Parkia ...

    African Journals Online (AJOL)

    Epicotyl and hypocotyl explants of Parkia biglobosa (Locust bean) were cultured in vitro to investigate their callogenic capacity. Established cultures were obtained and maintained on MS medium supplemented with either 2,4-D or NAA, each of concentration range of 0.4 – 1.0 mg/L. In general, while higher concentrations of ...

  5. 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.

  6. Comparison of regeneration potentials in tissue cultures of primitive and cultivated tomato species (Lycopersicon sp.

    Directory of Open Access Journals (Sweden)

    M. Lech

    2014-01-01

    Full Text Available Regeneration capacities of two tomato cultivars: Potentat and Rutgers, and of three accessions of wild tomato species: Lycopersicon peruvianum PI 128650, L. peruvianum var. dentatum PI 128655 and L. glandulosum were studied using an universal medium suitable for regeneration of those plants from leaf pieces in tissue culture. Fragments of leaf blades were taken from plants raised in greenhouse conditions and placed on a modified MS medium containing 0.3 mg/l IAA and 3.0 mg/l BAP solidified with 1% agar. The explants were transferred every 4-5 weeks on fresh medium of the same composition. It was shown that all the three primitive tomato species revealed much higher multiplication coefficients than the two cultivars. Appropriate values were: 11 - for L. glandulosum, 8 - for L. peruvianum, 7 - for L. peruvianum var. dentatum, 4 - for L. esculentum cv. Potentat and 2 - cv. Rutgers. Completely regenerated plants were obtained from all the tested species, but organogenesis occurred almost two weeks earlier in wild tomatoes than in the culitivated varieties of L. esculentum.

  7. Micropropagation of Eucalyptus grandis and nitens using tissue culture techniques

    Energy Technology Data Exchange (ETDEWEB)

    Furze, M.J.; Creswell, C.F.

    1985-01-01

    Experiments with nodal explants of E. grandis and E. nitens seedlings and E. grandis coppice shoots showed that a large number of plants can be produced from a single explant using micropropagation. The percentage of micropropagated shoots that formed roots was about 90% for E. grandis and 80% for E. nitens. For both species, about 90% of the rooted shoots survived after hardening off. 9 references.

  8. Carboxylesterase-dependent cytotoxicity of dibasic esters (DBE) in rat nasal explants.

    Science.gov (United States)

    Trela, B A; Bogdanffy, M S

    1991-02-01

    Dibasic esters (DBE) are a solvent mixture of dimethyl adipate (DMA), dimethyl glutarate (DMG), and dimethyl succinate (DMS) used in the paint and coating industry. Subchronic inhalation toxicity studies have demonstrated that DBE induce a mild degeneration of the olfactory, but not the respiratory, epithelium of the rat nasal cavity. Carboxylesterase-mediated hydrolysis of the individual dibasic esters is more efficient in olfactory than in respiratory mucosal homogenates. In the present study, an in vitro system of cultured rat nasal explants was utilized to determine if DBE toxicity is dependent on a metabolic activation by nonspecific carboxylesterase. Explants from both the olfactory and the respiratory regions of the female rat nasal cavity were incubated for 2 hr in Williams' medium E containing 10-100 mM DMA, DMG, or DMS. DBE caused a dose-related increase in nasal explant acid phosphatase release, a biochemical index of cytotoxicity. HPLC analysis demonstrated parallel increases in the carboxylesterase-mediated formation of monomethyl ester metabolites. Diacid metabolite production in the nasal explant system was not entirely concentration-dependent. Metabolite concentrations and acid phosphatase release were generally greater in olfactory than respiratory tissues. DBE-induced cytotoxicity and acid metabolite production were markedly attenuated in nasal tissue excised from rats which were pretreated with bis(p-nitrophenyl)phosphate, a carboxylesterase inhibitor. This study presents a viable in vitro method for assessing organic ester cytotoxicity in the rat nasal cavity. It was shown that DBE are weak nasal toxicants under the conditions of this system. It was further demonstrated that DBE toxicity is dependent on a carboxylesterase-mediated activation. A similar mechanism was proposed for the nasal toxicity induced by other organic esters following inhalation exposure.

  9. 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.

  10. 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.

  11. 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...

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. Study on Effect of Type of Explant and Hormone on Callus Induction and Regeneration in Saffron (Crocus sativus L.

    Directory of Open Access Journals (Sweden)

    Mohsen Sajjadi

    2015-10-01

    Full Text Available Saffron (Crocus sativus L. is one of the medicinal plants that contain active components and medicinal materials. Tissue culture of saffron can improve the quality and quantity of the saffron product, increase its export and the farmers’ income. In this study, 36 different types of hormone combinations in the dark and 9 different treatments of hormone combinations in cold (4°C, using different saffron explants (bulb, leaf, scales around leaf and distal parts of the leaf were studied in tissue culture. To investigate the growth of corms, the callus formation and the regeneration rate, three replications for each treatment were used and the length of shoot (cm, the callus formation percentage and the regeneration percentage were measured and statistical analysis was performed. Among the types of explants, only explants from bulbs produced the callus on MS medium containing 2 mg.l-1 BAP and 1 mg.l-1 IBA in both the dark and cold conditions. The highest percentage of regeneration was obtained in MS medium with hormonal composition of 0.3 mg.l-1 TDZ, 1 mg.l-1 BAP, 2 mg.l-1 IBA and 0.01 mg.l-1 GA3 in the cold conditions.

  19. 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.

  20. 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.

  1. 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.

  2. Relaxin's induction of metalloproteinases is associated with the loss of collagen and glycosaminoglycans in synovial joint fibrocartilaginous explants

    Science.gov (United States)

    Naqvi, Tabassum; Duong, Trang T; Hashem, Gihan; Shiga, Momotoshi; Zhang, Qin; Kapila, Sunil

    2005-01-01

    Diseases of specific fibrocartilaginous joints are especially common in women of reproductive age, suggesting that female hormones contribute to their etiopathogenesis. Previously, we showed that relaxin dose-dependently induces matrix metalloproteinase (MMP) expression in isolated joint fibrocartilaginous cells. Here we determined the effects of relaxin with or without β-estradiol on the modulation of MMPs in joint fibrocartilaginous explants, and assessed the contribution of these proteinases to the loss of collagen and glycosaminoglycan (GAG) in this tissue. Fibrocartilaginous discs from temporomandibular joints of female rabbits were cultured in medium alone or in medium containing relaxin (0.1 ng/ml) or β-estradiol (20 ng/ml) or relaxin plus β-estradiol. Additional experiments were done in the presence of the MMP inhibitor GM6001 or its control analog. After 48 hours of culture, the medium was assayed for MMPs and the discs were analyzed for collagen and GAG concentrations. Relaxin and β-estradiol plus relaxin induced the MMPs collagenase-1 and stromelysin-1 in fibrocartilaginous explants – a finding similar to that which we observed in pubic symphysis fibrocartilage, but not in articular cartilage explants. The induction of these proteinases by relaxin or β-estradiol plus relaxin was accompanied by a loss of GAGs and collagen in joint fibrocartilage. None of the hormone treatments altered the synthesis of GAGs, suggesting that the loss of this matrix molecule probably resulted from increased matrix degradation. Indeed, fibrocartilaginous explants cultured in the presence of GM6001 showed an inhibition of relaxin-induced and β-estradiol plus relaxin-induced collagenase and stromelysin activities to control baseline levels that were accompanied by the maintenance of collagen or GAG content at control levels. These findings show for the first time that relaxin has degradative effects on non-reproductive synovial joint fibrocartilaginous tissue and

  3. 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)

  4. Reprogramming of H3K27me3 is critical for acquisition of pluripotency from cultured Arabidopsis tissues.

    Directory of Open Access Journals (Sweden)

    Chongsheng He

    2012-08-01

    Full Text Available In plants, multiple detached tissues are capable of forming a pluripotent cell mass, termed callus, when cultured on media containing appropriate plant hormones. Recent studies demonstrated that callus resembles the root-tip meristem, even if it is derived from aerial organs. This finding improves our understanding of the regeneration process of plant cells; however, the molecular mechanism that guides cells of different tissue types to form a callus still remains elusive. Here, we show that genome-wide reprogramming of histone H3 lysine 27 trimethylation (H3K27me3 is a critical step in the leaf-to-callus transition. The Polycomb Repressive Complex 2 (PRC2 is known to function in establishing H3K27me3. By analyzing callus formation of mutants corresponding to different histone modification pathways, we found that leaf blades and/or cotyledons of the PRC2 mutants curly leaf swinger (clf swn and embryonic flower2 (emf2 were defective in callus formation. We identified the H3K27me3-covered loci in leaves and calli by a ChIP-chip assay, and we found that in the callus H3K27me3 levels decreased first at certain auxin-pathway genes. The levels were then increased at specific leaf genes but decreased at a number of root-regulatory genes. Changes in H3K27me3 levels were negatively correlated with expression levels of the corresponding genes. One possible role of PRC2-mediated H3K27me3 in the leaf-to-callus transition might relate to elimination of leaf features by silencing leaf-regulatory genes, as most leaf-preferentially expressed regulatory genes could not be silenced in the leaf explants of clf swn. In contrast to the leaf explants, the root explants of both clf swn and emf2 formed calli normally, possibly because the root-to-callus transition bypasses the leaf gene silencing process. Furthermore, our data show that PRC2-mediated H3K27me3 and H3K27 demethylation act in parallel in the reprogramming of H3K27me3 during the leaf-to-callus transition

  5. In vitro propagation of Alstroemeria using rhizome explants derived ...

    African Journals Online (AJOL)

    SERVER

    2007-09-19

    Sep 19, 2007 ... Single in vitro and in vivo rhizome bud were excised (3 -7 mm) using a sharp knife and cultured on MS basal medium with 3 different composition of growth regulators. Culture media. The explants were planted in culture media as M1 (MS + 1 mg l-1. BA + 0.2 mg l-1 NAA) Han et al. (1994), M2 (MS + 1 mg l-1 ...

  6. 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.

  7. 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)

  8. 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.

  9. In vitro response from cotyledon and hypocotyls explants in tomato ...

    African Journals Online (AJOL)

    USER

    2010-07-26

    Jul 26, 2010 ... 1Institute of Tropical Agriculture, University of Putra Malaysia, 43400 Serdang, Selangor, Malaysia. 2Faculty of Agriculture ... shoot length was significantly different between cotyledon and hypocotyls derived explants. No adventitious shoots ... growth regulator used in culture medium (Bhatia et al.,. 2004a).

  10. Somatic embryogenesis and plant regeneration from leaf explants of ...

    African Journals Online (AJOL)

    An attempt was made to study the somatic embryogenesis and plant regeneration from the in vitro leaf explants of Rumex vesicarius L. a renowned medicinal plant, which belongs to polygonaceae family. Effective in vitro regeneration of R. vesicarius was achieved via young leaf derived somatic embryo cultures.

  11. All-trans retinoic acid inhibits craniopharyngioma cell growth: study on an explant cell model.

    Science.gov (United States)

    Li, Qiang; You, Chao; Zhou, Liangxue; Sima, Xiutian; Liu, Zhiyong; Liu, Hao; Xu, Jianguo

    2013-05-01

    The ratio between FABP5 and CRABPII determines cellular response to physiological level of retinoic acid; tumor cells undergo proliferation with high level of FABP5 and apoptosis with high level of CRABPII. We intended to study FABP5 and CRABPII expression in craniopharyngiomas, to establish craniopharyngioma cell model using explants method, and to study the effect of pharmacological dose of retinoic acid on craniopharyngioma cells. Expression of FABP5 and CRABPII in craniopharyngioma tissue from 20 patients was studied using immunohistochemistry. Primary craniopharyngioma cell cultures were established using tissue explants method. Craniopharyngioma cells were treated using various concentrations of all-trans retinoic acid, and cell growth curve, apoptosis, expression of FABP5, CRABPII and NF-κB were assayed in different groups. FABP5/CRABPII ratio was significantly higher in adamatinomatous group than that in papillary group. Cell cultures were established in 19 cases (95 %). Pharmacological level retinoic acid inhibited cell growth and induced cellular apoptosis in dose dependent manner, and apoptosis rate cells treated with 30 μM retinoic acid for 24 h was 43 %. Also, retinoic acid increased CRABPII, and decreased FABP5 and NF-κB expression in craniopharyngioma cells. High FABP5/CRABPII ratio is observed in adamatinomatous craniopharyngioma. Retinoic acid at pharmacological level induced craniopharyngioma cell apoptosis via increasing FABP5/CRABPII ratio and inhibiting NF-κB signaling pathway. Our study demonstrated that all-trans retinoic acid might be a candidate for craniopharyngioma adjuvant chemotherapy in future.

  12. High levels of stable p53 protein and the expression of c-myc in cultured human epithelial tissue after cobalt-60 irradiation

    International Nuclear Information System (INIS)

    Mothersill, C.; Seymour, C.B.; Harney, J.; Hennessy, T.P.

    1994-01-01

    When explants of human uroepithelium or esophageal epithelium are exposed to acute doses of radiation (cobalt-60), the cells which grow out to form the primary cultures show a number of abnormal features. These include the development of characteristic nonsenescent foci. These foci have previously been shown to be c-myc positive and to have an abnormal, tumor-like ultrastructure. Expression of c-myc and the level of stable p53 proteins have now been examined in these cultures 2 weeks after irradiation. Both proteins occurred in dividing cells at the growing edge of the explant and in the foci. The expression of c-myc appeared to be correlated with growth. As expected, variation between individual cultures of normal human cells was noted in the expression of stable p53 protein. Most control uroepithelial cell cultures were negative, but a small cohort showed a wide range of values. The control cultures from the esophageal tissues had high expression of p53, and this decreased marginally after irradiation. Cells positive for p53 were always in cycle and were usually positive for c-myc as well. It would appear from these results that the expression of c-myc and the stable form of the p53 protein occur in irradiated primary cultures of normal human cells both in foci which also express a number of abnormalities and in open-quotes edgeclose quotes cells which are dividing. Cultures of unirradiated cells from esophagus and a small number of uroepithelial samples had high levels of p53. Possible reasons for this are discussed. 33 refs., 2 figs., 3 tabs

  13. Micropropagation of Origanum acutidens (HAND.-MAZZ.) IETSWAART using stem node explants.

    Science.gov (United States)

    Yildirim, Mehmet Ugur

    2013-01-01

    Origanum acutidens (HAND.-MAZZ.) IETSWAART is a promising ornamental plant that can be widely used in landscape management. It is endemic to Eastern Anatolian region of Turkey. Tissue culture has not been used to micropropagate it. The study reports stem node explants from one-week-old seedlings of the plant for successful micropropagation. The stem nodes were cultured on MS medium containing 0.6, 1.2, 1.8, and 2.4 mg/L BAP with 0.2 mg/L NAA. Visible effects of culture media on shoot proliferation were recorded. Shoot regeneration rate was maximum on MS medium containing 1.80 mg/L BAP-0.2 mg/L NAA. The micropropagated shoots were rooted on MS medium containing 0.2 mg/L NAA. All microrooted plantlets survived during acclimatisation on peat moss. It was concluded that O. acutidens can be successfully micropropagated under in vitro conditions.

  14. 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.

  15. Behavioural properties of chick somitic mesoderm and lateral plate when explanted in vitro.

    Science.gov (United States)

    Bellairs, R; Sanders, E J; Portch, P A

    1980-04-01

    Tissue culture, time-lapse cinematographic and electron microscopic techniques have been used to study the properties of chick mesoderm at several stages of differentiation. Lateral plate, unsegmented mesoderm (segmental plate), and newly formed somites were dissected from stage-12 embryos, whilst dermo-myotomes and sclerotomes were dissected from stage-18 embryos. Each type of mesoderm was found to exhibit a characteristic pattern of behaviour. The explants from the unsegmented mesoderm from the newly formed somites and from the older embryos could be placed in a developmental sequence; with increasing differentiation they settled and spread on the substrate more readily, whether explanted as pieces of tissue or as individual cells, and it was concluded that this implied an increased adhesion to the substrate. Similarly, with increasing differentiation, the cells segmented at a faster rate. No significant differences could be discerned in the internal structure of the different types of cells, although differences in the general shape were apparent. The lateral plate mesoderm cells, which bear some resemblances to the unsegmented mesoderm cells in the embryo, also show some morphological resemblances to them in vitro. However, the lateral plate cells had a much greater success in attaching to glass or platic substrates. They were also found to have the highest speed of locomotion of all the tissues studied, whereas the unsegmented had the lowest. It is concluded therefore, that although cells may look similar to one another morphologically, their behaviour may differ greatly, probably because they are already partially determined.

  16. Production of Gymnemic Acid Depends on Medium, Explants, PGRs, Color Lights, Temperature, Photoperiod, and Sucrose Sources in Batch Culture of Gymnema sylvestre

    Directory of Open Access Journals (Sweden)

    A. Bakrudeen Ali Ahmed

    2012-01-01

    Full Text Available Gymnema sylvestre (R.Br. is an important diabetic medicinal plant which yields pharmaceutically active compounds called gymnemic acid (GA. The present study describes callus induction and the subsequent batch culture optimization and GA quantification determined by linearity, precision, accuracy, and recovery. Best callus induction of GA was noticed in MS medium combined with 2,4-D (1.5 mg/L and KN (0.5 mg/L. Evaluation and isolation of GA from the calluses derived from different plant parts, namely, leaf, stem and petioles have been done in the present case for the first time. Factors such as light, temperature, sucrose, and photoperiod were studied to observe their effect on GA production. Temperature conditions completely inhibited GA production. Out of the different sucrose concentrations tested, the highest yield (35.4 mg/g d.w was found at 5% sucrose followed by 12 h photoperiod (26.86 mg/g d.w. Maximum GA production (58.28 mg/g d.w was observed in blue light. The results showed that physical and chemical factors greatly influence the production of GA in callus cultures of G. sylvestre. The factors optimized for in vitro production of GA during the present study can successfully be employed for their large-scale production in bioreactors.

  17. Effect of sucrose concentrations on Stevia rebaudiana Bertoni tissue culture and gene expression.

    Science.gov (United States)

    Ghorbani, T; Kahrizi, D; Saeidi, M; Arji, I

    2017-08-30

    Stevia rebaudiana (Bert.) Bertoni is known as sweet plant which it contains a high level of steviol glycosides in the leaves.  This plant has been used from centuries ago as a sweetener for tea. One of the most important steviol glycosides is stevioside that is attractive for diabetic persons. Tissue culture is the only rapid process for the mass propagation of stevia. One of the most important factors in the medium is sucrose that is a necessary for plant growth. In the present study, we use nodal segments of the stem as explants in mediums with different sucrose concentration (50 mM, 100mM and 150mM). Several morphological traits were measured in a 28 day period. Results analysis showed a significant variation between treatments. The highest growth rate, rooting and leaf production was obtained in medium with 100mM sucrose. The correlation between measured traits was significant at the 0.01 level. To investigation of UGT74G1, UGT76G1, UGT85C2 and KS genes expression that are involved in the synthesis of SGs, RT- PCR was done with the housekeeping gene of as internal control. There were significant differences between all media. The results showed thatsucrose 100 mM containing media was more desirable than others for expression of UGT76G1 and UGT85C2 genes. Whereas, the best medium for expression of UGT74G1 was sucrose 150 mM and sucrose 50 mM for KS gene. Totally, it seems that sucrose at a concentration of 100 mMprovides the best condition for stevia growth and steviol glycosides production.

  18. 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

  19. 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.

  20. A stable explant culture of HER2/neu invasive carcinoma supported by alpha-Smooth Muscle Actin expressing stromal cells to evaluate therapeutic agents

    Directory of Open Access Journals (Sweden)

    Piechocki Marie P

    2008-04-01

    Full Text Available Abstract Background To gain a better understanding of the effects of therapeutic agents on the tumor microenvironment in invasive cancers, we developed a co-culture model from an invasive lobular carcinoma. Tumor cells expressing HER2/neu organize in nests surrounded by alpha-Smooth Muscle Actin (α-SMA expressing tumor stroma to resemble the morphology of an invading tumor. This co-culture, Mammary Adenocarcinoma Model (MAM-1 maintains a 1:1 ratio of HER2/neu positive tumor cells to α-SMA-reactive stromal cells and renews this configuration for over 20 passages in vitro. Methods We characterized the cellular elements of the MAM-1 model by microarray analysis, and immunocytochemistry. We developed flow cytometric assays to evaluate the relative responses of the tumor and stroma to the tyrosine kinase inhibitor, Iressa. Results The MAM-1 gene expression profile contains clusters that represent the ErbB-2 breast cancer signature and stroma-specific clusters associated with invasive breast cancers. The stability of this model and the ability to antigenically label the tumor and stromal fractions allowed us to determine the specificity of Iressa, a receptor tyrosine kinase inhibitor, for targeting the tumor cell population. Treatment resulted in a selective dose-dependent reduction in phospho-pMEK1/2 and pp44/42MAPK in tumor cells. Within 24 h the tumor cell fraction was reduced 1.9-fold while the stromal cell fraction increased >3-fold, consistent with specific reductions in phospho-pp44/42 MAPK, MEK1/2 and PCNA in tumor cells and reciprocal increases in the stromal cells. Erosion of the tumor cell nests and augmented growth of the stromal cells resembled a fibrotic response. Conclusion This model demonstrates the specificity of Iressa for HER2/neu expressing tumor cells versus the tumor associated myofibroblasts and is appropriate for delineating effects of therapy on signal transduction in the breast tumor microenvironment and improving

  1. A stable explant culture of HER2/neu invasive carcinoma supported by alpha-Smooth Muscle Actin expressing stromal cells to evaluate therapeutic agents

    International Nuclear Information System (INIS)

    Piechocki, Marie P

    2008-01-01

    To gain a better understanding of the effects of therapeutic agents on the tumor microenvironment in invasive cancers, we developed a co-culture model from an invasive lobular carcinoma. Tumor cells expressing HER2/neu organize in nests surrounded by alpha-Smooth Muscle Actin (α-SMA) expressing tumor stroma to resemble the morphology of an invading tumor. This co-culture, Mammary Adenocarcinoma Model (MAM-1) maintains a 1:1 ratio of HER2/neu positive tumor cells to α-SMA-reactive stromal cells and renews this configuration for over 20 passages in vitro. We characterized the cellular elements of the MAM-1 model by microarray analysis, and immunocytochemistry. We developed flow cytometric assays to evaluate the relative responses of the tumor and stroma to the tyrosine kinase inhibitor, Iressa. The MAM-1 gene expression profile contains clusters that represent the ErbB-2 breast cancer signature and stroma-specific clusters associated with invasive breast cancers. The stability of this model and the ability to antigenically label the tumor and stromal fractions allowed us to determine the specificity of Iressa, a receptor tyrosine kinase inhibitor, for targeting the tumor cell population. Treatment resulted in a selective dose-dependent reduction in phospho-pMEK1/2 and pp44/42MAPK in tumor cells. Within 24 h the tumor cell fraction was reduced 1.9-fold while the stromal cell fraction increased >3-fold, consistent with specific reductions in phospho-pp44/42 MAPK, MEK1/2 and PCNA in tumor cells and reciprocal increases in the stromal cells. Erosion of the tumor cell nests and augmented growth of the stromal cells resembled a fibrotic response. This model demonstrates the specificity of Iressa for HER2/neu expressing tumor cells versus the tumor associated myofibroblasts and is appropriate for delineating effects of therapy on signal transduction in the breast tumor microenvironment and improving strategies that can dually or differentially target the tumor and stromal

  2. 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.

  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. 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

  5. In vitro culture of higher plants as a tool in the propagation of horticultural crops.

    NARCIS (Netherlands)

    Pierik, R.L.M.

    1988-01-01

    In vitro culture of higher plants is the culture, under sterile conditions, of plants, seeds, embryos, organs, explants, tissues, cells and protoplasts on nutrient media. This type of culture has shown spectacular development since 1975, resulting in the production and regeneration of viable

  6. Generation and characterisation of human umbilical cord derived mesenchymal stem cells by explant method.

    Science.gov (United States)

    Yusoff, Z; Maqbool, M; George, E; Hassan, R; Ramasamy, R

    2016-06-01

    Mesenchymal stem cells (MSCs) derived from human umbilical cord (UC) have been considered as an important tool for treating various malignancies, tissue repair and organ regeneration. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are better alternative to MSCs that derived from bone marrow (BM-MSCs) as they are regarded as medical waste with little ethical concern for research and easily culture-expanded. In this present study, the foetal distal end of human UC was utilised to generate MSC by explant method. Upon in vitro culture, adherent cells with fibroblastic morphology were generated with rapid growth kinetics. Under the respective inductive conditions, these cells were capable of differentiating into adipocytes and osteocytes; express an array of standard MSC's surface markers CD29, CD73, CD90, CD106 and MHC-class I. Further assessment of immunosuppression activity revealed that MSCs generated from UC had profoundly inhibited the proliferation of mitogen-activated T lymphocytes in a dosedependent manner. The current laboratory findings have reinforced the application of explant method to generate UCMSCs thus, exploring an ideal platform to fulfil the increasing demand of MSCs for research and potential clinical use.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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....

  14. 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

  15. 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.

  16. Stable genetic transformation of Jatropha curcas via Agrobacterium tumefaciens-mediated gene transfer using leaf explants

    KAUST Repository

    Kumar, Nitish; Vijay Anand, K.G.; Pamidimarri, D.V.N. Sudheer; Sarkar, Tanmoy; Reddy, Muppala P.; Radhakrishnan, T.; Kaul, Tanushri; Reddy, M.K.; Sopori, Sudhir K.

    2010-01-01

    Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel crop. A simple and reproducible protocol was developed for Agrobacterium tumefaciens-mediated stable genetic transformation of J. curcas using leaf explains. Agrobacterium strain LBA 4404 harbouring the binary vector pCAMBIA 1304 having sense-dehydration responsive element binding (S-DREB2A), beta-glucuronidase (gus), and hygromycin-phosphotransferase (hpt) genes were used for gene transfer. A number of parameters such as preculture of explains, wounding of leaf explants, Agrobacterium growth phase (OD), infection duration, co-cultivation period, co-cultivation medium pH, and acetosyringone, were studied to optimized transformation efficiency. The highest transformation efficiency was achieved using 4-day precultured, non-wounded leaf explants infected with Agrobacterium culture corresponding to OD(600)=0.6 for 20 min, followed by co-cultivation for 4 days in a co-cultivation medium containing 100 mu M acetosyringone, pH 5.7. Co-cultivated leaf explants were initially cultured on Murashige and Skoog (MS) medium supplemented with 2.27 mu M thidiazuron (TDZ) for regeneration of shoot buds, followed by selection on same medium with 5 mu g ml(-1) hygromycin. Selected shoot buds were transferred to MS medium containing 10 mu M kinetin (Kn), 4.5 mu M 6-benzyl aminopurine (BA), and 5.5 mu M alpha-naphthaleneacetic acid (NAA) for proliferation. The proliferated shoots were elongated on MS medium supplemented with 2.25 mu M BA and 8.5 mu M indole-3-acetic acid (IAA). The elongated shoots were rooted on half strength MS medium supplemented with 15 mu M indole-3-butyric acid (IBA), 5.7 mu M IAA, 5.5 mu M NAA, and 0.25 mg l(-1) activated charcoal. GUS histochemical analysis of the transgenic tissues further confirmed the transformation event. PCR and DNA gel blot hybridization were performed to confirm the presence of transgene. A transformation efficiency of 29% was

  17. Stable genetic transformation of Jatropha curcas via Agrobacterium tumefaciens-mediated gene transfer using leaf explants

    KAUST Repository

    Kumar, Nitish

    2010-07-01

    Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel crop. A simple and reproducible protocol was developed for Agrobacterium tumefaciens-mediated stable genetic transformation of J. curcas using leaf explains. Agrobacterium strain LBA 4404 harbouring the binary vector pCAMBIA 1304 having sense-dehydration responsive element binding (S-DREB2A), beta-glucuronidase (gus), and hygromycin-phosphotransferase (hpt) genes were used for gene transfer. A number of parameters such as preculture of explains, wounding of leaf explants, Agrobacterium growth phase (OD), infection duration, co-cultivation period, co-cultivation medium pH, and acetosyringone, were studied to optimized transformation efficiency. The highest transformation efficiency was achieved using 4-day precultured, non-wounded leaf explants infected with Agrobacterium culture corresponding to OD(600)=0.6 for 20 min, followed by co-cultivation for 4 days in a co-cultivation medium containing 100 mu M acetosyringone, pH 5.7. Co-cultivated leaf explants were initially cultured on Murashige and Skoog (MS) medium supplemented with 2.27 mu M thidiazuron (TDZ) for regeneration of shoot buds, followed by selection on same medium with 5 mu g ml(-1) hygromycin. Selected shoot buds were transferred to MS medium containing 10 mu M kinetin (Kn), 4.5 mu M 6-benzyl aminopurine (BA), and 5.5 mu M alpha-naphthaleneacetic acid (NAA) for proliferation. The proliferated shoots were elongated on MS medium supplemented with 2.25 mu M BA and 8.5 mu M indole-3-acetic acid (IAA). The elongated shoots were rooted on half strength MS medium supplemented with 15 mu M indole-3-butyric acid (IBA), 5.7 mu M IAA, 5.5 mu M NAA, and 0.25 mg l(-1) activated charcoal. GUS histochemical analysis of the transgenic tissues further confirmed the transformation event. PCR and DNA gel blot hybridization were performed to confirm the presence of transgene. A transformation efficiency of 29% was

  18. 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

  19. 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

  20. Mutation breeding in sugar-cane (Saccharum sp. hybrid) by gamma irradiation of cuttings and tissue cultures

    International Nuclear Information System (INIS)

    Siddiqui, S.H.; Javed, M.

    1982-01-01

    The sugar-cane variety Co 547, which is highly susceptible to smut disease (Ustilago scitaminea Syd.) and is also late maturing, was exposed to different doses of gamma radiation to study its radiosensitivity and to induce smut disease resistance and early maturity. The radiosensitivity of the variety showed that an optimum dose (LD 50 ) was 2.0 kR and the working dose range was found to be 1.5-3.0 kR, whereas doses higher than 4.0 kR drastically affected the growth and germination. A broad spectrum of variability in reaction to disease resistance was observed after radiation exposure. This facilitated the isolation of disease-resistant mutants. Twenty-three mutants showing varied reaction to smut under field infection conditions were tested for two years by artificial inoculation using the dip method. From these studies 15 stable mutants were isolated. Of 15 mutants, seven showed promising performance in cane yield and sucrose contents. The tissue culture technique was used to determine the potential of different commercial clones for callusing. In test explants callusing was achieved readily and proliferation of callus was fairly good in all the clones. (author)

  1. 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).

  2. 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.

  3. Nonparenchymal cells cultivated from explants of fibrotic liver resemble endothelial and smooth muscle cells from blood vessel walls

    International Nuclear Information System (INIS)

    Voss, B.; Rauterberg, J.; Pott, G.; Brehmer, U.; Allam, S.; Lehmann, R.; von Bassewitz, D.B.

    1982-01-01

    Tissue specimens from human fibrotic liver obtained by needle biopsy were cultured. Two cell types emerged from the tissue explants. From their morphology and biosynthetic products they resembled smooth muscle cells and endothelial cells from blood vessel walls. In the endothelial cells, factor VIII-associated protein was demonstrated by indirect immunofluorescence. Synthesis of collagen types I and III, basement membrane collagen types IV and V, and fibronectin by both cell types was observed by immunofluorescence microscopy. Homogeneous cultures of smooth muscle cells were observed in subcultures. After incubation with [ 14 C]glycine, collagen was isolated and characterized by CM cellulose chromatography, and consisted mainly of types I and III. These data suggest involvement of mesenchymal cells in hepatic fibrosis; they presumably originate from blood vessel or sinusoidal walls

  4. 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.

  5. Absorção de macronutrientes por explantes de bananeira in vitro Macronutrient absorption by banana explants in vitro

    Directory of Open Access Journals (Sweden)

    Josefa Diva Nogueira Diniz

    1999-07-01

    Full Text Available Com o objetivo de estudar a absorção de macronutrientes (N, P, K, Ca, Mg e S em explantes de bananeira cv. Prata Anã, foram utilizados explantes de plantas estabelecidas in vitro, inoculados em meio básico de Murashige & Skoog (1962 contendo sacarose (30 g/L, e BAP (3,5 mg/L com sete tratamentos, representados pelos períodos de 0, 10, 20, 30, 40, 50 e 60 dias de cultivo e três repetições. As quantidades de macronutrientes totais absorvidas pelos explantes seguiram a ordem: K > N > Ca > ou = P > Mg @ S. O P foi o nutriente absorvido mais rapidamente pelos explantes, com 75% extraído do meio de cultivo nos primeiros 30 dias, cessando sua absorção aos 50 dias, restando ainda 9% no meio de cultivo. A absorção do S cessou também aos 50 dias, quando 66% deste nutriente ainda permanecia no meio de cultivo. Este resultado sugere haver uma relação, quanto à absorção, entre esses dois nutrientes. As maiores taxas de absorção de todos os nutrientes foram verificadas nos primeiros 20 dias. O rizoma, o pseudocaule e as folhas, se diferenciaram quanto à concentração e extração ou acúmulo de nutrientes.The absorption of the nutrients (N, P, K, Ca, Mg and S by banana (Musa sp. cv. Prata Anã explants on the basic medium of Murashige & Skoog (1962 supplemented with sucrose (30 g/L and BAP (3.5 mg/L were evaluated at 0, 10, 20, 30, 40, 50 and 60 days after inoculation. The seven treatments were arranged on a completely randomized design with three replicates. The sequence of nutrient absorption by the explants was K > N > Ca > or = P > Mg @ S. The P was the nutrient with the fastest absorption rate and at the 30th day the explants had already absorbed 75% of the P from the medium. The P absorption stopped by the 50th day. The S absorption stopped at the 50th day with 66% of it remaining in the medium. The results suggested a close relationship between these two nutrients. The highest rates of nutrient absorption were observed during the

  6. Induction of shoot regeneration in cotyledon explants of the oilseed crop Sesamum indicum L.

    Directory of Open Access Journals (Sweden)

    Masochon Zimik

    2017-12-01

    Full Text Available Sesamum indicum is an ancient oilseed crop known for its high quality edible oil and its medicinally important lignans. The crop is said to be recalcitrant to plant tissue culture thus limiting the use of modern biotechnology for its genetic improvement. We present here a protocol describing plant regeneration through adventitious shoot formation from cotyledons dissected from sesame seeds soaked for four hours in water. Subculturing of the cotyledons after two weeks of culture on to a fresh Murashige and Skoog medium leads to differentiation of adventitious shoots from the proximal cut end of the explant. Culture of cotyledons on a medium containing 9% sucrose for a couple of weeks prior to transfer to MS medium supplemented with 3% sucrose induced a higher frequency of shoot regeneration. The highest frequency of 25% adventitious shoot regeneration was observed for S. indicum variety UMA. This variety also turned out to be the best among the ten genotypes tested for shoot regeneration through tissue culture. While addition of IAA marginally improved regeneration, silver nitrate was found essential for enhancing the frequency of shoot regeneration. The regenerated shoots formed roots on full strength MS medium supplemented with 1 mg/l IBA and the rooted plants were established in soil.

  7. 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

  8. 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

  9. 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.

  10. NMR-based metabolomics of mammalian cell and tissue cultures

    International Nuclear Information System (INIS)

    Aranibar, Nelly; Borys, Michael; Mackin, Nancy A.; Ly, Van; Abu-Absi, Nicholas; Abu-Absi, Susan; Niemitz, Matthias; Schilling, Bernhard; Li, Zheng Jian; Brock, Barry; Russell, Reb J.; Tymiak, Adrienne; Reily, Michael D.

    2011-01-01

    NMR spectroscopy was used to evaluate growth media and the cellular metabolome in two systems of interest to biomedical research. The first of these was a Chinese hamster ovary cell line engineered to express a recombinant protein. Here, NMR spectroscopy and a quantum mechanical total line shape analysis were utilized to quantify 30 metabolites such as amino acids, Krebs cycle intermediates, activated sugars, cofactors, and others in both media and cell extracts. The impact of bioreactor scale and addition of anti-apoptotic agents to the media on the extracellular and intracellular metabolome indicated changes in metabolic pathways of energy utilization. These results shed light into culture parameters that can be manipulated to optimize growth and protein production. Second, metabolomic analysis was performed on the superfusion media in a common model used for drug metabolism and toxicology studies, in vitro liver slices. In this study, it is demonstrated that two of the 48 standard media components, choline and histidine are depleted at a faster rate than many other nutrients. Augmenting the starting media with extra choline and histidine improves the long-term liver slice viability as measured by higher tissues levels of lactate dehydrogenase (LDH), glutathione and ATP, as well as lower LDH levels in the media at time points out to 94 h after initiation of incubation. In both models, media components and cellular metabolites are measured over time and correlated with currently accepted endpoint measures.

  11. 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.

  12. NMR-based metabolomics of mammalian cell and tissue cultures

    Energy Technology Data Exchange (ETDEWEB)

    Aranibar, Nelly; Borys, Michael; Mackin, Nancy A.; Ly, Van; Abu-Absi, Nicholas; Abu-Absi, Susan [Bristol-Myers Squibb Company (United States); Niemitz, Matthias [PERCH Solutions Ltd. (Finland); Schilling, Bernhard; Li, Zheng Jian; Brock, Barry; Russell, Reb J.; Tymiak, Adrienne; Reily, Michael D., E-mail: michael.reily@bms.com [Bristol-Myers Squibb Company (United States)

    2011-04-15

    NMR spectroscopy was used to evaluate growth media and the cellular metabolome in two systems of interest to biomedical research. The first of these was a Chinese hamster ovary cell line engineered to express a recombinant protein. Here, NMR spectroscopy and a quantum mechanical total line shape analysis were utilized to quantify 30 metabolites such as amino acids, Krebs cycle intermediates, activated sugars, cofactors, and others in both media and cell extracts. The impact of bioreactor scale and addition of anti-apoptotic agents to the media on the extracellular and intracellular metabolome indicated changes in metabolic pathways of energy utilization. These results shed light into culture parameters that can be manipulated to optimize growth and protein production. Second, metabolomic analysis was performed on the superfusion media in a common model used for drug metabolism and toxicology studies, in vitro liver slices. In this study, it is demonstrated that two of the 48 standard media components, choline and histidine are depleted at a faster rate than many other nutrients. Augmenting the starting media with extra choline and histidine improves the long-term liver slice viability as measured by higher tissues levels of lactate dehydrogenase (LDH), glutathione and ATP, as well as lower LDH levels in the media at time points out to 94 h after initiation of incubation. In both models, media components and cellular metabolites are measured over time and correlated with currently accepted endpoint measures.

  13. Lichen explants and natural occurrence of lichens

    Energy Technology Data Exchange (ETDEWEB)

    Kirschbaum, A; Klee, R

    1971-01-01

    Studies with lichen explants and with naturally occurring lichens, conducted in the Lower Main region in West Germany within the framework of an air hydgienic and meteorologic model study of that region, are described. Parmelia physodes explants from oak trees growing in nonpolluted areas were exposed in polluted areas, such as in an industrial area, an airport, a petroleum refinery, and near a large chemical plant. The degree of air pollution in the exposure site was evaluated by the degree of the lichen damage in seven grades. The large-scale average distribution of air pollution in the survey area was studied by surveying the natural occurrence of lichen species on 10 apple trees in area units of 6.25 sq km each. The lichen explant and lichen survey methods compared by the study of naturally occurring lichens were near the exposure site of lichen explants.

  14. Induction of mutations in garlic by combined use of gamma-rays and tissue culture

    Energy Technology Data Exchange (ETDEWEB)

    Rou, Zhen Hai [Horticultural Research Inst., Shanghai Academy of Agricultural Sciences, Shanghai (China)

    1997-07-01

    Callus cultures were initiated from leaf explants of garlic on MS medium supplemented with 1.0 mg/l KIN + 1.0 mg/l IAA + 2.0 mg/l 2,4-D. Plantlets were induced from leaf calli on MS medium with 2.0 mg/l BA + 2.0 mg/l IAA. Bulblets were induced from plantlets on MS medium containing 3.0 mg/l ITA. Callus growth and plantlets induction were remarkably inhibited with irradiation doses of 8 and 10 Gy. It was found that doses of 3 and 5 Gy were suitable to induce variation. Somatic embryos were induced on MS medium supplemented with 2.0 mg/l 2,4-D + 500 mg/l casein, 1000 mg/l yeast extract and 3 to 5% sucrose. (author). 11 tabs.

  15. Induction of mutations in garlic by combined use of gamma-rays and tissue culture

    International Nuclear Information System (INIS)

    Zhen Hai Rou

    1997-01-01

    Callus cultures were initiated from leaf explants of garlic on MS medium supplemented with 1.0 mg/l KIN + 1.0 mg/l IAA + 2.0 mg/l 2,4-D. Plantlets were induced from leaf calli on MS medium with 2.0 mg/l BA + 2.0 mg/l IAA. Bulblets were induced from plantlets on MS medium containing 3.0 mg/l ITA. Callus growth and plantlets induction were remarkably inhibited with irradiation doses of 8 and 10 Gy. It was found that doses of 3 and 5 Gy were suitable to induce variation. Somatic embryos were induced on MS medium supplemented with 2.0 mg/l 2,4-D + 500 mg/l casein, 1000 mg/l yeast extract and 3 to 5% sucrose. (author). 11 tabs

  16. Induction of recessive mutations in potato using tissue culture techniques

    International Nuclear Information System (INIS)

    Enckevort, L.J.G. van; Hoogkamp, T.J.H.; Bergervoet, J.E.M.; Visser, R.G.F.; Jacobsen, E.; Stiekma, W.J.; Pereira, A.

    2001-01-01

    In potato, two different in vitro approaches were used to generate recessive mutants. In the first method, monoploid plant material was irradiated to isolate and identify amylose-free (amf) mutants in potato. For isolating secondary mutants in the amf background new monoploids of the amf type were developed. A few selected amf monoploids showed excellent vigour in vitro, large leave; and microtuber formation. A diploid and a monoploid were tested for in vitro mutation induction and irradiated with 0 to 16 Gy X rays. The optimal dose for survival and mutation induction was between 4 and 8 Gy and plants were regenerated from irradiated leaf explants. In the second approach, mutants were induced by insertion of transposable elements in the diploids. This method was used to mutate R genes for resistance to Phytophthora infestans. Diploid heterozygous Rr plants with the immobilised Ds element, closely linked to one of the R genes, were selected. Mobilisation of Ds using Ac element transposase resulted in the selection of plants with active somatic Ds excision frequency of about 10%. In vitro protoplast isolation and plant regeneration from such plants enabled the selection of regenerants with new independent Ds insertions. Hygromycin selection (Ds excision marker on the T-DNA) during protoplast regeneration increased the frequency of Ds excision regenerants to 56%. A total of 582 hygromycin resistant plants were regenerated and selected in vitro. Preliminary analysis of the regenerants showed re-insertions of Ds in the predicted coding sequences of genes. (author)

  17. 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...

  18. 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...

  19. 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.

  20. Late graft explants in endovascular aneurysm repair.

    Science.gov (United States)

    Turney, Eric J; Steenberge, Sean P; Lyden, Sean P; Eagleton, Matthew J; Srivastava, Sunita D; Sarac, Timur P; Kelso, Rebecca L; Clair, Daniel G

    2014-04-01

    With more than a decade of use of endovascular aneurysm repair (EVAR), we expect to see a rise in the number of failing endografts. We review a single-center experience with EVAR explants to identify patterns of presentation and understand operative outcomes that may alter clinical management. A retrospective analysis of EVARs requiring late explants, >1 month after implant, was performed. Patient demographics, type of graft, duration of implant, reason for removal, operative technique, length of stay, complications, and in-hospital and late mortality were reviewed. During 1999 to 2012, 100 patients (91% men) required EVAR explant, of which 61 were placed at another institution. The average age was 75 years (range, 50-93 years). The median length of time since implantation was 41 months (range, 1-144 months). Explanted grafts included 25 AneuRx (Medtronic, Minneapolis, Minn), 25 Excluder (W. L. Gore & Associates, Flagstaff, Ariz), 17 Zenith (Cook Medical, Bloomington, Ind), 15 Talent (Medtronic), 10 Ancure (Guidant, Indianapolis, Ind), 4 Powerlink (Endologix, Irvine, Calif), 1 Endurant (Medtronic), 1 Quantum LP (Cordis, Miami Lakes, Fla), 1 Aorta Uni Iliac Rupture Graft (Cook Medical, Bloomington, Ind), and 1 homemade tube graft. Overall 30-day mortality was 17%, with an elective case mortality of 9.9%, nonelective case mortality of 37%, and 56% mortality for ruptures. Endoleak was the most common indication for explant, with one or more endoleaks present in 82% (type I, 40%; II, 30%; III, 22%; endotension, 6%; multiple, 16%). Other reasons for explant included infection (13%), acute thrombosis (4%), and claudication (1%). In the first 12 months, 23 patients required explants, with type I endoleak (48%) and infection (35%) the most frequent indication. Conversely, 22 patients required explants after 5 years, with type I (36%) and type III (32%) endoleak responsible for most indications. The rate of EVAR late explants has increased during the past decade at our

  1. 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.

  2. 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)

  3. Full-thickness human skin explants for testing the toxicity of topically applied chemicals

    International Nuclear Information System (INIS)

    Nakamura, M.; Rikimaru, T.; Yano, T.; Moore, K.G.; Pula, P.J.; Schofield, B.H.; Dannenberg, A.M. Jr.

    1990-01-01

    This report describes a model organ-culture system for testing the toxicity of chemical substances that are topically applied to human skin. In this system, the viable keratinocytes in the full-thickness skin explants are protected by the same keratinized layer as skin remaining on the donor, and toxicity can be assessed microscopically and/or biochemically. The human skin specimens were discards from a variety of surgical procedures. They were cut into full-thickness 1.0-cm2 explants, and briefly exposed to the military vesicant sulfur mustard (SM), which was used as a model toxicant. The explants were then organ cultured in small Petri dishes for 24 h at 36 degrees C. In the 0.03-1.0% dosage range, a straight-line dose-response relationship occurred between the concentration of SM applied and the number of paranuclear vacuoles seen histologically in the epidermis. Within the same SM dosage range, there was also a proportional decrease in 14C-leucine incorporation by the explants. Thus, the number of paranuclear vacuoles reflected decreases in protein synthesis by the injured epidermal cells. The epidermis of full-thickness untreated (control) human skin explants usually remained viable for 7 d when stored at 4 degrees C in culture medium. During storage, a relatively small number of paranuclear vacuoles developed within the epidermis, but the explants were still quite satisfactory for testing SM toxicity. Incubation (for 4 or 24 h at 36 degrees C) of such control skin explants reduced (often by 50%) the small number of paranuclear vacuoles produced during 4-7 d of storage. This reduction was probably caused by autolysis of many of the vacuolated cells. Two types of paranuclear vacuoles could be identified by both light and electron microscopy: a storage type and a toxicant type. The storage type seemed to be caused by autolysis of cell components

  4. Full-thickness human skin explants for testing the toxicity of topically applied chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Nakamura, M.; Rikimaru, T.; Yano, T.; Moore, K.G.; Pula, P.J.; Schofield, B.H.; Dannenberg, A.M. Jr. (Johns Hopkins Univ., Baltimore, MD (USA))

    1990-09-01

    This report describes a model organ-culture system for testing the toxicity of chemical substances that are topically applied to human skin. In this system, the viable keratinocytes in the full-thickness skin explants are protected by the same keratinized layer as skin remaining on the donor, and toxicity can be assessed microscopically and/or biochemically. The human skin specimens were discards from a variety of surgical procedures. They were cut into full-thickness 1.0-cm2 explants, and briefly exposed to the military vesicant sulfur mustard (SM), which was used as a model toxicant. The explants were then organ cultured in small Petri dishes for 24 h at 36 degrees C. In the 0.03-1.0% dosage range, a straight-line dose-response relationship occurred between the concentration of SM applied and the number of paranuclear vacuoles seen histologically in the epidermis. Within the same SM dosage range, there was also a proportional decrease in 14C-leucine incorporation by the explants. Thus, the number of paranuclear vacuoles reflected decreases in protein synthesis by the injured epidermal cells. The epidermis of full-thickness untreated (control) human skin explants usually remained viable for 7 d when stored at 4 degrees C in culture medium. During storage, a relatively small number of paranuclear vacuoles developed within the epidermis, but the explants were still quite satisfactory for testing SM toxicity. Incubation (for 4 or 24 h at 36{degrees}C) of such control skin explants reduced (often by 50%) the small number of paranuclear vacuoles produced during 4-7 d of storage. This reduction was probably caused by autolysis of many of the vacuolated cells. Two types of paranuclear vacuoles could be identified by both light and electron microscopy: a storage type and a toxicant type. The storage type seemed to be caused by autolysis of cell components.

  5. 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.

  6. 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.

  7. Enhanced regeneration in explants of tomato (Lycopersicon ...

    African Journals Online (AJOL)

    USER

    2010-06-14

    Jun 14, 2010 ... The development of a cost effective and efficient protocol for mass propagation of high quality tomato seedlings via tissue culture could help to reduce the price per seedling. A good in vitro plant regeneration system may also assist in further improvement of the commercially important cultivars for disease.

  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. Proliferation of Female Inflorescences explants of Date Palm

    International Nuclear Information System (INIS)

    Sidky, R.A; Eldawyati, M.M

    2012-01-01

    This study was conducted to determine the effect of Abscisic acid (ABA) and Ancymidol on proliferation of female inflorescences explants of date palm. In the first experiment two lengths of spath at (5-7 cm) or at (7-10 cm) were cultured on nutrient media which consists of half macro and full micro salts of MS medium supplemented with gradual decreasing in concentration of Abscisic acid (ABA) and Ancymidol from 4.5, 3.0, 1.5 to 0.5 mg -1 . In the second experiment two phases of nutrient medium (solid and liquid) and two source of carbon were investigated. Gradual decreasing of ABA concentrations from 4.5 mg -1 to 1.5 mg -1 in culture medium, stimulated the production of direct somatic embryos and accelerated callus initiation, but at last decrement (0.5 mg -1 ) of Ancymidol concentration few embryos were produced. Callus initiation from inflorescences explants gave high production and well development of somatic embryos when cultured on liquid medium supplemented with 40 g -1 sucrose. All direct or indirect somatic embryos obtained in these experiments were converted successfully to healthy normal plantlets which could be transferred to acclimatization stage.

  10. 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.

  11. Cultura in vitro de embriões e de gemas de mudas de pau-rosa (Aniba rosaeodora Ducke) Culture in vitro of rosewood (Aniba rosaeodora Ducke) embryos´seeds and buds explants

    OpenAIRE

    Lucia Handa; Paulo de Tarso B. Sampaio; Regina Caetano Quisen

    2005-01-01

    Este trabalho teve como objetivo o estabelecimento in vitro de embriões e de gemas de mudas de pau-rosa (Aniba rosaeodora Ducke) livres de contaminações e de oxidação fenólica. As gemas foram obtidas da rebrota de mudas cultivadas em viveiro e os embriões a partir de sementes em diversos estágios de maturação. Para a assepsia dos explantes foram utilizados dois antibiótico (Ampicilina e Agrimicina), etanol (70%) e hipoclorito de sódio, em concentrações e tempo de exposição variando em função ...

  12. Somatic embryogenesis from leaf explants of Australian fan flower, Scaevola aemula R. Br.

    Science.gov (United States)

    Wang, Y-H; Bhalla, P L

    2004-01-01

    Somatic embryogenesis from leaf explants of Scaevola aemula R. Br. was achieved. Somatic embryos were induced from explants cultured on MS medium supplemented with 0.2 mg/ 2,4-dichlorophenoxyacetic acid and 0.2-0.5 mg/l 6-benzylaminopurine (BAP). Various developmental stages of somatic embryos were found on this medium-from globular embryos to germinated embryos. The transfer of globular embryos to MS medium containing 0.5 mg/l BAP resulted in a high frequency of shoot regeneration. Leaf explants cultured on MS medium containing different combinations of BAP and alpha-naphthaleneacetic acid formed adventitious shoots and roots. Histological examination confirmed the process of somatic embryogenesis. Induction of somatic embryogenesis in Scaevola provides a system for studying embryogenesis in Australian native plants and will facilitate the improvement of these plants using genetic transformation techniques.

  13. 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.

  14. 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....

  15. NAA-Induced Direct Organogenesis from Female Immature Inflorescence Explants of Date Palm.

    Science.gov (United States)

    Khierallah, Hussam S M; Bader, Saleh M; Al-Khafaji, Makki A

    2017-01-01

    Micropropagation has great potential for the multiplication of female and male date palms of commercially grown cultivars by using inflorescences. This approach is simple, convenient, and much faster than the conventional method of using shoot-tip explants. We describe here a stepwise micropropagation procedure using inflorescence explants of Iraqi date palm cultivar Maktoom. Cultured explants were derived from 0.5-cm-long spike segments excised from 8 to 10-cm-long spathes. About 70% formed adventitious buds on Murashige and Skoog (MS) medium supplemented with 2 mg/L naphthalene acetic acid (NAA), 4 mg/L benzylaminopurine (BAP), and 40 g/L sucrose and maintained in the dark for 16 weeks before transferring to normal light conditions. The best multiplication rate was achieved with 3 mg/L 2ip and 2 mg/L; for shoot elongation, the best medium is MS containing 0.5 mg/L BAP, 0.5 mg/L 2ip, and 1 mg/L GA 3 . Well-developed shoots were cultured for rooting in half MS medium amended with 1 mg/L NAA and 45 g/L sucrose. Plantlets with well-developed roots were successfully hardened in the greenhouse. Inflorescence explants proved to be a promising alternative explant source for micropropagation of date palm cultivars.

  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. 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

    /or broad-range PCR-positive specimens (n = 96, 74 (77% were monomicrobial and 22 (23% polymicrobial, whereas the rate of polymicrobial samples was higher in broad-range PCR-positive specimens (93%.Conclusions: Combined cultures and broad-range 16S rRNA gene PCR from periprosthetic tissue and/or explanted vascular grafts increased the diagnostic accuracy in VGI, particularly in patients already under antimicrobial treatment at the time of redo surgery. Ideally, antimicrobial treatment should be withheld until surgical sampling in order to optimize microbiological diagnostics.Clinical trials.gov identifier: NCT01821664

  18. Evidence for CB2 receptor involvement in LPS-induced reduction of cAMP intracellular levels in uterine explants from pregnant mice: pathophysiological implications.

    Science.gov (United States)

    Salazar, Ana Inés; Carozzo, Alejandro; Correa, Fernando; Davio, Carlos; Franchi, Ana María

    2017-07-01

    What is the role of the endocannabinoid system (eCS) on the lipopolysaccharide (LPS) effects on uterine explants from 7-day pregnant mice in a murine model of endotoxin-induced miscarriage? We found evidence for cannabinoid receptor type2 (CB2) involvement in LPS-induced increased prostaglandin-F2α (PGF2α) synthesis and diminished cyclic adenosine monophosphate (cAMP) intracellular content in uterine explants from early pregnant mice. Genital tract infections by Gram-negative bacteria are a common complication of human pregnancy that results in an increased risk of pregnancy loss. LPS, the main component of the Gram-negative bacterial wall, elicits a strong maternal inflammatory response that results in embryotoxicity and embryo resorption in a murine model endotoxin-induced early pregnancy loss. We have previously shown that the eCS mediates the embryotoxic effects of LPS, mainly via CB1 receptor activation. An in vitro study of mice uterine explants was performed to investigate the eCS in mediating the effects of LPS on PGF2α production and cAMP intracellular content. Eight to 12-week-old virgin female BALB/c or CD1 (wild-type [WT] or CB1-knockout [CB1-KO]) mice were paired with 8- to 12-week-old BALB/c or CD1 (WT or CB1-KO) males, respectively. On day 7 of pregnancy, BALB/c, CD1 WT or CD1 CB1-KO mice were euthanized, the uteri were excised, implantation sites were removed and the uterine tissues were separated from decidual and embryo tissues. Uterine explants were cultured and exposed for an appropriate amount of time to different pharmacological treatments. The tissues were then collected for cAMP assay and PGF2α content determination by radioimmunoassay. In vitro treatment of uteri explants from 7-day pregnant BALB/c or CD1 (WT or CB1-KO) mice with LPS induced an increased production of PGF2α (P Investigaciones Científicas y Técnicas (PIP 2012/0061). Dr Carlos Davio was funded by Agencia Nacional para la Promoción Científica y Tecnológica (PICT 2013

  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. 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)

  1. 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

    days in culture or directly as dissociated cell suspensions, and compared with regard to neuronal survival and ability to normalize rotational behavior in adult rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. Other lesioned rats received injections of cell-free medium and served as controls...... 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...... improvements in terms of significant reductions in amphetamine-induced rotations were observed in rats grafted with FFRT cultures (127%) and rats grafted with cell suspensions (122%), while control animals showed no normalization of rotational behavior. At 84 days after transplantation, there were similar...

  2. 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.

  3. 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

  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. Adventitious bud formation from bulb-scale explants of Lilium speciosum Thunb. in vitro

    NARCIS (Netherlands)

    Aartrijk, van J.

    1984-01-01

    In this thesis the interactive effects are described of tissue, medium, and other environmental factors on the process of adventitious bud formation in vitro from bulb-scale explants of Lilium speciosum Thunb. Besides, results are presented of experiments

  6. 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.

  7. [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.

  8. Plant Regeneration Through Tissue Culture Of Pear Millet ...

    African Journals Online (AJOL)

    1. 1. 2,5), MS(5) and N6(1.100.25) culture media, calli embryogenic potential and fertile plants regeneration were conserved for more than 12 months. Characteristics of regenerated plants were similar to control. It appears that dissected shoot ...

  9. Organoid culture systems for prostate epithelial and cancer tissue

    NARCIS (Netherlands)

    Drost, Jarno; Karthaus, Wouter R; Gao, Dong; Driehuis, Else; Sawyers, Charles L; Chen, Yu; Clevers, Hans

    This protocol describes a strategy for the generation of 3D prostate organoid cultures from healthy mouse and human prostate cells (either bulk or FACS-sorted single luminal and basal cells), metastatic prostate cancer lesions and circulating tumor cells. Organoids derived from healthy material

  10. 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 ...

  11. Rat fetal ventral mesencephalon grown as solid tissue cultures

    DEFF Research Database (Denmark)

    Höglinger, G U; Sautter, J; Meyer, Morten

    1998-01-01

    in vitro (DIV) in the presence or absence (controls) of BDNF [100 ng/ml]. The dopamine content in the culture medium, analyzed by HPLC, was significantly higher (4-5 fold) in the BDNF group at DIV 8 and DIV 12 compared to the corresponding control levels (40 pg/ml). The number of tyrosine hydroxylase...

  12. 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.

  13. Effect of explant age, hormones on somatic embryogenesis and ...

    African Journals Online (AJOL)

    Effect of explant age, hormones on somatic embryogenesis and production of multiple shoot from cotyledonary leaf explants of Solanum trilobatum L. VNC Dhavala, RD Tejeswara, VR Yechuri, K Prabavathi ...

  14. Inhibition of proliferative activity in tissue culture in vivo of esophagus and stomach tumour cells under preoperative irradiation

    International Nuclear Information System (INIS)

    Zinchenko, V.A.; Okulov, L.V.; Gol'dshmid, B.Ya.

    1988-01-01

    Inhibition of proliferative activity of tumor cells as a result of radiation effect. Tumor tissue taken from patiets with preoperative tumor irradiation by 30 Gy cumulative dose (5 Gy per a session) and from patients whose tumors were not subjected to irradiation (control) was used. The tumor tissue was cultivated in the diffusion chamber and then implanted to the abdominal cavity of the non-inbred male rats. On preparations in the growth area pathomorphological changes were evaluated, the share of mitotically dividing and DNA-synthesizing cells was determined. The absence of growth area around the explant, obvious reduction of mitotic activity and DNA-synthesizing function of cells in preparations of irradiated tumors in 88 % of cases testify to the inhibition of the stomach cardial section and esophagus tumor tissue repopulation after radiation effect. The investigation results confirm the advisability of preoperative irradiation of patients with tumors of the given localization

  15. Sprifermin (rhFGF18) modulates extracellular matrix turnover in cartilage explants ex vivo

    DEFF Research Database (Denmark)

    Reker, Ditte; Kjelgaard-Petersen, Cecilie Freja; Siebuhr, Anne Sofie

    2017-01-01

    (ECM) production. To gain further insight into the process of sprifermin in the cartilage tissue, this study aimed at investigating the ECM turnover of articular cartilage explants in a longitudinal manner. Methods: Bovine full-depth articular cartilage explants were stimulated with sprifermin...... by immuno-histochemical detection of proliferating cell nuclear antigen. ECM turnover was quantified by biomarker ELISAs; ProC2 reflecting type II collagen formation, CS846 reflecting aggrecan formation, active MMP9, C2M and AGNx2 reflecting matrix metalloproteinase activity, and AGNx1 reflecting......, active MMP9 was slightly decreased, and AGNx1 was slightly increased. Over the course of treatment, the temporal order of ECM turnover responses was AGNx1, then ProC2, followed by CS846 and MMP9. Pro-inflammatory activation of the explants diminished the ECM turnover responses otherwise observed under...

  16. [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.

  17. 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.

  18. 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)

  19. 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.

  20. Indução de calos embriogênicos em explantes de cupuaçuzeiro Induction of embryogenics calli in cupuassu explants

    Directory of Open Access Journals (Sweden)

    Maria das Graças Rodrigues Ferreira

    2004-08-01

    Full Text Available Objetivou-se a indução de calos embriogênicos em cupuaçuzeiro, em função do tipo de explante e meio de cultura. Foram testados como explantes, segmentos cotiledonares e eixos embrionários divididos em três partes: região da plúmula, radícula e hipocótilo. Os explantes foram cultivados em 2 diferentes meios de cultura: 1 MS suplementado com 2,4-D (1 mg L-1 e Cinetina (0,25 mg L-1; 2 MS acrescido de ANA (5 mg L-1 e Cinetina (0,25 mg L-1. Constatou-se que a região do hipocótilo foi a parte mais responsiva do eixo embrionário, formando calos com aspecto branco e friável. As auxinas testadas nos meios não estimularam o processo embriogênico em calos de cupuaçuzeiro.It was studied the induction of embryogenics calli in cupuassu, in function of kind of explant and culture medium. Cotyledons segments and embryonic axes were tested and divided in three parts: region of plumule, radicule and hypocotile. The explants were cultivated in two different culture medium: 1 MS supplemented with 2,4-D (1 mg L-1 and Kinetin (0,25 mg L-1; 2 MS supplemented with NAA (5 mg L-1 and Kinetin (0,25 mg L-1. The hypocotile region demonstrated to be more responsive segment of the embryonic axe, forming callus with white and friable aspect. No somatic embryogenesis was evidenced in callus of cupuassu with auxines testeds in the medium.

  1. Tissue culture regeneration and radiation induced mutagenesis in banana

    International Nuclear Information System (INIS)

    Kulkarni, V.M.; Ganapathi, T.R.

    2009-01-01

    Radiation induced mutagenesis is an important tool for banana genetic improvement. At BARC, protocols for shoo-tip multiplication of commercial banana varieties have been developed and transferred to user agencies for commercial production. Excellent embryogenic cell suspensions were established in banana cvs. Rasthali and Rajeli, and were maintained at low temperatures for long-term storage. Normal plantlets were successfully regenerated from these cell suspensions. The cell suspensions and shoot-tip cultures were gamma-irradiated for mutagenesis. The mutagenized populations were field screened and a few interesting mutants have been isolated. The existence of genetic variation was confirmed using DNA markers. Further evaluation of these mutants is in progress. (author)

  2. 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

  3. 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

  4. Single molecule microscopy in 3D cell cultures and tissues.

    Science.gov (United States)

    Lauer, Florian M; Kaemmerer, Elke; Meckel, Tobias

    2014-12-15

    From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Antibiotic effects against periodontal bacteria in organ cultured tissue.

    Science.gov (United States)

    Takeshita, Masaaki; Haraguchi, Akira; Miura, Mayumi; Hamachi, Takafumi; Fukuda, Takao; Sanui, Terukazu; Takano, Aiko; Nishimura, Fusanori

    2017-02-01

    Mechanical reduction of infectious bacteria by using physical instruments is considered the principal therapeutic strategy for periodontal disease; addition of antibiotics is adjunctive. However, local antibiotic treatment, combined with conventional mechanical debridement, has recently been shown to be more effective in periodontitis subjects with type 2 diabetes. This suggests that some bacteria may invade the inflamed inner gingival epithelium, and mechanical debridement alone will be unable to reduce these bacteria completely. Therefore, we tried to establish infected organ culture models that mimic the inner gingival epithelium and aimed to see the effects of antibiotics in these established models. Mouse dorsal skin epithelia were isolated, and periodontal bacteria were injected into the epithelia. Infected epithelia were incubated with test antibiotics, and colony-forming ability was evaluated. Results indicated that effective antibiotics differed according to injected bacteria and the bacterial combinations tested. Overall, in organ culture model, the combination of amoxicillin or cefdinir and metronidazole compensate for the effects of less effective bacterial combinations on each other. This in vitro study would suggest effective periodontal treatment regimens, especially for severe periodontitis.

  6. 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

  7. 非洲紫罗兰组织培养体系的建立%Establishing the Tissue Culture System for Saintpaulia Ionantha

    Institute of Scientific and Technical Information of China (English)

    尹立辉; 武术杰; 刘亚亮; 王雪松

    2016-01-01

    以紫色白边单瓣花的非洲紫罗兰叶片为试材进行了组织培养技术的研究。结果表明:以叶片为外植体进行组织培养时,愈伤组织诱导最适培养基为MS+0.2 mg/L NAA+0.5 mg/L6-BA,培养3周左右诱导率达100%;继代增殖时最适培养基为MS+0.02 mg/L NAA+0.1 mg/L 6-BA;无需生根培养,直接瓶外炼苗,栽植于以m (草炭土)∶m(珍珠岩)=1∶1的基质中生长最好,成活率可达95%以上。%We studied the tissue culture technology of Saintpaulia Ionantha with the purple petals of white edge and single flower . The tissue culture was conducted with the leaves as explants .The inducing medium for callus was MS+0.2 mg/L NAA+0.5 mg/L 6-BA.The induction rate after three weeks was100 %.The most suitable medium for subculture was MS+0.02 mg/L NAA+0.1 mg/L 6-BA.The outk-tube rooting rate were relatively better without taking root in the bottle .The best matrix for planting effect was peat soil and perlite with the mixed proportion of 1∶1 and the survival rate of >95%.

  8. Effect of explant plant source and acetosyringone concentration on ...

    African Journals Online (AJOL)

    Jane

    2011-08-15

    Aug 15, 2011 ... a number of factors including tissue culture conditions, acetosyringone .... immature embryo culture, callus induction and in vitro salt stress. Plant Cell ... phenotypical characterization of transgenic tomato plants: Solanum.

  9. 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.

  10. 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.

  11. Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection.

    Science.gov (United States)

    Fornaro, Michele; Sharthiya, Harsh; Tiwari, Vaibhav

    2018-03-09

    This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory neurons and glial satellite cells. These are useful and versatile models to investigate a variety of biological responses associated with physiological and pathological conditions of the peripheral nervous system (PNS) ranging from neuron-glial interaction, neuroplasticity, neuroinflammation, and viral infection. The usage of DRG explant is scientifically advantageous compared to simplistic single cells models for multiple reasons. For instance, as an organotypic culture, the DRG explant allows ex vivo transfer of an entire neuronal network including the extracellular microenvironment that play a significant role in all the neuronal and glial functions. Further, DRG explants can also be maintained ex vivo for several days and the culture conditions can be perturbed as desired. In addition, the harvested DRG can be further dissociated into an in vitro co-culture of primary sensory neurons and satellite glial cells to investigate neuronal-glial interaction, neuritogenesis, axonal cone interaction with the extracellular microenvironment, and more general, any aspect associated with the neuronal metabolism. Therefore, the DRG-explant system offers a great deal of flexibility to study a wide array of events related to biological, physiological, and pathological conditions in a cost-effective manner.

  12. 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

  13. Studies on the effect of genotype and explant type on callogenesis and organogenesis in indica rice

    International Nuclear Information System (INIS)

    Noor, A.; Rashid, H.; Khan, M.H.; Chaudhry, Z.

    2011-01-01

    The purpose of the research study is to select the best rice explant and variety for Agrobacterium mediated genetic transformation studies. In vitro callus induction and regeneration frequency of two explants i.e. immature embryo and media supplemented mature grains of 9 rice varieties (Oryza sativa L.) was investigated by culturing explants source on N/sub 6/ with 2, 4-D at 2 mg/l for callus induction. Results indicated that mature seed explant produced significantly high number of calli as compared to immature embryo. Two types of calli were distinguished, designated as type-I calli and type-II calli. Regarding the quality of callus, type-I calli (produced from scutellum of mature grains) which were embryogenic produced higher plant regeneration frequency than type-II calli which were non-embryogenic. Mature-seed scutella calli and immature embryo-derived calli (after three weeks of culture) of rice varieties were transferred on regeneration medium i.e., MS salts and vitamins, 3% sucrose, 3% sorbitol, 2 g/l casine hydrolysate, NAA 1.0 mg/l, kin 2.5 mg /l and BAP at 0.5mg/l. The highest regeneration capacity was observed in DR-83 from mature seed derived calli followed by Basmati 385. After hardening the plantlets were transferred to soil. (author)

  14. 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

  15. 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

  16. 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

  17. 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.

  18. Host DNA synthesis-suppressing factor in culture fluid of tissue cultures infected with measles virus

    International Nuclear Information System (INIS)

    Minagawa, T.; Nakaya, C.; Iida, H.

    1974-01-01

    Host DNA synthesis is suppressed by the culture fluid of cell cultures infected with measles virus. This activity in the culture fluid is initiated somewhat later than the growth of infectious virus. Ninety percent of host DNA synthesis in HeLa cells is inhibited by culture fluid of 3-day-old cell cultures of Vero or HeLa cells infected with measles virus. This suppressing activity is not a property of the virion, but is due to nonvirion-associated componentnent which shows none of the activities of measles virus such as hemagglutination, hemolysis, or cell fusion nor does it have the antigenicity of measles virus as tested by complement-fixation or hemagglutination-inhibiting antibody blocking tests. Neutralization of the activity of this component is not attained with the pooled sera of convalescent measles patients. This component has molecular weights of about 45,000, 20,000, and 3,000 and appears to be a heat-stable protein. The production of host DNA suppressing factor (DSF) is blocked by cycloheximide. Neither uv-inactivated nor antiserum-neutralized measles virus produce DSF. Furthermore, such activity of nonvirion-associated component is not detected in the culture fluid of cultures infected with other RNA viruses such as poliovirus, vesicular stomatitis virus, or Sindbis virus. (auth)

  19. 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.

  20. 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

  1. 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)

  2. 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

  3. Optimization of callus induction of Zataria multiflora under the effect of different plant growth regulators and explant source

    Directory of Open Access Journals (Sweden)

    N. Mosavat*

    2017-11-01

    Full Text Available Background and objectives: The Lamiaceae family is rich in favorable secondary metabolites which have different medicinal properties and also use in food, cosmetic and sanitary industry. Zataria multiflora Boiss. is an aromatic and bushy plant containing specific pharmaceutical components which is only distributed in certain regions of Iran. Tissue culture technologies could be suitable for in vitro production of Zataria. Methods: In this study, callus production and callus related traits of Zataria was evaluated at in vitro condition. Callus induction was performed on Murashige and Skoog (MS medium containing different levels of plant growth regulators including different cytokinins (Kinetin, benzyl amino purine and auxins (2,4 dichlorophenoxyacetic acid and naphtalen acetic acid and two different explant (hypocotyl and leaf. Results: The friable calli with yellow-green color only appeared from leaf explants on three different treatments including: 1: 2.5 (mg/L 2,4-D; 2: 2 (mg/L  2,4-D; 3: [2 (mg/L 2,4-D+ 1 (mg/L Kin].  The best callus induction (75% was obtained at 2,4-D (2 mg/L + Kin (1 mg/L after 2 month of incubation under the photoperiod of 16/8 (light/dark. The highest callus growth rate (CGR (0.072 mm/day and callus fresh weight (0.135 g were denoted to the treatment of 2 mg/L (2,4-D.  Conclusion: The benefits of the protocol described here include the possibility of its use throughout the callus culture for commercial production of suitable secondary metabolites of Zataria in rapid time and huge scale.

  4. 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

  5. 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.

  6. Induction of hairy roots by various strains of Agrobacterium rhizogenes in different types of Capsicum species explants.

    Science.gov (United States)

    Md Setamam, Nursuria; Jaafar Sidik, Norrizah; Abdul Rahman, Zainon; Che Mohd Zain, Che Radziah

    2014-06-30

    Capsicum annuum and Capsicum frutescens, also known as "chilies", belong to the Solanaceae family and have tremendous beneficial properties. The application of hairy root culture may become an alternative method for future development of these species by adding value, such as by increasing secondary metabolites and improving genetic and biochemical stability compared with normal Capsicum plants. Therefore, in this research, different types of explants of both species were infected with various Agrobacterium rhizogenes strains to provide more information about the morphology and induction efficiency of hairy roots. After 2 weeks of in vitro seed germination, young seedling explants were cut into three segments; the cotyledon, hypocotyl, and radical. Then, the explants were co-cultured with four isolated A. rhizogenes strains in Murashige & Skoog culture media (MS) containing decreasing carbenicillin disodium concentrations for one month. In this experiment, thick and short hairy roots were induced at all induction sites of C. annuum while thin, elongated hairy roots appeared mostly at wound sites of C. frutescens. Overall, the hairy root induction percentages of C. frutescens were higher compared with C. annuum. Hairy root initiation was observed earliest using radicles (1st week), followed by cotyledons (2nd week), and hypocotyls (3rd week). Cotyledon explants of both species had the highest induction frequency with all strains compared with the other explants types. Strains ATCC 13333 and ATCC 15834 were the most favourable for C. frutescens while ATCC 43056 and ATCC 43057 were the most favourable for C. annuum. The interactions between the different explants and strains showed significant differences with p-values Capsicum species. Both Capsicum species were amenable to A. rhizogenes infection and hairy root induction is recommended for use as an alternative explants in future plant-based studies.

  7. 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.

  8. 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

  9. 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.}

  10. 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.

  11. 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

  12. 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 ...

  13. 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 ...

  14. 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.

  15. 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

  16. [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.

  17. 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 ...

  18. Indirect organogenesis in milkweed ( Calotropis procera from mature zygotic embryo explants

    Directory of Open Access Journals (Sweden)

    Hojatollah Abbasi

    2018-01-01

    Full Text Available Milkweed ( Calotropis procera is a valuable medicinal plant which grows in many regions of Iran. Its significant medicinal properties have made it an important crop which is cultivated commercially. This plant is propagated from seeds as well as root and shoot cuttings. Due to problems in the usage of these reproduction methods, new propagation methods such as tissue culturing should be developed. This study was aimed at obtaining appropriate concentrations of plant hormones for indirect organogenesis of milkweed. The experiment was arranged in a completely randomized design (CRD with 3 replications. The effects of various concentrations of (2,4-dichlorophenoxyacetic acid 2,4-D (0.1, 0.5, 1, 2 and 3 mg/l were studied in terms of callus induction and shoot regeneration on an MS based medium supplemented with BA (benzyl amino purine and NAA (naphthalene acetic acid at the same concentration. Mature embryos were used as explants and morphological traits such as embryo size, callus size, number and size of shoots and roots were recorded. The results showed that 2,4-D significantly increased the size of cultured embryos (P < 0.05. The largest embryo volume was observed in cultures treated with 3 mg/l 2,4-D. The highest callusing was recorded in 2 mg/l 2,4-D. The effects of BA and NAA concentrations on shoot regeneration were significant and the highest values were observed for a combination of 1 mg/l BA and 2 mg/l NAA. 1 mg/l IBA (Indole 3-butyric acid was able to induce the highest number of better quality roots and shoots.

  19. 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

  20. 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.

  1. 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

  2. Evaluation on the Effects of Culture Medium on Regeneration of Oil Palm Plantlets from Immature Embryos (IE)

    International Nuclear Information System (INIS)

    Wan Nur Syuhada, W.S.; Rasid, O.A.; Parveez, G.K.A

    2016-01-01

    Oil palm (Elaeis guineensis) tissue culture is slow, inefficient and genotype dependent. Therefore, efforts to improve the regeneration system are essential and thereby on-going. Immature embryos (IE) have been reported to be more responsive to in vitro culture as compared to other explant types. The objective of this study is to evaluate the most efficient culture media for the regeneration of whole plants from IE. IE were cultured on three media, namely N6, MY3 and MS +Y3. Initial observation showed that calli were induced as early as two weeks after culture. They started to produce friable calli around three to four months later. IE were shown to respond differently to the tested culture media. This initial result will be useful to identify the most responsive culture media for IE as explant. (author)

  3. 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

  4. 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.

  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. 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.

  7. Researches about selecting resistant melon types to fusarium oxyporum f. sp.melonis race 1,2 by using tissue culture and mutation techniques

    International Nuclear Information System (INIS)

    2009-01-01

    breeding was quickly understood. In vitro techniques for crop improvement first consisted of micropropagation and plant regeneration, and then in vitro methods were also found to be useful for eliminating disease and selecting for resistant cells or explants. In vitro selection using specific chemical compounds and pathogens is another useful aspect of tissue culture. More recently, in vitro techniques were combined to mutation induction for generating genetic variation, including novel disease resistant mutants. Mutation induction can be caused by chemical or physical mutagens that alter the structure of the DNA. Treatment of in vitro tissues with physical or chemical mutagens may increase the frequency of genetic variation considerably. The physical mutagens most commonly used are X-rays, gamma rays and UV light, whereas Ethyl Methane- Sulphonate (EMS) is the chemical mutagen most used in crop improvement. Irradiation treatments may be a suitable choice of mutagen for a number of reasons because the application is fast and, in contrast to chemical mutagens, there is no risk that residues remain in the medium. In this research we are going to determine the resistant cells, which will come from irradiated and non-irradiated explants by using races of F. oxysporum f. sp. melonis culture filtrates in vitro conditions. If we will get successful results by this method we will be able to develop new melon cultivars, which will be resistant to F. oxysporum f. sp. melonis. The present work was performed with in vitro plantlets obtained from seeds of melon cv. Yuva which is an important commercial cultivar in Turkey. According to our observations filtrate and gamma ray treatment have an important effect on tolerant plantlet and callus formation. In this research, we show a method for mass-selection of melon mutants resistant to Fusarium wilt. In vitro selection of resistant cells, which are come from irradiated and non-irradiated explants, is done using culture filtrates of

  8. Hypoxia inhibits hypertrophic differentiation and endochondral ossification in explanted tibiae.

    Directory of Open Access Journals (Sweden)

    Jeroen C H Leijten

    Full Text Available Hypertrophic differentiation of growth plate chondrocytes induces angiogenesis which alleviates hypoxia normally present in cartilage. In the current study, we aim to determine whether alleviation of hypoxia is merely a downstream effect of hypertrophic differentiation as previously described or whether alleviation of hypoxia and consequent changes in oxygen tension mediated signaling events also plays an active role in regulating the hypertrophic differentiation process itself.Fetal mouse tibiae (E17.5 explants were cultured up to 21 days under normoxic or hypoxic conditions (21% and 2.5% oxygen respectively. Tibiae were analyzed on growth kinetics, histology, gene expression and protein secretion.The oxygen level had a strong influence on the development of explanted fetal tibiae. Compared to hypoxia, normoxia increased the length of the tibiae, length of the hypertrophic zone, calcification of the cartilage and mRNA levels of hypertrophic differentiation-related genes e.g. MMP9, MMP13, RUNX2, COL10A1 and ALPL. Compared to normoxia, hypoxia increased the size of the cartilaginous epiphysis, length of the resting zone, calcification of the bone and mRNA levels of hyaline cartilage-related genes e.g. ACAN, COL2A1 and SOX9. Additionally, hypoxia enhanced the mRNA and protein expression of the secreted articular cartilage markers GREM1, FRZB and DKK1, which are able to inhibit hypertrophic differentiation.Collectively our data suggests that oxygen levels play an active role in the regulation of hypertrophic differentiation of hyaline chondrocytes. Normoxia stimulates hypertrophic differentiation evidenced by the expression of hypertrophic differentiation related genes. In contrast, hypoxia suppresses hypertrophic differentiation of chondrocytes, which might be at least partially explained by the induction of GREM1, FRZB and DKK1 expression.

  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. Gamma of effects radiation on the production of secondary metabolites by tissues of Jatropha curcas L. cultured in-vitro

    International Nuclear Information System (INIS)

    Nyembo, K.; Luyindula, N.; Onyembe, P.M.L.; Makosso, N.; Muambi, N.

    2005-01-01

    Petiole explants of Jatropha curcas L. were irradiated with gamma rays at doses ranging from 5 to 100 Gy from a Cs source and cultured in vitro during 60 days. Callus formation decreased markedly with increasing gamma rays dose. The extraction's yield of hydrosoluble components from irradiated material at 5Gy (29,3%) is higher than of the control (17,2%). The phytochemical screening has revealed presence of saponins, alkaloids, amino-compounds, steroids and terpenoids in a large amount in both irradiated and control materials. The antibacterial activity against the strains of Proteus mirabilis and Escherichia coli is highly significant. This activity is depending upon one or more of phytochemical groups identified.

  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. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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…

  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. 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

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. Reproducible simulation of respiratory motion in porcine lung explants

    Energy Technology Data Exchange (ETDEWEB)

    Biederer, J. [Dept. of Diagnostic Radiology, Univ. Hospital Schleswig-Holstein, Campus Kiel (Germany); Dept. of Radiology, German Cancer Research Center, Heidelberg (Germany); Plathow, C. [Dept. of Diagnostic Radiology, Eberhard-Karls-Univ. Tuebingen (Germany); Dept. of Radiology, German Cancer Research Center, Heidelberg (Germany); Schoebinger, M.; Meinzer, H.P. [Dept. of Medical and Biological Informatics, German Cancer Research Center, Heidelberg (Germany); Tetzlaff, R.; Puderbach, M.; Zaporozhan, J.; Kauczor, H.U. [Dept. of Radiology, German Cancer Research Center, Heidelberg (Germany); Bolte, H.; Heller, M. [Dept. of Diagnostic Radiology, Univ. Hospital Schleswig-Holstein, Campus Kiel (Germany)

    2006-11-15

    Purpose: To develop a model for exactly reproducible respiration motion simulations of animal lung explants inside an MR-compatible chest phantom. Materials and Methods: The materials included a piston pump and a flexible silicone reconstruction of a porcine diaphragm and were used in combination with an established MR-compatible chest phantom for porcine heart-lung preparations. The rhythmic inflation and deflation of the diaphragm at the bottom of the artificial thorax with water (1-1.5 L) induced lung tissue displacement resembling diaphragmatic breathing. This system was tested on five porcine heart-lung preparations using 1.5T MRI with transverse and coronal 3D-GRE (TR/TE=3.63/1.58, 256 x 256 matrix, 350 mm FOV, 4 mm slices) and half Fourier T2-FSE (TR/TE=545/29, 256 x 192, 350 mm, 6 mm) as well as multiple row detector CT (16 x 1 mm collimation, pitch 1.5, FOV 400 mm, 120 mAs) acquired at five fixed inspiration levels. Dynamic CT scans and coronal MRI with dynamic 2D-GRE and 2D-SS-GRE sequences (image frequencies of 10/sec and 3/sec, respectively) were acquired during continuous 'breathing' (7/minute). The position of the piston pump was visually correlated with the respiratory motion visible through the transparent wall of the phantom and with dynamic displays of CT and MR images. An elastic body splines analysis of the respiratory motion was performed using CT data. Results: Visual evaluation of MRI and CT showed three-dimensional movement of the lung tissue throughout the respiration cycle. Local tissue displacement inside the lung explants was documented with motion maps calculated from CT. The maximum displacement at the top of the diaphragm (mean 26.26 [SD 1.9] mm on CT and 27.16 [SD 1.5] mm on MRI, respectively [p=0.25; Wilcoxon test]) was in the range of tidal breathing in human patients. Conclusion: The chest phantom with a diaphragmatic pump is a promising platform for multi-modality imaging studies of the effects of respiratory lung

  4. Reproducible simulation of respiratory motion in porcine lung explants

    International Nuclear Information System (INIS)

    Biederer, J.; Plathow, C.; Schoebinger, M.; Meinzer, H.P.; Tetzlaff, R.; Puderbach, M.; Zaporozhan, J.; Kauczor, H.U.; Bolte, H.; Heller, M.

    2006-01-01

    Purpose: To develop a model for exactly reproducible respiration motion simulations of animal lung explants inside an MR-compatible chest phantom. Materials and Methods: The materials included a piston pump and a flexible silicone reconstruction of a porcine diaphragm and were used in combination with an established MR-compatible chest phantom for porcine heart-lung preparations. The rhythmic inflation and deflation of the diaphragm at the bottom of the artificial thorax with water (1-1.5 L) induced lung tissue displacement resembling diaphragmatic breathing. This system was tested on five porcine heart-lung preparations using 1.5T MRI with transverse and coronal 3D-GRE (TR/TE=3.63/1.58, 256 x 256 matrix, 350 mm FOV, 4 mm slices) and half Fourier T2-FSE (TR/TE=545/29, 256 x 192, 350 mm, 6 mm) as well as multiple row detector CT (16 x 1 mm collimation, pitch 1.5, FOV 400 mm, 120 mAs) acquired at five fixed inspiration levels. Dynamic CT scans and coronal MRI with dynamic 2D-GRE and 2D-SS-GRE sequences (image frequencies of 10/sec and 3/sec, respectively) were acquired during continuous 'breathing' (7/minute). The position of the piston pump was visually correlated with the respiratory motion visible through the transparent wall of the phantom and with dynamic displays of CT and MR images. An elastic body splines analysis of the respiratory motion was performed using CT data. Results: Visual evaluation of MRI and CT showed three-dimensional movement of the lung tissue throughout the respiration cycle. Local tissue displacement inside the lung explants was documented with motion maps calculated from CT. The maximum displacement at the top of the diaphragm (mean 26.26 [SD 1.9] mm on CT and 27.16 [SD 1.5] mm on MRI, respectively [p=0.25; Wilcoxon test]) was in the range of tidal breathing in human patients. Conclusion: The chest phantom with a diaphragmatic pump is a promising platform for multi-modality imaging studies of the effects of respiratory lung motion. (orig.)

  5. 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.

  6. 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.

  7. Effect of glutaraldehyde fixation on the frictional response of immature bovine articular cartilage explants.

    Science.gov (United States)

    Oungoulian, Sevan R; Hehir, Kristin E; Zhu, Kaicen; Willis, Callen E; Marinescu, Anca G; Merali, Natasha; Ahmad, Christopher S; Hung, Clark T; Ateshian, Gerard A

    2014-02-07

    This study examined functional properties and biocompatibility of glutaraldehyde-fixed bovine articular cartilage over several weeks of incubation at body temperature to investigate its potential use as a resurfacing material in joint arthroplasty. In the first experiment, treated cartilage disks were fixed using 0.02, 0.20 and 0.60% glutaraldehyde for 24h then incubated, along with an untreated control group, in saline for up to 28d at 37°C. Both the equilibrium compressive and tensile moduli increased nearly twofold in treated samples compared to day 0 control, and remained at that level from day 1 to 28; the equilibrium friction coefficient against glass rose nearly twofold immediately after fixation (day 1) but returned to control values after day 7. Live explants co-cultured with fixed explants showed no quantitative difference in cell viability over 28d. In general, no significant differences were observed between 0.20 and 0.60% groups, so 0.20% was deemed sufficient for complete fixation. In the second experiment, cartilage-on-cartilage frictional measurements were performed under a migrating contact configuration. In the treated group, one explant was fixed using 0.20% glutaraldehyde while the apposing explant was left untreated; in the control group both explants were left untreated. From day 1 to 28, the treated group exhibited either no significant difference or slightly lower friction coefficient than the untreated group. These results suggest that a properly titrated glutaraldehyde treatment can reproduce the desired functional properties of native articular cartilage and maintain these properties for at least 28d at body temperature. © 2013 Published by Elsevier Ltd.

  8. 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...

  9. 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

  10. [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.

  11. 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)

  12. Low light and low ammonium are key factors for guayule leaf tissue shoot organogenesis and transformation.

    Science.gov (United States)

    Dong, Niu; Montanez, Belen; Creelman, Robert A; Cornish, Katrina

    2006-02-01

    A new method has been developed for guayule tissue culture and transformation. Guayule leaf explants have a poor survival rate when placed on normal MS medium and under normal culture room light conditions. Low light and low ammonium treatment greatly improved shoot organogenesis and transformation from leaf tissues. Using this method, a 35S promoter driven BAR gene and an ubiquitin-3 promoter driven GUS gene (with intron) have been successfully introduced into guayule. These transgenic guayule plants were resistant to the herbicide ammonium-glufosinate and were positive to GUS staining. Molecular analysis showed the expected band and signal in all GUS positive transformants. The transformation efficiency with glufosinate selection ranged from 3 to 6%. Transformation with a pBIN19-based plasmid containing a NPTII gene and then selection with kanamycin also works well using this method. The ratio of kanamycin-resistant calli to total starting explants reached 50% in some experiments.

  13. Effect of plant growth regulators, explants type and efficient plantlet ...

    African Journals Online (AJOL)

    use

    2011-12-05

    Dec 5, 2011 ... Plant Pathology, Tissue Culture and Biotechnology Laboratory, Department of Botany,. University of ... variability in response to growth regulators. In vitro rooting ..... an adult tree Wrightia tomentosa through enhanced axillary.

  14. Direct and indirect plant regeneration from various explants of ...

    African Journals Online (AJOL)

    user

    2011-04-18

    Apr 18, 2011 ... Populus species are important resource for certain branches of industry and have special roles for ... Key words: Poplar, tissue culture, regeneration, organogenesis. .... the best (90 to 100%) adaptation ratio in the plant growth.

  15. Callus regeneration from stem explants of Pseudarthira viscida (L ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-09-01

    Sep 1, 2009 ... regeneration frequency have come from tissue culture work done in ... Table 1. In vitro responses from stem callus of Psudarthria viscida Wight & Arn. Growth .... plantlets regeneration from cotyledonary callus of Tomato.

  16. Direct organogenesis of seaside heliotrope (Heliotropium crassavicum) using stem explants.

    Science.gov (United States)

    Satyavani, K; Dheepak, V; Gurudeeban, S; Ramanathan, T

    2013-10-15

    Heliotropium crassavicum L. is a sand binder salt marsh herb with enormous traditional value and widely found in South Asia America and Europe. In the direct method of regeneration from stem explants, we observed the maximum number of shoot regeneration after four weeks culture of MS elongation medium with 2.0 mg L(-1) of 2, 4-D (17.27 +/- 0.51). It was clear that MS medium with 2.0 mg mL(-1) 2, 4-D alone suitable for shoot multiplication as well as shoot elongation then compared to other combination of auxin and cytokinin. In vitro shoots were excised from shoot clumps and transferred to rooting medium containing 2, 4-dichlorophenoxy acetic acid (0.5-3.0 mg L(-1)). The maximum number of root regeneration (6.4 +/- 0.416) and root length (6.08 +/- 0.07) were observed in MS rooting medium fortified with 2.5 mg L(-1) of 2, 4-D after 2 weeks of culture. 85% of in vitro raised plantlets with well-developed shoots and roots were transferred to ex vivo conditions into polythene bag containing sterile compost with ratio (v/v/v) of organic fertilizer: sand: peat (1:2:2; 3:1:0 or 2:2:1). Sixty five percent of acclimated plants were transferred to the pots under full sun where they grew well without any detectable phenotypic variations.

  17. 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.

  18. 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)

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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)

  4. 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)

  5. 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

  6. 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

  7. Callogenesis in root explants of four species of the family Solanaceae after inducing by Agrobacterium rhizogenes

    Directory of Open Access Journals (Sweden)

    Zahra Shakeran

    2015-09-01

    Full Text Available Studying explants affected by Agrobacterium rhizogenes shows that in addition to possible formation of hairy roots, it is likely that callogenesis can be induced in these tissues. The T-DNA region of A. rhizogenes codes enzymes that participate in biosynthesis of plants growth hormones. These hormones also affect callogenesis, hence, the formation of various calluses with different morphological properties are possible. It is very likely that the level of biosynthetic growth hormone, the plasmid carried by each bacteria strain, the position of T-DNA, and the level of gene expression contribute to this morphologic variation. In this study, the root explants of four species of the family Solanaceae namely Atropa belladonna, Datura metel, D. stramonium and Hyoscyamus niger were induced by using different strains of A. rhizogenes (A4, A7, AR15834, AR318, AR9402 and AR9543. Some of these explants entered callus phase and formed various calluses with different colors and shapes. Moreover, in some callus samples hairy roots were also appeared. These variations were probably caused by variations in the levels and ratios of auxin and cytokinine hormons after the induction. As shown in previous studies, the amount of secondary metabolites is reduced due to undifferentiated tissue produced in the callogenesis process.

  8. Plant regeneration via direct somatic embryogenesis from leaf explants of Tolumnia Louise Elmore 'Elsa'.

    Science.gov (United States)

    Shen, Hui-Ju; Chen, Jen-Tsung; Chung, Hsiao-Hang; Chang, Wei-Chin

    2018-01-22

    Tolumnia genus (equitant Oncidium) is a group of small orchids with vivid flower color. Thousands of hybrids have been registered on Royal Horticulture Society and showed great potential for ornamental plant market. The aim of this study is to establish an efficient method for in vitro propagation. Leaf explants taken from in vitro-grown plants were used to induce direct somatic embryogenesis on a modified 1/2 MS medium supplemented with five kinds of cytokinins, 2iP, BA, kinetin, TDZ and zeatin at 0.3, 1 and 3 mg l -1 in darkness. TDZ at 3 mg l -1 gave the highest percentage of explants with somatic globular embryos after 90 days of culture. It was found that 2,4-D and light regime highly retarded direct somatic embryogenesis and showed 95-100% of explant browning. Histological observations revealed that the leaf cells divided into meristematic cells firstly, followed by somatic proembryos, and then somatic globular embryos. Eventually, somatic embryos developed a bipolar structure with the shoot apical meristem and the root meristem. Scanning electron microscopy observations showed that the direct somatic embryogenesis from leaf explants was asynchronously. The somatic embryos were found on the leaf tip, the adaxial surface and also the mesophyll through a cleft, and it reflected the heterogeneity of the explant. The 90-day-old globular embryos were detached from the parent explants and transferred onto a hormone-free 1/2 MS medium in light condition for about 1 month to obtain 1-cm-height plantlets. After another 3 months for growth, the plantlets were potted with Sphagnum moss and were acclimatized in a shaded greenhouse. After 1 month of culture, the survival rate was 100%. In this report, a protocol for efficient regenerating a Tolumnia orchid, Louise Elmore 'Elsa', was established via direct somatic embryogenesis and might reveal an alternative approach for mass propagation of Tolumnia genus in orchid industry.

  9. Pistacia lentiscus fruit oil reduces oxidative stress in human skin explants caused by hydrogen peroxide.

    Science.gov (United States)

    Ben Khedir, S; Moalla, D; Jardak, N; Mzid, M; Sahnoun, Z; Rebai, T

    2016-10-01

    We investigated the efficacy of Pistacia lentiscus fruit oil (PLFO) for protecting human skin from damage due to oxidative stress. PLFO contains natural antioxidants including polyphenols, sterols and tocopherols. We compared the antioxidant potential of PLFO with extra virgin olive oil (EVOO). Explants of healthy adult human skin were grown in culture with either PLFO or EVOO before adding hydrogen peroxide (H 2 O 2 ). We also used cultured skin explants to investigate the effects of PLFO on lipid oxidation and depletion of endogenous antioxidant defense enzymes including glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) one day after 2 h exposure to H 2 O 2 . We found that PLFO scavenged radicals and protected skin against oxidative injury. PLFO exhibited greater antioxidant and free radical scavenging activity than EVOO. Skin explants treated with PLFO inhibited H 2 O 2 induced MDA formation by inhibition of lipid oxidation. In addition, the oil inhibited H 2 O 2 induced depletion of antioxidant defense enzymes including GPx, SOD and CAT. We found that treatment with PLFO repaired skin damage owing to its antioxidant properties.

  10. [Explantation method of isolating a persistent tick-borne encephalitis virus from the organs of infected monkeys].

    Science.gov (United States)

    Levina, L S; Pogodina, V V

    1981-01-01

    The method of explantation was used to examine 63 organs from M. rhesus monkeys 92-783 days after intracerebral and subcutaneous inoculation with the Vasilchenko, Aina/1448 and 41/65 strains of tick-borne encephalitis virus. The optimal time for examination of the explants by tests of the hemagglutinating, cytopathogenic activity of the virus and its pathogenicity for mice was found to be the 15th day of cultivation. A comparative study of the properties of 3 isolates obtained from explants of the spleen, liver and subcortical cerebral ganglia 202 and 307 days after inoculation of monkeys was carried out. The isolates differed from the parental TBE virus strains by their capacity to form small plaques in PEKV cell cultures (pig embryo kidney cells in versen medium).

  11. Acetylcholine causes rooting in leaf explants of in vitro raised tomato (Lycopersicon esculentum Miller) seedlings.

    Science.gov (United States)

    Bamel, Kiran; Gupta, Shrish Chandra; Gupta, Rajendra

    2007-05-30

    The animal neurotransmitter acetylcholine (ACh) induces rooting and promotes secondary root formation in leaf explants of tomato (Lycopersicon esculentum Miller var. Pusa Ruby), cultured in vitro on Murashige and Skoog's medium. The roots originate from the midrib of leaf explants and resemble taproot. ACh at 10(-5) M was found to be the optimum over a wide range of effective concentrations between 10(-7) and 10(-3) M. The breakdown products, choline and acetate were ineffective even at 10(-3) M concentration. ACh appears to have a natural role in tomato rhizogenesis because exogenous application of neostigmine, an inhibitor of ACh hydrolysis, could mimic the effect of ACh. Neostigmine, if applied in combination with ACh, potentiated the ACh effect.

  12. 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.

  13. 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.

  14. 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.)

  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. 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

  17. 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

  18. Peritumoral adipose tissue as a source of inflammatory and angiogenic factors in colorectal cancer.

    Science.gov (United States)

    Amor, S; Iglesias-de la Cruz, M C; Ferrero, E; García-Villar, O; Barrios, V; Fernandez, N; Monge, L; García-Villalón, A L; Granado, M

    2016-02-01

    Obesity is a risk factor for the development of human colorectal cancer (CC). The aim of this work is to report the inflammatory and angiogenic scenario in lean (BMI  30 kg/m2) patients with and without CC and to assess the role of peritumoral adipose tissue in CC-induced inflammation. Patients were divided in four experimental groups: obese patients with CC (OB-CC), lean patients with CC (LEAN-CC), obese patients without CC (OB), and lean patients without CC (LEAN). Plasma levels of pro-inflammatory cytokines (interleukin (IL)-6, IL-4, IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in OB-CC patients. Peritumoral adipose tissue (TF) explants and cultured mature adipocytes secreted higher amounts of nitrites and nitrates than did control and non-tumoral (NTF) adipose tissue both alone and in response to lipopolysaccharide (LPS). Nitrite and nitrate secretion was also increased in TF explants from OB-CC patients compared with that from LEAN-CC patients. Gene expression of adiponectin, tumor necrosis factor alpha (TNF-α), insulin-like growth factor type I (IGF-I), cyclooxygenase-2 (COX-2), and peroxisome proliferator-activated receptor γ (PPAR-γ) was increased in TF explants from CC patients. LPS increased the gene expression of IL-6, IL-10, TNF-α, vascular endothelial growth factor (VEGF), and COX-2 in OB and in TF explants from OB-CC patients. COX-2 and PPAR-γ inhibition further increased LPS-induced release of nitrites and nitrates in TF explants and adipocytes from OB-CC patients. In conclusion, OB-CC patients have increased plasma levels of pro-inflammatory and angiogenic factors. TF from OB-CC patients shows an increased secretion of inflammatory markers compared with both TF from LEAN-CC and non-tumoral adipose tissue (AT) through a COX-2- and PPAR-γ-independent mechanism.

  19. 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

  20. Making post-mortem implantable cardioverter defibrillator explantation safe

    DEFF Research Database (Denmark)

    Räder, Sune B E W; Zeijlemaker, Volkert; Pehrson, Steen

    2009-01-01

    that the resting voltage over the operating person would not exceed 50 V. CONCLUSION: The use of intact medical gloves made of latex, neoprene, or plastic eliminates the potential electrical risk during explantation of an ICD. Two gloves on each hand offer sufficient protection. We will recommend the use......AIMS: The aim of this study is to investigate whether protection with rubber or plastic gloves during post-mortem explantation of an implantable cardioverter defibrillator (ICD) offers enough protection for the explanting operator during a worst-case scenario (i.e. ICD shock). METHODS AND RESULTS...

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. Establishment of an efficient protocol for micropropagation of some pakistani cultivars of date palm (phonex dactylifera l.) using novel inflorescence explants

    International Nuclear Information System (INIS)

    Jatoi, M. A.; Markhand, G. S.; Solangi, N.

    2015-01-01

    An efficient protocol for rapid and large scale In vitro propagation of some Pakistani cultivars of date palm has been established using inflorescence explants at Date Palm Research Institute (DPRI), Shah Abdul Latif University (SALU), Khairpur, Pakistan. Immature inflorescences of desired cultivars of date palm detached from mother palms followed by surface sterilization with low torrent of current tap water and then 30 percentage NaOCl/sub 2/ solution, the outer cover were removed in order to get spike explants and cut into the 2-3 cm small pieces and cultured on modified MS medium supplemented with 0.1 mg l-1 2, 4-D + 0.1 mg l-1 IAA + 5.0 mg l-1 NAA for initiation and establishment of cultures. The obtained somatic embryos were subjected to multiplication medium involved 0.1 mg l-1 NAA + 0.05 mg l-1BA. Rooting was achieved using quarter strength MS medium containing 0.1 mg l-1NAA without activated charcoal (AC) initially and then with 3 g l-1 AC. Strong rooted plantlets with 2-3 leaves were transferred to pots contained sand and peat moss mixture (1:1 v/v) with more than 95 percentage success in acclimatization. The acclimatized plants with at least one compound leaf were shifted to the open field conditions at SALU campus for further studying morphological and fruit characterization to ensure the true-to-type nature of tissue culture derived plantlets. High multiplication efficiency and survival percentage with no any somaclonal variation ensured the efficacy of the protocol developed for the production of elite cultivars of date palm of Pakistan and can be used to optimize production of other cultivars of date palm worldwide. (author)

  7. 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

  8. A survey of the effect of explants type, plant growth regulators and activated charcoal on callus induction in Papaver bracteatum

    Directory of Open Access Journals (Sweden)

    Bahman Hosseini

    2015-09-01

    Full Text Available Callus culture is necessary for production of suspension cell culture in plant breeding programs. Regarding to the application of Papaver bracteatum as an important medicinal plant in production of benzophenantridine alkaloids, this study was performed to find the most suitable hormone combination and explant type for achieving to high percentage of callus induction fresh weight and somatic embryogenesis in this plant. For this purpose, hypocotyl explants were cultured in ½MS media containing active charcoal (2 and 4 mgL-1 in combination of different concentrations of NAA, 2,4-D (0, 1, 2, 3 and 5 mgL-1 and BA (0, 0.1 and 0.5 mgL-1. The seed explants were cultured in same treatments without active charcoal. Also, somatic embryogesis induction using seed explants in ½MS media containing different concentrations of NAA and 2,4-D (0, 0.5, 1 and 2 mgL-1 with BA 0.5 mgL-1 were investigated. The results showed that the highest percentage of callus induction (43.6%, 54% in hypocotyls explants were obtained in the ½MS media containing 2 mgL-1 active charcoal and 2 mgL-1 2,4-D and 5 mgL-1 NAA in companion with BA 0.5 mgL-1 respectively. The maximum callus induction (84% was obtained in ½MS medium with 1 mgL-1 2,4-D without active charcoal. The highest callus fresh weight (0.35% was obtained in MS media with 0.5 mgL-1 2,4-D andthe maximum rate of somatic embryogenesis induction (77% was observed in ½MS media containing 1 mgL-1 2,4-D with 0.5 mgL-1 BA.

  9. 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.

  10. 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

  11. 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.

  12. 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)

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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....

  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. 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.

  20. Dedifferentiation of leaf explants and antileukemia activity of an ...

    African Journals Online (AJOL)

    user

    2011-04-04

    Apr 4, 2011 ... known as drumstick tree or horseradish tree (Little and. Wadsworth, 1964; Morton ... Leaves explants (Figure 1a) obtained from 21 day-old seedlings were sterilized by sodium ... Sweden), according to Harbeck et al. (1982).

  1. Simple, effective and economical explant-surface sterilization ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-19

    Oct 19, 2009 ... recommend this technique due to its simplicity and economy. Key words: Explant ... mercuric chloride, hydrogen peroxide, silver nitrate and bromine water (Rai ... actually within the structure that is being surface steri- lized.

  2. 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.

  3. Phytic acid decreases deoxynivalenol and fumonisin B1-induced changes on swine jejunal explants

    Directory of Open Access Journals (Sweden)

    Elisângela Olegário da Silva

    2014-01-01

    Full Text Available The purpose of the present study was to investigate the effects of phytic acid (IP6 on morphological and immunohistochemical parameters on intestinal explants exposed to deoxynivalenol (DON and fumonisin B1 (FB1. The jejunal explants were exposed for 4 h to different treatments: control, DON (10 μM, DON plus 2.5 mM or 5 mM IP6, FB1 (70 μM, and FB1 plus 2.5 mM or 5 mM IP6. Both mycotoxins induced significant intestinal lesions and decreased villi height. The presence of 2.5 mM and 5 mM IP6 significantly inhibited the morphological changes caused by the mycotoxins. DON induced a significant increase in caspase-3 (83% and cyclooxygenase-2 (71.3% expression compared with the control. The presence of 5 mM IP6 induced a significant decrease in caspase-3 (43.7% and Cox-2 (48% expression compared with the DON group. FB1 induced a significant increase in caspase-3 expression (47% compared to the control, whereas IP6 induced no significant change in this expression. A significant decrease in cell proliferation was observed when explants were exposed to 5 mM of IP6 in comparison with the DON and FB1 groups. The present data provide evidence that phytic acid modulates the toxic effects induced by DON and FB1 on intestinal tissue.

  4. Electron microscopic evaluation of a gold glaucoma micro shunt after explantation.

    Science.gov (United States)

    Berk, Thomas A; Tam, Diamond Y; Werner, Liliana; Mamalis, Nick; Ahmed, Iqbal Ike K

    2015-03-01

    We present a case of an explanted gold glaucoma micro shunt (GMS Plus) and the subsequent light and electron microscopic analyses. The shunt was implanted in a patient with medically refractive glaucoma. The intraocular pressure (IOP) was stable at 12 mm Hg 6 months postoperatively but spiked to 26 mm Hg 6 months later; membranous growth was visible on the implant gonioscopically. A second gold micro shunt was placed 2 years after the first. The IOP was 7 mm Hg 1 week postoperatively but increased to 23 mm Hg 3 weeks later; similar membranous growth was visible on this implant. One of the shunts was explanted, and light and scanning electron microscopic analyses revealed encapsulation around the shunt exterior and connective tissue invasion of the microstructure. This represents the first electron microscopic analysis of an explanted gold glaucoma micro shunt and the first unequivocal images of the fibrotic pseudo-capsule traversing its microchannels and fenestrations. Dr. Ahmed is a consultant to and has received research grants from Solx, Inc. No other author has a financial or proprietary interest in any material or method mentioned. Copyright © 2015 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

  5. 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

  6. Clinicopathologic features of hepatic neoplasms in explanted livers: a single institution experience

    International Nuclear Information System (INIS)

    Mourad, W.; Tulbah, A.; Al-Omari, M.; Al-Mana, H.; Khalaf, H.; Neiamatallah, M.

    2007-01-01

    Hepatic neoplasms can be the primary indication for hepatic transplantation. The tumors can also be incidentally identified in explanted livers. We explored the clinicopathologic features of hepatic neoplasms identified in explanted livers. All explanted livers resected between 2001 and 2006 were evaluated for the presence of neoplasms and their clinicopathologic features were examined. In 198 liver transplants, 15 neoplasms (15.3%) were identified. Patient ages ranged from 5 to 63 years (median, 56 years). The primary etiology of hepatic disease was hepatitis C virus in 12 cases, hepatitis B virus in 1 case, cryptogenic cirrhosis in 1 case and congenital hepatic fibrosis in 1 case. Serum alpha-fetoprotein was significantly elevated (>400 U/L) in only 2 cases. CA19-9 was not elevated in any of the cases. The tumors included hepatocellular carcinoma (HCC) in 13 cases, 1 case of cholangiocarcinoma and 1 case of combined HCC and hepatoblastoma. The tumors ranged in size from 0.5 to 5 cm (median 1.4 cm) and were multifocal in 5 of the cases (33%). Tissue alpha-fetoprotein expression was only seen in the cases associated with elevated serum levels. In our institution hepatic neoplasma are seen in more than 15% of explanted livers. They can be incidentally identified, are frequently not associated with elevated serum levels of alpha-fetoprotein and CA19-9, are commonly multifocal but small and are associated with good prognosis. Elevated serum alpha-fetoprotein, albeit specific, is not a very sensitive marker in the detection of hepatic neoplasms. (author)

  7. „IN VITRO” EFFECT OF SOME INDUSTRIAL BY-PRODUCTS ON LAVANDULA ANGUSTIFOLIA MILL. EXPLANT GROWTH

    Directory of Open Access Journals (Sweden)

    Corneliu Tanase

    2013-12-01

    Full Text Available After many studies, it was observed that lavender has many therapeutic effects, such as sedation, activities spasmolytic, antiviral, antibacterial. Thus, given the importance of lavender in different areas of human life, in the present study, we studied the influence of natural products bioregulatoars separated from industrial by-products on some lavender stems explants. These explants were inoculated in vitro on MS nutrient media. In these culture media were added polyphenolic extracts obtained from spruce bark and hemp shives, and evaluated their influence on lavender stems explants. The results obtained were compared with those obtained for the control variant, where MS culture medium was used as standard. It was found that the addition of aqueous extract from spruce bark of concentration of 130 mg GAE / L, in the growth of explants of Lavandula angustifolia Mill, an increase in the elongation of the main stem, number of leaves formed, the amount of photoassimilating pigments synthesized and causes the phenomenon of shoots formation. At a higher concentration of the extract (26 mgGAE/100g values are lower.

  8. Enamel formation in vitro in mouse molar explants exposed to amelogenin polypeptides ATMP and LRAP on enamel development.

    Science.gov (United States)

    Ravindranath, Rajeswari M H; Devarajan, Asokan; Bringas, Pablo

    2007-12-01

    The enamel matrix contains amelogenin, leucine-rich amelogenin-polypeptide (LRAP), resulting from alternative splicing of the primary amelogenin-RNA transcript and tyrosine-rich amelogenin-polypeptide (TRAP), a proteolytic product of amelogenin. Presence of amelogenin-trityrosyl-motif peptide (ATMP) distinguishes TRAP from LRAP. The roles of these polypeptides in the formation of enamel remain to be elucidated. The mouse in vitro molar tooth-organ developed from bud stage (E16) was exposed to LRAP, ATMP, and mutated ATMP (T-ATMP, third proline replaced by threonine). The histology and morphometry of the explants on day-12 in culture was examined using Mallory's stain. Guanidine-HCl soluble protein concentrations of explants were compared. The enamel width and protein solubility indicate that the explant on day-12 is comparable to postnatal molar on day-3 in vivo. The enamel of both untreated explants as well as that in vivo is fuchinophilic (acid fuchsin, AF+). ATMP reduced the ameloblast-height, accumulated AF+ spherules at the apical end of ameloblasts, and disrupted enamel-dentin bonding. T-ATMP abrogated deposition of AF+ material on the aniline blue positive (AB+) enamel matrix. LRAP reduced ameloblast-height, increased the enamel-width without disruption (at 17.25 nmol) and increased the density of AF+ dentinal tubules. AF+ substance from the tubules is released onto the surface of the dentin. The Guanidine-HCl-soluble protein is elevated in ATMP-treated explants but decreased in LRAP-treated explants. Exogenous ATMP, T-ATMP and LRAP have divergent effects on developing enamel. Exogenous ATMP, but not LRAP, abrogates enamel-dentin bonding at 17.25 nmol. LRAP may play a role in the differentiation of ameloblasts, growth of enamel and formation of dentinal tubules.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. In vitro plant regeneration from leaf explants of Solanum pimpinellifolium L.

    Directory of Open Access Journals (Sweden)

    Shirley Valderrama-Alfaro

    2011-01-01

    Full Text Available Tomato is a species of agricultural importance. Besides, it is a source of vitamins, minerals and antioxidant compounds. Therefore it is essential to obtain varieties resistant to diseases. Solanum pimpinellifolium L. ‘Tomatillo wild’, one of the wild relatives of tomato, is considered multipurpose reservoir of genes. This characteristic must be exploited. In vitro plantlets from leaves of ‘wild tomatillo’ were regenerated with this objective. A system for in vitro germination of seeds obtained from ex vitro sources was created. The use of seed allowed obtaining adequate seedlings in a short time to start regeneration. Thein vitro response of explants was evaluated in four treatments. The Murashige and Skoog (MS basal culture medium supplemented with a-naphthaleneacetic acid (NAA and 6-benzyl aminopurine (BAP was used in different combinations. The higher percentage (30% of shoot induction was achieved with 0.1 mg l1 NAA / 1 mg l1 BAP. The presence of callus and roots was observed after seven days of culture in the combination treatment with 1 mg l1 NAA / 0.1 mg l1 BAP. Callus showed 1 or 2 adventitious shoots per explant after 30 days of culture and 3 to 6 shoots after seventy days. Furthermore, the presence of fully formed adventitious plantlets (shoot and root was observed after the fifth week of culture. Therefore, the best combination for in vitro regeneration is NAA 0.1 mg l1 / 1 mg l1 BAP. Keywords: auxin, cytokinins, indirect organogenesis

  19. 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

  20. 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.

  1. 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.

  2. Radiation transformation in differentiated human cells in culture

    International Nuclear Information System (INIS)

    Mothersill, C.; Seymour, C.; Moriarty, M.; Malone, J.; Byrne, P.; Hennessy, T.

    1986-01-01

    A tissue culture technique is described for human thyroid tissue as an approach to studying mechanisms of human radiation carcinogenesis. Normal human tissue obtained from surgery is treated in one of two ways, depending upon size of specimen. Large pieces are completely digested in trypsin/ collagenase solution to a single cell suspension. Small pieces of tissue are plated as explants following partial digestion in trypsin/collagenase solution. Following irradiation of the primary differentiated monolayers (normally 10 days after plating), the development of transformed characteristics is monitored in the subsequent subcultures. A very high level of morphological and functional differentiation is apparent in the primary cultures. Over a period of approx. 6 months, the irradiated surviving cells continue to grow in culture, unlike the unirradiated controls which senesce after 2-3 subcultures. (UK)

  3. Somatic embryogenesis and plant regeneration in tissue cultures of sweet potato (Ipomea batatas Poir.).

    Science.gov (United States)

    Liu, J R; Cantliffe, D J

    1984-06-01

    Leaf, shoot-tip, stem, and root explants of sweet potato (Ipomea batatas Poir.) gave rise to two kinds of callus on nutrient agar medium containing 0.5 to 2.0 mg/l 2,4-D. One callus, bright- to pale-yellow, was compact and organized, while the other was dull-yellow and friable. The former callus gave rise to numerous globular and heart-shaped embryoids. When transferred onto hormone-free medium, the embryoids readily developed into a torpedo-shape before germination. The plantlets were transplanted to soil where they flowered and formed storage roots at maturity.

  4. High frequency organogenesis in hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica), an important vegetable crop.

    Science.gov (United States)

    Kumar, Pankaj; Srivastava, D K

    2015-04-01

    Broccoli (Brassica oleracea L. var. italica) is an important, nutritionally rich vegetable crop, but severely affected by environmental stresses, pests and diseases which cause massive yield and quality losses. Genetic manipulation is becoming an important method for broccoli improvement. In the present study, a reproducible and highly efficient protocol for obtaining organogenesis from hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica cv. Solan green head) has been developed. Hypocotyl and cotyledon explants were used from 10 to 12 days old aseptically grown seedlings whereas leaf and petiole explants were excised from 18 to 20 days old green house grown seedlings and surface sterilized. These explants were cultured on shoot induction medium containing different concentration and combination of BAP and NAA. High efficiency shoot regeneration has been achieved in hypocotyl (83.33 %), cotyledon (90.11 %), leaf (62.96 %) and petiole (91.10 %) explants on MS medium supplemented with 3.5 mg/l BAP + 0.019 mg/l NAA 2.5 mg/l BAP + 0.5 mg/l NAA, 4.0 mg/l BAP + 0.5 mg/l NAA and 4.5 mg/l BAP + 0.019 mg/l NAA respectively. Petiole explants showed maximum shoot regeneration response as compared to other explants. MS medium supplemented with 0.10 mg/l NAA was found best for root regeneration (100 %) from in vitro developed shoots. The regenerated complete plantlets were transferred to the pots containing cocopeat and successfully acclimatized. This optimized regeneration protocol can be efficiently used for genetic transformation in broccoli. This is the first comparative report on multiple shoot induction using four different types of explants viz. hypocotyl, cotyledon, leaf and petiole.

  5. 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)

  6. 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

  7. 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

  8. High frequency plant regeneration from leaf explants derived callus of evening primrose (oenothera biennis)

    International Nuclear Information System (INIS)

    Ghauri, E.G.; Shafi, N.; Ghani, S.; Fatima, A.

    2008-01-01

    The seeds of Evening primrose were aseptically grown and leaf explants were used for establishment of callus culture. The Excellent growth in callus biomass was achieved on MS medium supplemented with 2, 4, -D and TDZ. For optimal growth of bud and shoot regeneration, fortification of IAA along with TDZ, or BAP was found to be essential. Rooting (70%) could be inducted on hormone free MS-medium. This percentage improved to 98 when NAA was added to the medium. The plantlets thus obtained were transferred to the field successfully after passing through the process of hardening. (author)

  9. Histological and molecular evaluation of patient-derived colorectal cancer explants.

    Directory of Open Access Journals (Sweden)

    Joshua M Uronis

    Full Text Available Mouse models have been developed to investigate colorectal cancer etiology and evaluate new anti-cancer therapies. While genetically engineered and carcinogen-induced mouse models have provided important information with regard to the mechanisms underlying the oncogenic process, tumor xenograft models remain the standard for the evaluation of new chemotherapy and targeted drug treatments for clinical use. However, it remains unclear to what extent explanted colorectal tumor tissues retain inherent pathological features over time. In this study, we have generated a panel of 27 patient-derived colorectal cancer explants (PDCCEs by direct transplantation of human colorectal cancer tissues into NOD-SCID mice. Using this panel, we performed a comparison of histology, gene expression and mutation status between PDCCEs and the original human tissues from which they were derived. Our findings demonstrate that PDCCEs maintain key histological features, basic gene expression patterns and KRAS/BRAF mutation status through multiple passages. Altogether, these findings suggest that PDCCEs maintain similarity to the patient tumor from which they are derived and may have the potential to serve as a reliable preclinical model that can be incorporated into future strategies to optimize individual therapy for patients with colorectal cancer.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. A fluid dynamics approach to bioreactor design for cell and tissue culture.

    Science.gov (United States)

    Dusting, Jonathan; Sheridan, John; Hourigan, Kerry

    2006-08-20

    The problem of controlling cylindrical tank bioreactor conditions for cell and tissue culture purposes has been considered from a flow dynamics perspective. Simple laminar flows in the vortex breakdown region are proposed as being a suitable alternative to turbulent spinner flask flows and horizontally oriented rotational flows. Vortex breakdown flows have been measured using three-dimensional Stereoscopic particle image velocimetry, and non-dimensionalized velocity and stress distributions are presented. Regions of locally high principal stress occur in the vicinity of the impeller and the lower sidewall. Topological changes in the vortex breakdown region caused by an increase in Reynolds number are reflected in a redistribution of the peak stress regions. The inclusion of submerged scaffold models adds complexity to the flow, although vortex breakdown may still occur. Relatively large stresses occur along the edge of disks jutting into the boundary of the vortex breakdown region. Copyright 2006 Wiley Periodicals, Inc.

  17. 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

  18. 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.

  19. 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...

  20. Molecular characterization of three anther tissue culture varieties of tobaco (Nicotiana tabacum L. using RAPD analysis

    Directory of Open Access Journals (Sweden)

    Gloria Azucena Fernández B.

    2002-01-01

    Full Text Available Randomly Amplified Polymorphic DNA (RAPO analysis was used to characterize two new Flue Cured and one black tobacco type varieties derived from in vitro anther tissue culture technique. RAPOs are proposed as an appropriate complement of the morphoagronomic characteristics evaluations to fulfil international seed registration standards established for the identification of tobacco varieties. The identification of three tobacco varieties and their parents was carried out using the RAPO analysis with 64 random primers. Polymorphic products, 214 in number, were amplified only from 14 primers. Statistical analysis realized with the NTSYS program version 1.2 using the Jaccard similarity coefficient. The visual inspection revealed that five primers allowed the separation of the varieties in two groups, according to the type of tobacco: the Flue Cured and Black; while a group of nine primers separates each variety and establish its genetic relationship with their parents. The results obtained show that this technique is appropiated to establish genetic differences between tobacco varieties.

  1. 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.

  2. Effect of ZnO nanoparticles on Brassica nigra seedlings and stem explants: growth dynamics and antioxidative response

    Directory of Open Access Journals (Sweden)

    Hira eZafar

    2016-04-01

    Full Text Available Nanoparticles (NPs have diverse properties in comparison to respective chemicals due to structure, surface area ratio, morphology, and reactivity. Toxicological effects of metallic NPs to organisms including plants have been reported. However, to the authors’ knowledge there is no report on the effect of NPs on in vitro culture of plant explants. In this study, ZnO NPs at 500-1500 mg/L badly affected Brassica nigra seed germination and seedling growth and raised antioxidative activities and antioxidants concentrations. On the other hand, culturing the stem explants of B. nigra on Murashige and Skoog (MS medium in presence of low concentration of ZnO NPs (1-20 mg/L produced white thin roots with thick root hairs. At 10 mg/L ZnO NPs shoots emergence was also observed. The developed calli/roots showed 79% DPPH (2,2-diphenyl-1-picryl hydrazyl radical scavenging activity at 10 mg/L. While total antioxidant and reducing power potential were also significantly different in presence of ZnO NPs. Non enzymatic antioxidative molecules, phenolics (up to 0.15 µg GAE/mg FW and flavonoids (up to 0.22 µg QE/mg FW, also raised and found NPs concentration dependent. We state that ZnO NPs may induce roots from explants cultured on appropriate medium and can be cultured for production of valuable secondary metabolites.

  3. Integrin expression by human osteoblasts cultured on degradable polymeric materials applicable for tissue engineered bone.

    Science.gov (United States)

    El-Amin, Saadiq F; Attawia, Mohamed; Lu, Helen H; Shah, Asist K; Chang, Richard; Hickok, Noreen J; Tuan, Rocky S; Laurencin, Cato T

    2002-01-01

    The use of biodegradable polymers in the field of orthopaedic surgery has gained increased popularity, as surgical pins and screws, and as potential biological scaffolds for repairing cartilage and bone defects. One such group of polymers that has gained considerable attention are the polyesters, poly(lactide-co-glycolide) (PLAGA) and polylactic acid (PLA), because of their minimal tissue inflammatory response, favorable biocompatibility and degradation characteristics. The objective of this study was to evaluate human osteoblastic cell adherence and growth on PLAGA and PLA scaffolds by examining integrin receptor (alpha2, alpha3, alpha4, alpha5, alpha6 and beta1) expression. Primary human osteoblastic cells isolated from trabecular bone adhered efficiently to both PLAGA and PLA, with the rate of adherence on PLAGA comparable to that of control tissue culture polystyrene (TCPS), and significantly higher than on PLA polymers at 3, 6 and 12 h. Human osteoblastic phenotypic expression, alkaline phosphatase (ALP) activity was positive on both degradable matrices, whereas osteocalcin levels were significantly higher on cells grown on PLAGA than on PLA composites. Interestingly, the integrin subunits, alpha2, alpha3, alpha4, alpha5, alpha6 and beta1 were all expressed at higher levels by osteoblasts cultured on PLAGA than those on PLA as analyzed by westerns blots and by flow cytometry. Among the integrins, alpha2, beta5 and beta1 showed the greatest difference in levels between the two surfaces. Thus, both PLA and PLAGA support osteoblastic adhesion and its accompanying engagement of integrin receptor and expression of osteocalcin and ALP. However PLAGA consistently appeared to be a better substrate for osteoblastic cells based on these parameters. This study is one of the first to investigate the ability of primary human osteoblastic cells isolated from trabecular bone to adhere to the biodegradable polymers PLAGA and PLA, and to examine the expression of their key

  4. 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)

  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. Optimization of culture conditions (sucrose, pH, and photoperiod) for in vitro regeneration and early detection of somaclonal variation in ginger lime (Citrus assamensis).

    Science.gov (United States)

    Yaacob, Jamilah Syafawati; Mahmad, Noraini; Mat Taha, Rosna; Mohamed, Normadiha; Mad Yussof, Anis Idayu; Saleh, Azani

    2014-01-01

    Various explants (stem, leaf, and root) of Citrus assamensis were cultured on MS media supplemented with various combinations and concentrations (0.5-2.0 mg L(-1)) of NAA and BAP. Optimum shoot and root regeneration were obtained from stem cultures supplemented with 1.5 mg L(-1) NAA and 2.0 mg L(-1) BAP, respectively. Explant type affects the success of tissue culture of this species, whereby stem explants were observed to be the most responsive. Addition of 30 gL(-1) sucrose and pH of 5.8 was most optimum for in vitro regeneration of this species. Photoperiod of 16 hours of light and 8 hours of darkness was most optimum for shoot regeneration, but photoperiod of 24 hours of darkness was beneficial for production of callus. The morphology (macro and micro) and anatomy of in vivo and in vitro/ex vitro Citrus assamensis were also observed to elucidate any irregularities (or somaclonal variation) that may arise due to tissue culture protocols. Several minor micromorphological and anatomical differences were observed, possibly due to stress of tissue culture, but in vitro plantlets are expected to revert back to normal phenotype following full adaptation to the natural environment.

  7. Plant regeneration from cotyledonary explants of Eucalyptus camaldulensis dehn and histological study of organogenesis in vitro

    Directory of Open Access Journals (Sweden)

    Roberson Dibax

    2010-04-01

    Full Text Available The present work aimed at regenerating plants of Eucalyptus camaldulensis from the cotyledonary explants and describing the anatomy of the tissues during callogenesis and organogenesis processes, in order to determine the origin of the buds. The cotyledonary leaves of E. camaldulensis were cultured in Murashige and Skoog (MS, WPM and JADS media supplemented with 2.7 µM NAA and 4.44 µM BAP. The best results for bud regeneration were obtained on MS and WPM media (57.5 and 55% of calluses formed buds, respectively. Shoot elongation and rooting (80% were obtained on MS/2 medium (with half-strength salt concentration with 0.2% activated charcoal. Acclimatization was performed in the growth chamber for 48 h and then the plants were transferred to a soil:vermiculite mixture and cultured in a greenhouse. Histological studies revealed that the callogenesis initiated in palisade parenchyma cells and that the adventitious buds were formed from the calluses, indicating indirect organogenesis.Este trabalho teve como objetivo a obtenção de plantas de Eucalyptus camaldulensis a partir de folhas cotiledonares e o estudo da anatomia dos tecidos durante a calogênese e organogênese para determinar a origem das gemas. Folhas cotiledonares foram cultivadas em meios de cultura MS, WPM e JADS suplementados com 2,7 µM de ANA e 4,44 µM de BAP. Os melhores resultados para a regeneração de gemas foram obtidos com os meios MS e WPM. Para o alongamento e enraizamento, o meio de cultura MS/2 contendo 0,2% de carvão ativado apresentou-se eficiente para ambas as etapas. A aclimatização foi realizada mediante a abertura dos frascos na sala de crescimento por 48 horas, seguido da transferência para casa-de-vegetação com nebulização intermitente. Estudos histológicos foram conduzidos e revelaram que a calogênese teve início nas células do parênquima paliçádico e que as gemas adventícias formaram-se a partir dos calos, indicando a organogênese indireta.

  8. 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.

  9. Rapid detection of bacterial contamination in cell or tissue cultures based on Raman spectroscopy

    Science.gov (United States)

    Bolwien, Carsten; Sulz, Gerd; Becker, Sebastian; Thielecke, Hagen; Mertsching, Heike; Koch, Steffen

    2008-02-01

    Monitoring the sterility of cell or tissue cultures is an essential task, particularly in the fields of regenerative medicine and tissue engineering when implanting cells into the human body. We present a system based on a commercially available microscope equipped with a microfluidic cell that prepares the particles found in the solution for analysis, a Raman-spectrometer attachment optimized for non-destructive, rapid recording of Raman spectra, and a data acquisition and analysis tool for identification of the particles. In contrast to conventional sterility testing in which samples are incubated over weeks, our system is able to analyze milliliters of supernatant or cell suspension within hours by filtering relevant particles and placing them on a Raman-friendly substrate in the microfluidic cell. Identification of critical particles via microscopic imaging and subsequent image analysis is carried out before micro-Raman analysis of those particles is then carried out with an excitation wavelength of 785 nm. The potential of this setup is demonstrated by results of artificial contamination of samples with a pool of bacteria, fungi, and spores: single-channel spectra of the critical particles are automatically baseline-corrected without using background data and classified via hierarchical cluster analysis, showing great promise for accurate and rapid detection and identification of contaminants.

  10. Hormonal regulation of epithelial organization in a three-dimensional breast tissue culture model.

    Science.gov (United States)

    Speroni, Lucia; Whitt, Gregory S; Xylas, Joanna; Quinn, Kyle P; Jondeau-Cabaton, Adeline; Barnes, Clifford; Georgakoudi, Irene; Sonnenschein, Carlos; Soto, Ana M

    2014-01-01

    The establishment of hormone target breast cells in the 1970's resulted in suitable models for the study of hormone control of cell proliferation and gene expression using two-dimensional (2D) cultures. However, to study mammogenesis and breast tumor development in vitro, cells must be able to organize in three-dimensional (3D) structures like in the tissue. We now report the development of a hormone-sensitive 3D culture model for the study of mammogenesis and neoplastic development. Hormone-sensitive T47D breast cancer cells respond to estradiol in a dose-dependent manner by forming complex epithelial structures. Treatment with the synthetic progestagen promegestone, in the presence of estradiol, results in flat epithelial structures that display cytoplasmic projections, a phenomenon reported to precede side-branching. Additionally, as in the mammary gland, treatment with prolactin in the presence of estradiol induces budding structures. These changes in epithelial organization are accompanied by collagen remodeling. Collagen is the major acellular component of the breast stroma and an important player in tumor development and progression. Quantitative analysis of second harmonic generation of collagen fibers revealed that collagen density was more variable surrounding budding and irregularly shaped structures when compared to more regular structures; suggesting that fiber organization in the former is more anisotropic than in the latter. In sum, this new 3D model recapitulates morphogenetic events modulated by mammogenic hormones in the breast, and is suitable for the evaluation of therapeutic agents.

  11. 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.

  12. 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.

  13. Effect of flow on vascular endothelial cells grown in tissue culture on polytetrafluoroethylene grafts

    International Nuclear Information System (INIS)

    Sentissi, J.M.; Ramberg, K.; O'Donnell, T.F. Jr.; Connolly, R.J.; Callow, A.D.

    1986-01-01

    Vascular grafts lined with endothelial cells (EC) grown to confluence in culture before implantation may provide a thromboresistant flow surface. Growth of EC on and their adherence to currently available prosthetic materials under conditions of flow are two impediments remaining in the development of such a graft. To address these problems, 22 polytetrafluoroethylene grafts (PTFE) (5 cm by 4 mm inside diameter) were pretreated with collagen and fibronectin, seeded with 2 to 3 X 10(6) bovine aortic EC per graft, and placed in tissue culture (seeded grafts). Twenty-two grafts pretreated with collagen and fibronectin alone served as controls. After 2 weeks morphologic studies revealed that 20/22 seeded grafts were lined with a confluent endothelial layer. Indium 111-oxine was then used to label the EC-seeded grafts. After exposure to either low (25 ml/min) or high (200 ml/min) flow rates for 60 minutes in an in vitro circuit, examination of the luminal surface of the graft by light microscopy and scanning electron microscopy revealed minimal loss of EC. These findings were corroborated by radionuclide scans that showed an insignificant loss of the EC-associated indium label during exposure to flow (7% low flow, 11% high flow). Pretreatment of PTFE grafts with collagen and fibronectin thus promotes both attachment and adherence of EC even under flow conditions

  14. 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.

  15. Development and characterization of cell culture systems from Puntius (Tor) chelynoides (McClelland).

    Science.gov (United States)

    Goswami, M; Sharma, B S; Tripathi, A K; Yadav, Kamalendra; Bahuguna, S N; Nagpure, N S; Lakra, W S; Jena, J K

    2012-05-25

    Puntius (Tor) chelynoides, commonly known as dark mahseer, is a commercially important coldwater fish species which inhabits fast-flowing hill-streams of India and Nepal. Cell culture systems were developed from eye, fin, heart and swim bladder tissues of P. chelynoides using explant method. The cell culture system developed from eye has been maintained towards a continuous cell line designated as PCE. The cells were grown in 25cm(2) tissue culture flasks with Leibovitz' L-15 media supplemented with 20 % fetal bovine serum (FBS) at 24°C. The PCE cell line consists of predominantly fibroblast-like cells and showed high plating efficiency. The monolayer formed from the fin and heart explants were comprised of epithelial as well as fibroblast-like cells, a prominent and rhythmic heartbeat was also observed in heart explants. Monolayer formed from swim bladder explants showed the morphology of fibroblast-like cells. All the cells from different tissues are able to grow at an optimum temperature of 24°C and growth rate increased as the FBS concentration increased. The PCE cell line was characterized using amplification of mitochondrial cytochrome oxidase subunit I (COI) & 16S rRNA genes which confirmed that the cell line originated from P. chelynoides. Cytogenetic analysis of PCE cell line and cells from fin revealed a diploid count of 100 chromosomes. Upon transfection with pEGFP-C1 plasmid, bright fluorescent signals were observed, suggesting that this cell line can be used for transgenic and genetic manipulation studies. Further, genotoxicity assessment of PCE cells illustrated the utility of this cell line as an in vitro model for aquatic toxicological studies. The PCE cell line was successfully cryopreserved and revived at different passage levels. The cell line and culture systems are being maintained to develop continuous cell lines for further studies. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. 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.

  17. Efficient regeneration of sorghum, Sorghum bicolor (L.) Moench, from shoot-tip explant.

    Science.gov (United States)

    Syamala, D; Devi, Prathibha

    2003-12-01

    Novel protocols for production of multiple shoot-tip clumps and somatic embryos of Sorghum bicolor (L.) Moench were developed with long-term goal of crop improvement through genetic transformation. Multiple shoot-tip clumps were developed in vitro from shoot-tip explant of one-week old seedling, cultured on MS medium containing only BA (0.5, 1 or 2 mg/l) or both BA (1 or 2 mg/l) and 2,4-D (0.5 mg/l) with bi-weekly subculture. Somatic embryos were directly produced on the enlarged dome shaped growing structures that developed from the shoot-tips of one-week old seedling explants (without any callus formation) when cultured on MS medium supplemented with both 2,4-D (0.5 mg/l) and BA (0.5 mg/l). However, the supplementation of MS medium with only 2,4-D (0.5 mg/l) induced compact callus without any plantlet regeneration. Each multiple shoot-clump was capable of regenerating more than 80 shoots via an intensive differentiation of both axillary and adventitious shoot buds, the somatic embryos were capable of 90% germination, plant conversion and regeneration. The regenerated shoots could be efficiently rooted on MS medium containing indole-3-butyric acid (IBA 1 mg/l). The plants were successfully transplanted to glasshouse and grown to maturity with a survival rate of 98%. Morphogenetic response of the explants was found to be genotypically independent.

  18. 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.

  19. Tissue

    Directory of Open Access Journals (Sweden)

    David Morrissey

    2012-01-01

    Full Text Available Purpose. In vivo gene therapy directed at tissues of mesenchymal origin could potentially augment healing. We aimed to assess the duration and magnitude of transene expression in vivo in mice and ex vivo in human tissues. Methods. Using bioluminescence imaging, plasmid and adenoviral vector-based transgene expression in murine quadriceps in vivo was examined. Temporal control was assessed using a doxycycline-inducible system. An ex vivo model was developed and optimised using murine tissue, and applied in ex vivo human tissue. Results. In vivo plasmid-based transgene expression did not silence in murine muscle, unlike in liver. Although maximum luciferase expression was higher in muscle with adenoviral delivery compared with plasmid, expression reduced over time. The inducible promoter cassette successfully regulated gene expression with maximum levels a factor of 11 greater than baseline. Expression was re-induced to a similar level on a temporal basis. Luciferase expression was readily detected ex vivo in human muscle and tendon. Conclusions. Plasmid constructs resulted in long-term in vivo gene expression in skeletal muscle, in a controllable fashion utilising an inducible promoter in combination with oral agents. Successful plasmid gene transfection in human ex vivo mesenchymal tissue was demonstrated for the first time.

  20. 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...

  1. 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.

  2. Effect of gamma radiation on different explants of sweet potato (Ipomoea batatas L. (Lam)) to induce NaCl tolerance

    International Nuclear Information System (INIS)

    Gutierrez-Rosati, A.

    1997-01-01

    Clonal lines tolerant to NaCl were obtained by combining in vitro culture and gamma radiation in two Peruvian varieties 'Amarillo de Quillabamba' and 'Nemanete'. The most suitable explants were pedicel sections and leaf blades. Embryogenic callus was induced on basal MS basal medium containing 0.5 ppm 2,4-D. The embryogenic calli were irradiated with 5 Gy from a 137 Cs source. Several putative mutants appeared to be stable. (author). 9 refs, 3 tabs

  3. Effect of gamma radiation on different explants of sweet potato (Ipomoea batatas L. (Lam)) to induce NaCl tolerance

    Energy Technology Data Exchange (ETDEWEB)

    Gutierrez-Rosati, A [Biology Dept., National Agriculture Univ., La Molina, Lima (Peru)

    1997-07-01

    Clonal lines tolerant to NaCl were obtained by combining in vitro culture and gamma radiation in two Peruvian varieties `Amarillo de Quillabamba` and `Nemanete`. The most suitable explants were pedicel sections and leaf blades. Embryogenic callus was induced on basal MS basal medium containing 0.5 ppm 2,4-D. The embryogenic calli were irradiated with 5 Gy from a {sup 137}Cs source. Several putative mutants appeared to be stable. (author). 9 refs, 3 tabs.

  4. Effect Of Gamma Rays And Growth Regulators On Explants Excised From In Vitro Shoots And Greenhouse Seedlings, Of Pepper (Capsicum Annum L.)

    International Nuclear Information System (INIS)

    Maarouf, A. A.; Kassem, M.

    2004-01-01

    This experiment was conducted on pepper (Capsicum annum L.) to compare the ability of the in vitro explants with those of greenhouse grown seedlings on shoot proliferation and callus formation and their ability to form plantlets and the effect of gamma irradiation and growth regulators on the shoot tip, hypocotyls and leaf tissue was used as laboratory explants, leaf tissue nodes and internodes were taken from greenhouse seedlings. 6- benzyla-minopurine (BAP) in different concentrations was combined with Indoleacertic acid (IAA) to know their effect on shoot proliferation, 2,4 - Dichlorophenoxy acetic acid (2,4- D) was used for callus formation, and use stimulation effect of gamma irradiation, potassium nitrat (KNO 3 ), Thidaiazurom (TDZ) and casine hydrolysate (CH) for plantlet formation. The results showed that the highest percentage of callus was obtained by in vitro hypocotyls and greenhouse grown nodes followed by in vitro leaf tissue thereafter greenhouse leaf tissue. The shoot tips were the lowest efficient explants in producing callus in both in vitro and greenhouse ones. The highest percentage of shooting resulted from shoot tip, hypocotyls and leaf tissue of in vitro explants, followed by shoot tip, nodes and internodes of greenhouse grown explants and the lowest percentage was recorded by leaf tissue. Highest percentage of shoot number was obtained form greenhouse grown shoot tip followed by in vitro shoot tip, hypocotyls and leaf tissue of greenhouse grown seedlings the internodes were the lowest efficient in producing shoo