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Sample records for cell culture bioassay

  1. Determining UV Inactivation of Toxoplasma gondii Oocysts by Using Cell Culture and a Mouse Bioassay

    Science.gov (United States)

    The effect of UV exposure on Toxoplasma gondii oocysts has not been completely defined for use in water disinfection. This study evaluated UV irradiated oocysts by three assays: a SCID mouse bioassay, an in vitro T. gondii oocyst plaque assay (TOP-assay), and a quantitative reve...

  2. The apoptosis-inducing activity towards leukemia and lymphoma cells in a cyanobacterial culture collection is not associated with mouse bioassay toxicity.

    Science.gov (United States)

    Oftedal, Linn; Skjærven, Kaja H; Coyne, Rosie T; Edvardsen, Bente; Rohrlack, Thomas; Skulberg, Olav M; Døskeland, Stein Ove; Herfindal, Lars

    2011-04-01

    Cyanobacteria (83 strains and seven natural populations) were screened for content of apoptosis (cell death)-inducing activity towards neoplastic cells of the immune (jurkat acute T-cell lymphoma) and hematopoetic (acute myelogenic leukemia) lineage. Apoptogenic activity was frequent, even in strains cultured for decades, and was unrelated to whether the cyanobacteria had been collected from polar, temperate, or tropic environments. The activity was more abundant in the genera Anabaena and Microcystis compared to Nostoc, Phormidium, Planktothrix, and Pseudanabaena. Whereas the T-cell lymphoma apoptogens were frequent in organic extracts, the cell death-inducing activity towards leukemia cells resided mainly in aqueous extracts. The cyanobacteria were from a culture collection established for public health purposes to detect toxic cyanobacterial blooms, and 54 of them were tested for toxicity by the mouse bioassay. We found no correlation between the apoptogenic activity in the cyanobacterial isolates with their content of microcystin, nor with their ability to elicit a positive standard mouse bioassay. Several strains produced more than one apoptogen, differing in biophysical or biological activity. In fact, two strains contained microcystin in addition to one apoptogen specific for the AML cells, and one apoptogen specific for the T-cell lymphoma. This study shows the potential of cyanobacterial culture collections as libraries for bioactive compounds, since strains kept in cultures for decades produced apoptogens unrelated to the mouse bioassay detectable bloom-associated toxins. PMID:20689978

  3. Methods for the isolation and identification of polycyclic aromatic hydrocarbons found in complex mixtures and the determination of their possible toxicity by means of a host mediated bioassay technique. Progress report, July 1, 1976--February 1, 1977. [Cultured mouse leumemia cell bioassay system

    Energy Technology Data Exchange (ETDEWEB)

    Lipsky, S.R.; Alexander, G.; McMurray, W.; Capizzi, R.

    1977-02-01

    Techniques were developed to produce excellent high performance glass capillary columns for gas chromatographic analyses of a wide range of complex mixtures of organic compounds, including those containing a wide array of polycyclic aromatic hydrocarbons (PAH) derived from a coal liquefaction process. Work was begun to assess the potential mutogenicity and/or carcinogenicity of the various isolated PAH fractions utilizing a unique host mediated bioassay system. Preliminary results indicate that further efforts will be required to determine dose response parameters of cultured mouse leukemia cells, as well as suitable vehicles for the satisfactory introduction of certain PAH fractions into this particular bioassay system.

  4. Cell-based bioassays in microfluidic systems

    Science.gov (United States)

    Itle, Laura J.; Zguris, Jeanna C.; Pishko, Michael V.

    2004-12-01

    The development of cell-based bioassays for high throughput drug screening or the sensing of biotoxins is contingent on the development of whole cell sensors for specific changes in intracellular conditions and the integration of those systems into sample delivery devices. Here we show the feasibility of using a 5-(and-6)-carboxy SNARF-1, acetoxymethyl ester, acetate, a fluorescent dye capable of responding to changes in intracellular pH, as a detection method for the bacterial endotoxin, lipopolysaccharide. We used photolithography to entrap cells with this dye within poly(ethylene) glyocol diacrylate hydrogels in microfluidic channels. After 18 hours of exposure to lipopolysaccharide, we were able to see visible changes in the fluorescent pattern. This work shows the feasibility of using whole cell based biosensors within microfluidic networks to detect cellular changes in response to exogenous agents.

  5. Optimisation of the CT h4S bioassay for detection of human interleukin-4 secreted by mononuclear cells stimulated by phytohaemaglutinin or by human leukocyte antigen mismatched mixed lymphocyte culture

    DEFF Research Database (Denmark)

    Petersen, Søren Lykke; Russell, Charlotte Astrid; Bendtzen, Klaus;

    2002-01-01

    Limiting dilution analysis has been used in the context of allogeneic bone marrow transplantation to determine anti-recipient interleukin-2 (IL-2) producing helper T lymphocyte precursor (HTLp) frequencies, which in several studies have been predictive of graft-versus-host disease (GVHD). Recently......S bioassay detects 5 pg/ml of human recombinant IL-4 with no detection of IL-2 in concentrations below 500 pg/ml. We have found 72 h of culture optimal for detection of IL-2 and IL-4 produced by human mononuclear cells (MNC) in response to stimulation with phytohaemaglutinin and for detection of IL...

  6. The apoptosis-inducing activity towards leukemia and lymphoma cells in a cyanobacterial culture collection is not associated with mouse bioassay toxicity

    OpenAIRE

    Oftedal, Linn; Skjærven, Kaja H.; Coyne, Rosie T.; Edvardsen, Bente; Rohrlack, Thomas; Skulberg, Olav M.; Døskeland, Stein Ove; Herfindal, Lars

    2010-01-01

    Cyanobacteria (83 strains and seven natural populations) were screened for content of apoptosis (cell death)-inducing activity towards neoplastic cells of the immune (jurkat acute T-cell lymphoma) and hematopoetic (acute myelogenic leukemia) lineage. Apoptogenic activity was frequent, even in strains cultured for decades, and was unrelated to whether the cyanobacteria had been collected from polar, temperate, or tropic environments. The activity was more abundant in the genera Anabaena and Mi...

  7. Allelopathy in a leguminous mangrove plant, Derris indica: protoplast co-culture bioassay and rotenone effect.

    Science.gov (United States)

    Inoue, Aya; Mori, Daisuke; Minagawa, Reiko; Fujii, Yoshiharu; Sasamoto, Hamako

    2015-05-01

    To investigate allelopathic activity of a leguminous mangrove plant, Derris indica, the 'Protoplasts Co-culture Method' for bioassay of allelopathy was developed using suspension culture. A suspension culture was induced from immature seed and sub-cultured in Murashige and Skoog's (MS) basal medium containing 10 μM each of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA). The protoplasts were isolated using the separate wells method with 2% each of Cellulase RS, Driselase 20 and Macerozyme R10 in 0.4 M mannitol solution. Protoplast cultures of D. indica revealed that high concentrations of cytokinins, BA and thidiazuron, were effective for cell divisions. The co-cultures of D. indica protoplasts with recipient lettuce protoplasts using 96 multi-well culture plates were performed in MS basal medium containing 0.4 M mannitol solution and 1 μM 2,4-D and 0.1 μM BA. The protoplast density of D. indica used in co-culturing varied from 6 x 10(3) - 10(5) / mL. Very strong inhibitory allelopathic effects of D. indica protoplasts on lettuce protoplast growth were found. A similar strong inhibitory allelopathic activity of dried young leaves on lettuce seedling growth was also observed by using the sandwich method. Rotenone, which is a component of Derris root, dissolved in DMSO, was highly inhibitory on the growth of lettuce protoplasts in culture and this could be one of the causes of the strong allelopathic activity of D. indica. PMID:26058149

  8. Erythropoietin bioassays using fetal mouse liver cells: validations and technical improvements

    International Nuclear Information System (INIS)

    Studies are described which were designed to compare an erythropoietin (Ep) bioassay using the uptake of 125I-deoxyuridine (125-I-UdR) into whole cells cultured in micro-titer plates with an established method where the cells were cultured in 1 ml volumes using the incorporation of 59Fe into heme as the endpoint. The results demonstrate the validity of the substitution of 125I-UdR uptake into whole cells as a replacement for 59Fe incorporation into heme as an assay endpoint and the adaptation of the method to a semi-micro scale with automated cell harvesting. A culture time significantly shorter than 24 h is demonstrated not to be of practical benefit. Two methods are proposed to saturate the growth promoting effects of iron-containing transferrin. The performance of semi-micro, serum-containing bioassay employing untreated serum as the test material is shown to be superior to previous systems for the biological measurement of Ep

  9. Cell bioassay for paralytic shellfish poisoning (PSP): comparison with postcolumn derivatization liquid chromatographic analysis and application to the monitoring of PSP in shellfish.

    Science.gov (United States)

    Hayashi, Rumiko; Saito, Hiroshi; Okumura, Masanao; Kondo, Fumio

    2006-01-25

    We performed a neuroblastoma cell (Neuro2a) culture assay modified slightly from a method reported previously to provide a simple and sensitive evaluation of paralytic shellfish poisoning (PSP) toxicity in shellfish. The cell bioassay was just as sensitive for C-toxins as for gonyautoxins. The sensitivity of our cell bioassay was 4 times that of the current standard mouse bioassay. Using the cell bioassay, we evaluated PSP toxicity in 361 shellfish samples collected from Mikawa Bay and Ise Bay, Aichi Prefecture, Japan, from April 1999-March 2002. The results were compared with those obtained in a postcolumn derivatization liquid chromatographic analysis. PSP toxins were detected in 236/361 samples by both assays, and there was a fairly good correlation (r = 0.9001, n = 236, p < 0.001) between the results from the two assays. We applied this cell bioassay when short-necked clams in the bay turned poisonous in 2001. The chronological changes in PSP toxicity in the short-necked clams were analyzed and compared with those of the cell density of poisonous plankton (Alexandrium tamarense) occurring in the bay. The PSP toxicity in shellfish peaked 2 weeks after the cell density reached a maximum. We recommend using the cell bioassay for routine monitoring of PSP toxicity in shellfish living in natural marine environments. PMID:16417278

  10. Optimisation of the CT h4S bioassay for detection of human interleukin-4 secreted by mononuclear cells stimulated by phytohaemaglutinin or by human leukocyte antigen mismatched mixed lymphocyte culture

    DEFF Research Database (Denmark)

    Petersen, Søren Lykke; Russell, Charlotte Astrid; Bendtzen, Klaus;

    2002-01-01

    Limiting dilution analysis has been used in the context of allogeneic bone marrow transplantation to determine anti-recipient interleukin-2 (IL-2) producing helper T lymphocyte precursor (HTLp) frequencies, which in several studies have been predictive of graft-versus-host disease (GVHD). Recently...... high anti-recipient IL-4 producing HTLp frequencies have been reported and associated with a decreased risk of GVHD. The aim of the present study was to define the optimal conditions for combined determination of IL-2 and IL-4 producing anti-recipient HTLp frequencies. We have optimised the CT.h4S......-2 in human leukocyte antigen (HLA)-mismatched mixed leukocyte culture (MLC). An interindividual variation in cytokine accumulation was demonstrated for IL-4 but not for IL-2. With the use of 5x10(4) responder cells/well no IL-4 could be detected in HLA-mismatched MLC between days 1 and 16. The lack...

  11. Use of viral promoters in mammalian cell-based bioassays: How reliable?

    OpenAIRE

    Gill-Sharma Manjit; Choudhuri Jyoti; Betrabet Shrikant S

    2004-01-01

    Abstract Cell-based bioassays have been suggested for screening of hormones and drug bioactivities. They are a plausible alternative to animal based methods. The technique used is called receptor/reporter system. Receptor/reporter system was initially developed as a research technique to understand gene function. Often reporter constructs containing viral promoters were used because they could be expressed with very 'high' magnitude in a variety of cell types in the laboratory. On the other h...

  12. Fish Stem Cell Cultures

    OpenAIRE

    Ni Hong, Zhendong Li, Yunhan Hong

    2011-01-01

    Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is th...

  13. Optimizing stem cell culture.

    Science.gov (United States)

    van der Sanden, Boudewijn; Dhobb, Mehdi; Berger, François; Wion, Didier

    2010-11-01

    Stem cells always balance between self-renewal and differentiation. Hence, stem cell culture parameters are critical and need to be continuously refined according to progress in our stem cell biology understanding and the latest technological developments. In the past few years, major efforts have been made to define more precisely the medium composition in which stem cells grow or differentiate. This led to the progressive replacement of ill-defined additives such as serum or feeder cell layers by recombinant cytokines or growth factors. Another example is the control of the oxygen pressure. For many years cell cultures have been done under atmospheric oxygen pressure which is much higher than the one experienced by stem cells in vivo. A consequence of cell metabolism is that cell culture conditions are constantly changing. Therefore, the development of high sensitive monitoring processes and control algorithms is required for ensuring cell culture medium homeostasis. Stem cells also sense the physical constraints of their microenvironment. Rigidity, stiffness, and geometry of the culture substrate influence stem cell fate. Hence, nanotopography is probably as important as medium formulation in the optimization of stem cell culture conditions. Recent advances include the development of synthetic bioinformative substrates designed at the micro- and nanoscale level. On going research in many different fields including stem cell biology, nanotechnology, and bioengineering suggest that our current way to culture cells in Petri dish or flasks will soon be outdated as flying across the Atlantic Ocean in the Lindbergh's plane. PMID:20803548

  14. Cell Culture Made Easy.

    Science.gov (United States)

    Dye, Frank J.

    1985-01-01

    Outlines steps to generate cell samples for observation and experimentation. The procedures (which use ordinary laboratory equipment) will establish a short-term primary culture of normal mammalian cells. Information on culture vessels and cell division and a list of questions to generate student interest and involvement in the topics are…

  15. Development of a cell-based bioassay for phospholipase A2-triggered liposomal drug release.

    Directory of Open Access Journals (Sweden)

    Ahmad Arouri

    Full Text Available The feasibility of exploiting secretory phospholipase A2 (sPLA2 enzymes, which are overexpressed in tumors, to activate drug release from liposomes precisely at the tumor site has been demonstrated before. Although the efficacy of the developed formulations was evaluated using in vitro and in vivo models, the pattern of sPLA2-assisted drug release is unknown due to the lack of a suitable bio-relevant model. We report here on the development of a novel bioluminescence living-cell-based luciferase assay for the monitoring of sPLA2-triggered release of luciferin from liposomes. To this end, we engineered breast cancer cells to produce both luciferase and sPLA2 enzymes, where the latter is secreted to the extracellular medium. We report on setting up a robust and reproducible bioassay for testing sPLA2-sensitive, luciferin remote-loaded liposomal formulations, using 1,2-distearoyl-sn-glycero-3-phosphatidylcholine/1,2-distearoyl-sn-glycero-3-phosphatidylglycerol (DSPC/DSPG 7:3 and DSPC/DSPG/cholesterol 4:3:3 as initial test systems. Upon their addition to the cells, the liposomes were degraded almost instantaneously by sPLA2 releasing the encapsulated luciferin, which provided readout from the luciferase-expressing cells. Cholesterol enhanced the integrity of the formulation without affecting its susceptibility to sPLA2. PEGylation of the liposomes only moderately broadened the release profile of luciferin. The provided bioassay represents a useful tool for monitoring active drug release in situ in real time as well as for testing and optimizing of sPLA2-sensitive lipid formulations. In addition, the bioassay will pave the way for future in-depth in vitro and in vivo studies.

  16. Use of viral promoters in mammalian cell-based bioassays: How reliable?

    Directory of Open Access Journals (Sweden)

    Gill-Sharma Manjit

    2004-01-01

    Full Text Available Abstract Cell-based bioassays have been suggested for screening of hormones and drug bioactivities. They are a plausible alternative to animal based methods. The technique used is called receptor/reporter system. Receptor/reporter system was initially developed as a research technique to understand gene function. Often reporter constructs containing viral promoters were used because they could be expressed with very 'high' magnitude in a variety of cell types in the laboratory. On the other hand mammalian genes are expressed in a cell/tissue specific manner, which makes them (i.e. cells/tissues specialized for specific function in vivo. Therefore, if the receptor/reporter system is to be used as a cell-based screen for testing of hormones and drugs for human therapy then the choice of cell line as well as the promoter in the reporter module is of prime importance so as to get a realistic measure of the bioactivities of 'test' compounds. We evaluated two conventionally used viral promoters and a natural mammalian promoter, regulated by steroid hormone progesterone, in a cell-based receptor/reporter system. The promoters were spliced into vectors expressing enzyme CAT (chloramphenicol acetyl transferase, which served as a reporter of their magnitudes and consistencies in controlling gene expressions. They were introduced into breast cell lines T47D and MCF-7, which served as a cell-based source of progesterone receptors. The yardstick of their reliability was highest magnitude as well as consistency in CAT expression on induction by sequential doses of progesterone. All the promoters responded to induction by progesterone doses ranging from 10-12 to 10-6 molar by expressing CAT enzyme, albeit with varying magnitudes and consistencies. The natural mammalian promoter showed the most coherence in magnitude as well as dose dependent expression profile in both the cell lines. Our study casts doubts on use of viral promoters in a cell-based bioassay for

  17. Pharmacodynamics of TRPV1 Agonists in a Bioassay Using Human PC-3 Cells

    Directory of Open Access Journals (Sweden)

    Daniel Alvarez-Berdugo

    2014-01-01

    Full Text Available Purpose. TRPV1 is a multimodal channel mainly expressed in sensory neurons. We aimed to explore the pharmacodynamics of the TRPV1 agonists, capsaicin, natural capsaicinoids, and piperine in an in vitro bioassay using human PC-3 cells and to examine desensitization and the effect of the specific antagonist SB366791. Methods. PC-3 cells expressing TRPV1 were incubated with Fluo-4. Fluorescence emission changes following exposition to agonists with and without preincubation with antagonists were assessed and referred to maximal fluorescence following the addition of ionomycin. Concentration-response curves were fitted to the Hill equation. Results. Capsaicin and piperine had similar pharmacodynamics (Emax 204.8 ± 184.3% piperine versus 176.6 ± 35.83% capsaicin, P=0.8814, Hill coefficient 0.70 ± 0.50 piperine versus 1.59 ± 0.86 capsaicin, P=0.3752. In contrast, capsaicinoids had lower Emax (40.99 ± 6.14% capsaicinoids versus 176.6 ± 35.83% capsaicin, P<0.001. All the TRPV1 agonists showed significant desensitization after the second exposition and their effects were strongly inhibited by SB366791. Conclusion. TRPV1 receptor is successfully stimulated by capsaicin, piperine, and natural capsaicinoids. These agonists present desensitization and their effect is significantly reduced by a TRPV1-specific antagonist. In addition, PC-3 cell bioassays proved useful in the study of TRPV1 pharmacodynamics.

  18. Critical parameters in the MCF-7 cell proliferation bioassay (E-Screen)

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Høj; Nielsen, Jesper Bo

    2002-01-01

    The MCF-7 cell proliferation bioassay has grown in popularity as a rapid test for detecting potentially oestrogenic compounds. Several MCF-7 cell sublines with different sensitivities to oestrogens are currently used, with maximal proliferation responses ranging from two- to 10-fold above those of...... hormone-free controls. In the highly responsive MCF-7 BUS cell line, we evaluated critical assay parameters for test performance, including growth conditions, initial seeding densities and differences in growth stimulation in medium containing human serum or fetal calf serum as well as appropriate...... solvents for oestrogen-mimicking compounds. Modifications significantly reduced the labour-intensive steps and overall assay costs without affecting the sensitivity of the assay. Using this optimized test regimen, the responsiveness of treated MCF-7 BUS cells was consistently increased up to 11-fold over...

  19. Bacterial cell culture

    OpenAIRE

    sprotocols

    2014-01-01

    ### Materials 1. Glass culture tubes with metal caps and labels - Growth medium, from media room or customized - Glass pipette tubes - Parafilm ### Equipment 1. Vortexer - Fireboy or Bunsen burner - Motorized pipette - Micropipettes and sterile tips ### Procedure For a typical liquid culture, use 5 ml of appropriate medium. The amount in each tube does not have to be exact if you are just trying to culture cells for their precious DNA. 1. Streak an a...

  20. Mammalian Cell Culture Simplified.

    Science.gov (United States)

    Moss, Robert; Solomon, Sondra

    1991-01-01

    A tissue culture experiment that does not require elaborate equipment and that can be used to teach sterile technique, the principles of animal cell line maintenance, and the concept of cell growth curves is described. The differences between cancerous and normal cells can be highlighted. The procedure is included. (KR)

  1. Fish Stem Cell Cultures

    Directory of Open Access Journals (Sweden)

    Ni Hong, Zhendong Li, Yunhan Hong

    2011-01-01

    Full Text Available Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on “Fish Stem Cells and Nuclear Transfer”, we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer.

  2. Receptor-mediated endocytosis of diphtheria toxin by cells in culture.

    OpenAIRE

    Keen, J H; Maxfield, F R; Hardegree, M C; Habig, W H

    1982-01-01

    The binding and uptake of fluorescently labeled diphtheria toxin by cells in culture has been examined by using epifluorescence video intensification microscopy. Rhodamine-labeled diphtheria toxin retained significant toxicity on bioassay and in cell culture and was tested for uptake by human WI-38 and mouse 3T3 fibroblasts grown in culture. When added to cells at 37 degrees C, toxin was observed to become concentrated and internalized in discrete vesicles in both cell lines. The appearance o...

  3. A new in vitro screening bioassay for the ecotoxicological evaluation of the estrogenic responses of environmental chemicals using roach (Rutilus rutilus) liver explant culture.

    Science.gov (United States)

    Gerbron, Marie; Geraudie, Perrine; Rotchell, Jeanette; Minier, Christophe

    2010-10-01

    There is growing evidence that many chemicals released in the environment are able to disturb the normal endocrinology of organisms affecting the structure and function of their reproductive system. This has prompted the scientific community to develop appropriate testing methods to identify active compounds and elucidate mechanisms of action. Of particular interest are in vitro screening methods that can document the effects of these endocrine disrupting compounds on fish. In this study, an in vitro bioassay was developed in the roach (Rutilus rutilus) for evaluating the estrogenicity or antiestrogenicity potency of environmental pollutants by measuring vitellogenin (VTG) induction in cultured liver explants. The cell viability was assessed by the measurement of nonspecific esterase activity using a fluorescein diacetate hydrolysis assay. Results showed that explants could be cultured for 72 h without any significant loss of activity. Dose-dependent responses have been measured with estrogenic model compounds such as 17-β-estradiol (E2) and 17-α-ethynylestradiol (EE2) or antiestrogenic compounds such as tamoxifen. Lowest observable effective concentrations were 1 nM for E2, 1 nM for EE2, and 100 nM for tamoxifen, showing a good sensitivity of the test system. Estrogenicity of butyl 4-hydroxybenzoate, 4-nonylphenol, and bisphenol A was tested. bisphenol A (100 μM) or butylparaben induced a twofold increase in VTG production when compared with 100 nM E2, whereas this production was only 20% with 100 μM 4-nonylphenol. Overall, this study shows that the bioassay could provide valuable information on endocrine disrupting chemicals including metabolites and mixtures of compounds. PMID:20549626

  4. Optimizing stem cell culture.

    OpenAIRE

    van der Sanden, Boudewijn; Dhobb, Mehdi; Berger, François; Wion, Didier

    2010-01-01

    International audience Stem cells always balance between self-renewal and differentiation. Hence, stem cell culture parameters are critical and need to be continuously refined according to progress in our stem cell biology understanding and the latest technological developments. In the past few years, major efforts have been made to define more precisely the medium composition in which stem cells grow or differentiate. This led to the progressive replacement of ill-defined additives such a...

  5. Digital Microfluidic Cell Culture.

    Science.gov (United States)

    Ng, Alphonsus H C; Li, Bingyu Betty; Chamberlain, M Dean; Wheeler, Aaron R

    2015-01-01

    Digital microfluidics (DMF) is a droplet-based liquid-handling technology that has recently become popular for cell culture and analysis. In DMF, picoliter- to microliter-sized droplets are manipulated on a planar surface using electric fields, thus enabling software-reconfigurable operations on individual droplets, such as move, merge, split, and dispense from reservoirs. Using this technique, multistep cell-based processes can be carried out using simple and compact instrumentation, making DMF an attractive platform for eventual integration into routine biology workflows. In this review, we summarize the state-of-the-art in DMF cell culture, and describe design considerations, types of DMF cell culture, and cell-based applications of DMF. PMID:26643019

  6. Mycoplasmas detection in cells cultures

    OpenAIRE

    Rivera-Tapia José Antonio; Castillo-Viveros Linda Valeria; Sánchez-Hernández José Antonio

    2010-01-01

    INTRODUCTION. Cells cultures are widely used in both biomedical and biotechnological research centers and industry, as well as for diagnostic test in hospitals. Contaminations of cells cultures with microbial organisms as well as with virus or other eukaryotic cell lines are a major problem in cell culture related research.OBJECTIVE. Mycoplasmas detection in cells cultures came from biomedical laboratories.MATERIAL AND METHODS. The cells cultures screened for mycoplasmas by using of microbiol...

  7. Acute toxicity bioassays using Daphnia magna Straus (Cladocera, Daphniidae) maintained in a modified culture medium

    OpenAIRE

    Mónica Núñez; Jasmin Hurtado

    2013-01-01

    Daphnia magna is a test organism used in ecotoxicological assays of freshwater; however, traditional culture systems for this organism could result expensive, for that the aim of this research was to developed a new economic culture medium. With this purpose, 10 strains of D. magna were isolated, their population development was evaluated by total count of organisms and pregnant females using 3 different culture media: (A) alfalfa juice, (B) solved yeast and (C) a mixture of alfalfa juice plu...

  8. Perfusion based cell culture chips

    DEFF Research Database (Denmark)

    Heiskanen, Arto; Emnéus, Jenny; Dufva, Martin

    2010-01-01

    Performing cell culture in miniaturized perfusion chambers gives possibilities to experiment with cells under near in vivo like conditions. In contrast to traditional batch cultures, miniaturized perfusion systems provide precise control of medium composition, long term unattended cultures and ti...

  9. Gamma-interferon bioassay for detection of bovine tuberculosis in cattle: kinetics of production and dose response in whole blood culture

    International Nuclear Information System (INIS)

    Stimulation with mycobacterium bovis PPD sensitised lymphocytes (whole blood or peripheral blood lymphocytes) results in release of gamma-interferon that can be detected by simple bioassay. The optimum concentration of bovine PPD was 20 μg ml and the optimum incubation period was 24 hr for maximum production of gamma-interferon in whole blood culture (128 units/ml) and peripheral blood culture (64 units/ml). (author)

  10. Oscillating Cell Culture Bioreactor

    Science.gov (United States)

    Freed, Lisa E.; Cheng, Mingyu; Moretti, Matteo G.

    2010-01-01

    To better exploit the principles of gas transport and mass transport during the processes of cell seeding of 3D scaffolds and in vitro culture of 3D tissue engineered constructs, the oscillatory cell culture bioreactor provides a flow of cell suspensions and culture media directly through a porous 3D scaffold (during cell seeding) and a 3D construct (during subsequent cultivation) within a highly gas-permeable closed-loop tube. This design is simple, modular, and flexible, and its component parts are easy to assemble and operate, and are inexpensive. Chamber volume can be very low, but can be easily scaled up. This innovation is well suited to work with different biological specimens, particularly with cells having high oxygen requirements and/or shear sensitivity, and different scaffold structures and dimensions. The closed-loop changer is highly gas permeable to allow efficient gas exchange during the cell seeding/culturing process. A porous scaffold, which may be seeded with cells, is fixed by means of a scaffold holder to the chamber wall with scaffold/construct orientation with respect to the chamber determined by the geometry of the scaffold holder. A fluid, with/without biological specimens, is added to the chamber such that all, or most, of the air is displaced (i.e., with or without an enclosed air bubble). Motion is applied to the chamber within a controlled environment (e.g., oscillatory motion within a humidified 37 C incubator). Movement of the chamber induces relative motion of the scaffold/construct with respect to the fluid. In case the fluid is a cell suspension, cells will come into contact with the scaffold and eventually adhere to it. Alternatively, cells can be seeded on scaffolds by gel entrapment prior to bioreactor cultivation. Subsequently, the oscillatory cell culture bioreactor will provide efficient gas exchange (i.e., of oxygen and carbon dioxide, as required for viability of metabolically active cells) and controlled levels of fluid

  11. Detection of estrogen receptor endocrine disruptor potency of commonly used organochlorine pesticides using the LUMI-CELL ER bioassay

    Energy Technology Data Exchange (ETDEWEB)

    Gordon, J.D.; Chu, A.C.; Clark, G.C. [Xenobiotic Detection Systems, Inc., Durham, NC (United States); Chu, M.D. [Alta Analytical Perspectives, Wilmington, NC (United States); Denison, M.S. [Dept. of Environmental Toxicology, Univ. of California, Davis, CA (United States)

    2004-09-15

    In order to detect the endocrine disrupting potency of organochlorine pesticides and other compounds, BG-1 (human ovarian carcinoma) cells containing a stably transfected estrogenresponsive luciferase reporter gene plasmid (BG1Luc4E2), was used. This cell line, termed the LUMI-CELL trademark ER estrogenic cell bioassay system, responds in a time-, dose dependent- and chemical-specific manner with the induction of luciferase gene expression in response to exposure to estrogen (but not other steroid hormones) and estrogenic chemicals in a high-throughput screening (HTPS) format6. Here we describe studies in which the LUMI-CELL trademark ER estrogenic cell bioassay system was used for high throughput screening (HTPS) analysis of the estrogenic disrupting potency of several commonly used pesticides and organochlorines: p,p'DDT; p,p'-DDE; DDD; {alpha}a-chlordane; {psi}-chlordane; Kepone; Methoxychlor; Vinclozolin; Fenarimol; 2,4,5-Trichlorophenoxyacetic Acid; and Dieldrin. Our results demonstrate the utility of XDS's LUMI-CELL trademark ER bioassay HTPS system for screening chemicals for estrogenic activity.

  12. Acute toxicity bioassays using Daphnia magna Straus (Cladocera, Daphniidae maintained in a modified culture medium

    Directory of Open Access Journals (Sweden)

    Mónica Núñez

    2013-05-01

    Full Text Available Daphnia magna is a test organism used in ecotoxicological assays of freshwater; however, traditional culture systems for this organism could result expensive, for that the aim of this research was to developed a new economic culture medium. With this purpose, 10 strains of D. magna were isolated, their population development was evaluated by total count of organisms and pregnant females using 3 different culture media: (A alfalfa juice, (B solved yeast and (C a mixture of alfalfa juice plus solved yeast. Successful development of 4 strains was observed in the A medium, but the same strains failed to survive in the B and the C media. The 24h and 48h EC50 average values in acute ecotoxicological assays with potassium dicromate were 0,4045 mg/L ± 0,0389 and 0,1857 mg/L ± 0,0072 respectively. Also, acute ecotoxicological assays with these 4 strains were performed using potassium cyanide, which is a toxic reactive frequently used in mining operations. In this case 24h EC50 value was 1,5388 mg/L ± 0,1146 and 48h EC50 values were 0,6359 mg/L ± 0,0516. 48h EC50 values were lower than the cyanide permissible effluent values established by the Energy and Mining Authority.

  13. Development of a pneumatically driven active cover lid for multi-well microplates for use in perfusion three-dimensional cell culture

    OpenAIRE

    Song-Bin Huang; Dean Chou; Yu-Han Chang; Ke-Cing Li; Tzu-Keng Chiu; Yiannis Ventikos; Min-Hsien Wu

    2015-01-01

    Before microfluidic-based cell culture models can be practically utilized for bioassays, there is a need for a transitional cell culture technique that can improve conventional cell culture models. To address this, a hybrid cell culture system integrating an active cover lid and a multi-well microplate was proposed to achieve perfusion 3-D cell culture. In this system, a microfluidic-based pneumatically-driven liquid transport mechanism was integrated into the active cover lid to realize 6-un...

  14. Microfluidic Cell Culture Device

    Science.gov (United States)

    Takayama, Shuichi (Inventor); Cabrera, Lourdes Marcella (Inventor); Heo, Yun Seok (Inventor); Smith, Gary Daniel (Inventor)

    2014-01-01

    Microfluidic devices for cell culturing and methods for using the same are disclosed. One device includes a substrate and membrane. The substrate includes a reservoir in fluid communication with a passage. A bio-compatible fluid may be added to the reservoir and passage. The reservoir is configured to receive and retain at least a portion of a cell mass. The membrane acts as a barrier to evaporation of the bio-compatible fluid from the passage. A cover fluid may be added to cover the bio-compatible fluid to prevent evaporation of the bio-compatible fluid.

  15. Phytochemical and toxicity evaluation of Phaleria macrocarpa (Scheff. Boerl by MCF-7 cell line and brine shrimp lethality bioassay

    Directory of Open Access Journals (Sweden)

    Abul Kalam Azad

    2016-01-01

    Full Text Available Objective: To evaluate the cytotoxicity of Phaleria macrocarpa fruits extracts. Methods: The cytotoxicity test was carried out by in vitro MCF-7 cell line and in vivo brine shrimp lethality bioassay. Results: The preliminary phytochemical test showed the presence of alkaloids, carbohydrate, glycosides, saponin, terpene, steroids, phenols and flavonoids. The MTT-assay results showed that the highest percentage of cell viability was 106.23% at concentration of 1.25 µL and the lowest percentage was 13.04% at concentration of 10 µL. Conclusions: The MTT-assay and brine shrimp lethality bioassay results showed that the extract was non-toxic and it would be consumable as a herbal remedy.

  16. Phytochemical and toxicity evaluation ofPhaleria macrocarpa (Scheff.) Boerl by MCF-7 cell line and brine shrimp lethality bioassay

    Institute of Scientific and Technical Information of China (English)

    Abul Kalam Azad; Wan Mohd Azizi Wan Sulaiman; Nushrat Khan Sunzida

    2016-01-01

    Objective:To evaluate the cytotoxicity ofPhaleria macrocarpa fruits extracts. Methods: The cytotoxicity test was carried out byin vitroMCF-7 cell line andin vivo brine shrimp lethality bioassay. Results: The preliminary phytochemical test showed the presence of alkaloids, carbohydrate, glycosides, saponin, terpene, steroids, phenols and flavonoids. TheMTT-assay results showed that the highest percentage of cell viability was 106.23% at concentration of 1.25µL and the lowest percentage was 13.04% at concentration of 10µL. Conclusions:TheMTT-assay and brine shrimp lethality bioassay results showed that the extract was non-toxic and it would be consumable as a herbal remedy.

  17. Development and characterization of a green fluorescent protein-based rat cell bioassay system for detection of AH receptor ligands

    Energy Technology Data Exchange (ETDEWEB)

    Zhao Bin; Denison, M. [California Univ., Davis, CA (United States). Dept. of Environmental Toxicology

    2004-09-15

    Proper epidemiological, risk assessment and exposure analysis of TCDD and related HAHs requires accurate measurements of these chemicals both in the species of interest and in various exposure matrices (i.e. biological, environmental, food and feed). While high-resolution instrumental analysis techniques are established for these chemicals, these procedures are very costly, time-consuming and are impractical for large scale sampling studies. Accordingly, numerous bioanalytical methods have been developed for the detection of these chemicals in extracts from a variety of matrices, the majority of which take the advantage of the ability of these chemicals to activate one or more aspects of the AhR-dependent mechanism of action. One of the most sensitive bioassay systems developed to date is the so-called CALUX (Chemically Activated Luciferase Expression) assay, which is based on novel recombinant cell lines that contain a stably transfected dioxin (AhR)-responsive firefly luciferase gene. Treatment of these cells with TCDD and related HAHs and polycyclic aromatic hydrocarbons (PAHs), as well as other AhR ligands, results in induction of reporter gene expression in a time-, dose-, AhR-, and chemical-specific manner. The level of reporter gene expression correlates with the total concentration of the TCDD-like AhR inducers (agonists) present in the sample. Although the firefly luciferase reporter gene contributes to the high degree of sensitivity of the assay, it also has limitations with respect to our need for a rapid and inexpensive bioassay for high-throughput screening analysis. Accordingly, we previously developed a stably transfected murine cell line containing an AhRresponsive enhanced green fluorescent protein (EGFP) reporter gene. This cell line provided us with a high-throughput cell bioassay system for identification and characterization of AhR agonists and antagonists. Here we have extended these studies and describe the development, optimization, and

  18. Efficient screening of dioxins in food and feed using shape-selective pressurized liquid extraction and cell based bioassay analysis

    Energy Technology Data Exchange (ETDEWEB)

    Nording, M. [Swedish Defence Research Agency, Umea (Sweden)]|[Umea Univ. (Sweden). Environmental Chemistry, Dept. of Chemistry; Sporring, S.; Bjoerklund, E. [Lund Univ. (Sweden). Dept. of Analytical Chemistry; Wiberg, K.; Haglund, P. [Umea Univ. (Sweden). Environmental Chemistry, Dept. of Chemistry

    2004-09-15

    Cell based bioassays with enhanced green fluorescent protein (EGFP) detection are potential screening methods for determination of aryl hydrocarbon receptor (AhR) ligands, such as dioxins and similar compounds, in environmental samples. With this technique, it is possible to detect dioxins at levels normally found in food and feed, i.e. pg toxic equivalents (TEQ)/g. Since the signal from the bioassay might be caused by compounds other than dioxins binding to the AhR, determination of the dioxin TEQ generally involve extraction with organic solvents or solvent mixtures, e.g. using a Soxhlet apparatus, followed by clean-up with sulphuric acid or sulphuric acid impregnated silica gel and carbon fractionation in order to exclude possible interferences from the extracts. Until now, sample preparation has been time consuming and labour intensive, but alternatives to traditional methods have recently been developed, with the benefits of shorter analysis times and reduced organic solvent consumption. Pressurized liquid extraction (PLE) may, for instance, be used with a fat retainer in the PLE cell to selectively extract PCBs from food, feed, and biota matrices. In order to further streamline the sample preparation, new assemblies have been developed to fit into a commercially available PLE-equipment. The assemblies are packed with an activated carbon/celite mixture and the sample. In the subsequent extraction, the pollutants are fractionated into three fractions according to their planarity (shape-selective extraction). In the first fraction (I) bulk lipids and PCBs are eluted, in the second fraction (II) the majority of planar (non-ortho) PCBs, and in the third fraction (III), which is back-flushed, the dioxins are recovered. In this way, a pure dioxin fraction may be isolated and analysed separately with the cell based bioassay described above. This study was conducted to meet the imperative demands for dioxin monitoring. The aim was to develop a comprehensive method for

  19. Huanglongbing and psyllid cell cultures

    Science.gov (United States)

    We successfully established cell cultures of the Asian citrus psyllid, Diaphorina citri (Psyllidae: Hemiptera), DcHH-1. The cell culture also supported growth of Candidatus Liberibacter asiaticus. This bacterial pathogen is associated with Huanglongbing, known as citrus greening disease. Research on...

  20. Androgenic and estrogenic response of green mussel extracts from Singapore's coastal environment using a human cell-based bioassay.

    Science.gov (United States)

    Bayen, Stéphane; Gong, Yinhan; Chin, Hong Soon; Lee, Hian Kee; Leong, Yong Eu; Obbard, Jeffrey Philip

    2004-11-01

    In the last decade, evidence of endocrine disruption in biota exposed to environmental pollutants has raised serious concern. Human cell-based bioassays have been developed to evaluate induced androgenic and estrogenic activities of chemical compounds. However, bioassays have been sparsely applied to environmental samples. In this study we present data on sex hormone activities in the green mussel, Perna viridis, in Singapore's coastal waters. P.viridis is a common bioindicator of marine contamination, and this study is a follow-up to an earlier investigation that reported the presence of sex hormone activities in seawater samples from Singapore's coastal environment. Specimens were collected from eight locations around the Singapore coastline and analyzed for persistent organic pollutants (POPs) and heavy metals. Tissue extracts were then screened for activities on androgen receptors (ARs) and estrogen receptors (ER-alpha and ER-beta) using a reporter gene bioassay based on a HeLa human cell line. Mussel extracts alone did not exhibit AR activity, but in the presence of the reference androgenic hormone dihydrotestosterone (DHT), activities were up to 340% higher than those observed for DHT alone. Peak activities were observed in locations adjacent to industrial and shipping activities. Estrogenic activities of the mussel extract both alone and in the presence of reference hormone were positive. Correlations were statistically investigated between sex hormone activities, levels of pollutants in the mussel tissues, and various biological parameters (specimen size, sex ratio, lipid and moisture content). Significant correlations exist between AR activities, in the presence of DHT, and total concentration of POPs (r= 0.725, p < 0.05). PMID:15531429

  1. High density cell culture system

    Science.gov (United States)

    Spaulding, Glenn F. (Inventor)

    1994-01-01

    An annular culture vessel for growing mammalian cells is constructed in a one piece integral and annular configuration with an open end which is closed by an endcap. The culture vessel is rotatable about a horizontal axis by use of conventional roller systems commonly used in culture laboratories. The end wall of the endcap has tapered access ports to frictionally and sealingly receive the ends of hypodermic syringes. The syringes permit the introduction of fresh nutrient and withdrawal of spent nutrients. The walls are made of conventional polymeric cell culture material and are subjected to neutron bombardment to form minute gas permeable perforations in the walls.

  2. Cell culture purity issues and DFAT cells

    International Nuclear Information System (INIS)

    Highlights: •DFAT cells are progeny cells derived from dedifferentiated mature adipocytes. •Common problems in this research is potential cell contamination of initial cultures. •The initial cell culture purity is crucial in DFAT cell research field. -- Abstract: Dedifferentiation of mature adipocytes, in vitro, has been pursued/documented for over forty years. The subsequent progeny cells are named dedifferentiated adipocyte-derived progeny cells (DFAT cells). DFAT cells are proliferative and likely to possess mutilineage potential. As a consequence, DFAT cells and their progeny/daughter cells may be useful as a potential tool for various aspects of tissue engineering and as potential vectors for the alleviation of several disease states. Publications in this area have been increasing annually, but the purity of the initial culture of mature adipocytes has seldom been documented. Consequently, it is not always clear whether DFAT cells are derived from dedifferentiated mature (lipid filled) adipocytes or from contaminating cells that reside in an impure culture

  3. Aseptic technique for cell culture.

    Science.gov (United States)

    Coté, R J

    2001-05-01

    This unit describes some of the ways that a laboratory can deal with the constant threat of microbial contamination in cell cultures. A protocol on aseptic technique is described first. This catch-all term universally appears in any set of instructions pertaining to procedures in which noncontaminating conditions must be maintained. In reality, aseptic technique encompasses all aspects of environmental control, personal hygiene, equipment and media sterilization, and associated quality control procedures needed to ensure that a procedure is, indeed, performed with aseptic, noncontaminating technique. Although cell culture can theoretically be carried out on an open bench in a low-traffic area, most cell culture work is carried out using a horizontal laminar-flow clean bench or a vertical laminar-flow biosafety cabinet. Both are described here. PMID:18228291

  4. Cell culture compositions

    Science.gov (United States)

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yiao, Jian

    2014-03-18

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6 (SEQ ID NO:1 encodes the full length endoglucanase; SEQ ID NO:4 encodes the mature form), and the corresponding endoglucanase VI amino acid sequence ("EGVI"; SEQ ID NO:3 is the signal sequence; SEQ ID NO:2 is the mature sequence). The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

  5. Bioassay-Guided Isolation of DPP-4 Inhibitory Fractions from Extracts of Submerged Cultured of Inonotus obliquus

    Directory of Open Access Journals (Sweden)

    Jin-Song Shi

    2013-01-01

    Full Text Available Inonotus obliquus is a medicinal mushroom used in Russian and Eastern European folk medicine for the treatment of gastrointestinal cancer, cardiovascular disease and diabetes. Previous studies in our laboratory have demonstrated that the mycelium powders of I. obliquus possess significant antihyperglycemic effects in a mouse model of diabetic disease induced by alloxan. However, the active ingredients of mycelium powders responsible for the diabetes activity have not been identified. This study aims to identify the active ingredients of I. obliquus mycelium powders by a bioassay-guided fractionation approach and explore the mechanism of action of these active ingredients by using a well-established DPP-4 (an important enzyme as a new therapeutic target for diabetes inhibitory assay model. The results showed the chloroform extract of mycelium was potential inhibitory against DPP-4. Bioactivity guided fractionation led to the identification of 19 compounds using UPLC-Q-TOF-MS. Molecular docking between the compounds and DPP-4 revealed that compounds 5, 8, 9, 14, 15 may be the active components responsible for the DPP-4 inhibitory activity.

  6. Bioassay-guided isolation of DPP-4 inhibitory fractions from extracts of submerged cultured of Inonotus obliquus.

    Science.gov (United States)

    Geng, Yan; Lu, Zhen-Ming; Huang, Wei; Xu, Hong-Yu; Shi, Jin-Song; Xu, Zheng-Hong

    2013-01-01

    Inonotus obliquus is a medicinal mushroom used in Russian and Eastern European folk medicine for the treatment of gastrointestinal cancer, cardiovascular disease and diabetes. Previous studies in our laboratory have demonstrated that the mycelium powders of I. obliquus possess significant antihyperglycemic effects in a mouse model of diabetic disease induced by alloxan. However, the active ingredients of mycelium powders responsible for the diabetes activity have not been identified. This study aims to identify the active ingredients of I. obliquus mycelium powders by a bioassay-guided fractionation approach and explore the mechanism of action of these active ingredients by using a well-established DPP-4 (an important enzyme as a new therapeutic target for diabetes) inhibitory assay model. The results showed the chloroform extract of mycelium was potential inhibitory against DPP-4. Bioactivity guided fractionation led to the identification of 19 compounds using UPLC-Q-TOF-MS. Molecular docking between the compounds and DPP-4 revealed that compounds 5, 8, 9, 14, 15 may be the active components responsible for the DPP-4 inhibitory activity. PMID:23325103

  7. Dynamized Preparations in Cell Culture

    OpenAIRE

    Girija Kuttan; Korengath Chandran Preethi; Ramadasan Kuttan; Ellanzhiyil Surendran Sunila

    2009-01-01

    Although reports on the efficacy of homeopathic medicines in animal models are limited, there are even fewer reports on the in vitro action of these dynamized preparations. We have evaluated the cytotoxic activity of 30C and 200C potencies of ten dynamized medicines against Dalton's Lymphoma Ascites, Ehrlich's Ascites Carcinoma, lung fibroblast (L929) and Chinese Hamster Ovary (CHO) cell lines and compared activity with their mother tinctures during short-term and long-term cell culture. The ...

  8. Expression Patterns of Cancer Stem Cell Markers During Specific Celecoxib Therapy in Multistep Rat Colon Carcinogenesis Bioassays.

    Science.gov (United States)

    Salim, Elsayed I; Hegazi, Mona M; Kang, Jin Seok; Helmy, Hager M

    2016-01-01

    The purpose of this study was to investigate the role of colon cancer stem cells (CSCs) during chemicallyinduced rat multi-step colon carcinogenesis with or without the treatment with a specific cyclooxygenase-2 inhibitor drug (celecoxib). Two experiments were performed, the first, a short term 12 week colon carcinogenesis bioassay in which only surrogate markers for colon cancer, aberrant crypt foci (ACF) lesions, were formed. The other experiment was a medium term colon cancer rat assay in which tumors had developed after 32 weeks. Treatment with celecoxib lowered the numbers of ACF, as well as the tumor volumes and multiplicities after 32 weeks. Immunohistochemical proliferating cell nuclear antigen (PCNA) labeling indexes LI (%) were downregulated after treatment by celecoxib. Also different cell surface antigens known to associate with CSCs such as the epithelial cell adhesion molecule (EpCAM), CD44 and CD133 were compared between the two experiments and showed differential expression patterns depending on the stage of carcinogenesis and treatment with celecoxib. Flow cytometric analysis demonstrated that the numbers of CD133 cells were increased in the colonic epithelium after 12 weeks while those of CD44 but not CD133 cells were increased after 32 weeks. Moreover, aldehyde dehydrogenase-1 activity levels in the colonic epithelium (a known CSC marker) detected by ELISA assay were found down-regulated after 12 weeks, but were up-regulated after 32 weeks. The data have also shown that the protective effect of celecoxib on these specific markers and populations of CSCs and on other molecular processes such as apoptosis targeted by this drug may vary depending on the genetic and phenotypic stages of carcinogenesis. Therefore, uncovering these distinction roles of CSCs during different phases of carcinogenesis and during specific treatment could be useful for targeted therapy. PMID:27039721

  9. Impedimetric quantification of cells encapsulated in hydrogel cultured in a paper-based microchamber.

    Science.gov (United States)

    Lei, Kin Fong; Huang, Chia-Hao; Tsang, Ngan-Ming

    2016-01-15

    Recently, 3D cell culture technique was proposed to provide a more physiologically-meaningful environment for cell-based assays. With the development of microfluidics technology, cellular response can be quantified by impedance measurement technique in a real-time and non-invasive manner. However, handling of these microfluidic systems requires a trained engineering personnel and the operation is not compatible to traditional biological research laboratories. In this work, we incorporated the impedance measurement technique to paper-based 3D cell culture model and demonstrated non-invasive quantification of cells encapsulated in hydrogel during the culture course. A cellulose filter paper was patterned with an array of circular microchambers. Cells were encapsulated in hydrogel and loaded to the microchambers for culturing cells in 3D environment. At the preset schedule during the culture course, the paper was placed on a glass substrate with measurement electrodes for the impedance measurement. Cells in each microchamber was represented by impedance magnitude and cell proliferation could be studied over time. Also, conventional bio-assay was performed to further confirm the feasibility of the impedimetric quantification of cells encapsulated in hydrogel cultured in the paper-based microchamber. This technique provides a convenient, fast, and non-invasive approach to monitor cells cultured in 3D environment. It has potential to be developed for routine 3D cell culture protocol in biological research laboratories. PMID:26592655

  10. Insect Cell Culture and Biotechnology

    Institute of Scientific and Technical Information of China (English)

    Robert R.Granados; Guoxun Li; G.W.Blissard

    2007-01-01

    The continued development of new cell culture technology is essential for the future growth and application of insect cell and baculovirus biotechnology. The use of cell lines for academic research and for commercial applications is currently dominated by two cell lines; the Spodoptera frugiperda line, SF21 (and its clonal isolate, SF9), and the Trichoplusia ni line, BTI 5B1-4, commercially known as High Five cells. The long perceived prediction that the immense potential application of the baculovirus-insect cell system, as a tool in cell and molecular biology, agriculture, and animal health, has been achieved. The versatility and recent applications of this popular expression system has been demonstrated by both academia and industry and it is clear that this cell-based system has been widely accepted for biotechnological applications. Numerous small to midsize startup biotechnology companies in North America and the Europe are currently using the baculovirus-insect cell technology to produce custom recombinant proteins for research and commercial applications. The recent breakthroughs using the baculovirus-insect cell-based system for the development of several commercial products that will impact animal and human health will further enhance interest in this technology by pharma. Clearly, future progress in novel cell and engineering advances will lead to fundamental scientific discoveries and serve to enhance the utility and applications of this baculovirus-insect cell system.

  11. Comparison of the rat and mouse cell lines commercially available for CALUX bioassays

    Energy Technology Data Exchange (ETDEWEB)

    Goeyens, L.; Windal, I.; Wouwe, N. van [Institute of Public Health, Brussels (Belgium); Scippo, M.L.; Eppe, G.; Pauw, E. de; Maghin-Rogister, G. [Liege Univ. (Belgium)

    2004-09-15

    CALUX (Chemical-activated luciferase gene expression) is nowadays more and more widely used, both for the control of the norms applied to the food chain and to environmental contamination evaluation. In that purpose, two cell lines are commercially available: one rat cell line, commercialized by Bio Detection System (BDS, The Netherlands) and one mouse cell line, commercialized by Xenobiotic Detection System (XDS, USA). Both suppliers propose different clean-up methods and a slightly different method in the preparation, dosage and reading of the plates. Until now, almost no comparison of the cell lines has been performed, or the comparison includes many variables (extraction, purification, method of preparation, dosage and reading of the plate) so that it is difficult to evaluate which variables are mainly responsible of the observed differences. The objective of the research presented here is to perform a direct comparison of the 2 cell lines, and evaluate which variables can be responsible of the discrepancy observed between the results.

  12. Techniques for mammalian cell tissue culture.

    Science.gov (United States)

    Phelan, Mary C

    2006-05-01

    This unit opens with detailed discussions on the latest principles of sterile technique and preparation of culture media. Step-by-step protocols describe trypsinizing and subculturing monolayer cultures, passaging suspension cultures, freezing and thawing cells, counting cells using a hemacytometer, and preparing cells for transport. PMID:18770828

  13. QSAR modeling, synthesis and bioassay of diverse leukemia RPMI-8226 cell line active agents.

    Science.gov (United States)

    Katritzky, Alan R; Girgis, Adel S; Slavov, Svetoslav; Tala, Srinivasa R; Stoyanova-Slavova, Iva

    2010-11-01

    A rigorous QSAR modeling procedure employing CODESSA PRO descriptors has been utilized for the prediction of more efficient anti-leukemia agents. Experimental data concerning the effect on leukemia RPMI-8226 cell line tumor growth of 34 compounds (treated at a dose of 10 μM) was related to their chemical structures by a 4-descriptor QSAR model. Four bis(oxy)bis-urea and bis(sulfanediyl)bis-urea derivatives (4a, 4b, 8, 11a) predicted as active by this model, together with 11b predicted to be of low activity, were synthesized and screened for anti-tumor activity utilizing 55 different tumor cell lines. Compounds 8 and 11a showed anti-tumor properties against most of the adopted cell lines with growth inhibition exceeding 50%. The highly promising preliminary anti-tumor properties of compounds 8 and 11a, were screened at serial dilutions (10(-4)-10(-8) μM) for determination of their GI(50) and TGI against the screened human tumor cell lines. Compound 11a (GI(50) = 1.55, TGI = 8.68 μM) is more effective than compound 8 (GI(50)=58.30, TGI = > 100 μM) against the target leukemia RPMI-8226 cell line. Compound 11a also exhibits highly pronounced anti-tumor properties against NCI-H226, NCI-H23 (non-small cell lung cancer), COLO 205 (colon cancer), SNB-75 (CNS cancer), OVCAR-3, SK-OV-3 (ovarian cancer), A498 (renal cancer) MDA-MB-231/ATCC and MDA-MB-468 (breast cancer) cell lines (GI(50) = 1.95, 1.61, 1.38, 1.56, 1.30, 1.98, 1.18, 1.85, 1.08, TGI = 8.35, 6.01, 2.67, 8.59, 4.01, 7.01, 5.62, 6.38, 5.63 μM, respectively). Thus 11a could be a suitable lead towards the design of broad spectrum anti-tumor active agents targeting various human tumor cell lines. PMID:20843586

  14. Development of a cell-based bioassay for phospholipase A2-triggered liposomal drug release

    DEFF Research Database (Denmark)

    Arouri, Ahmad; Trojnar, Jakub; Schmidt, Steffen;

    2015-01-01

    models, the pattern of sPLA2-assisted drug release is unknown due to the lack of a suitable bio-relevant model. We report here on the development of a novel bioluminescence living-cell-based luciferase assay for the monitoring of sPLA2-triggered release of luciferin from liposomes. To this end, we...

  15. Design and construction of a whole cell bacterial 4-hydroxyphenylacetic acid and 2-phenylacetic acid bioassay

    Directory of Open Access Journals (Sweden)

    Seppe eDierckx

    2015-06-01

    Full Text Available IntroductionAuxins are hormones that regulate plant growth and development. To accurately quantify the low levels of auxins present in plant and soil samples, sensitive detection methods are needed. In this study, the design and construction of two different whole cell auxin biosensors is illustrated. Both use the auxin responsive element HpaA as an input module and differ in output module. The first biosensor is combined with the gfp gene to produce a fluorescent biosensor. Whereas the second one is combined with the genes textit and phzS to produce a pyocyanin producing biosensor whose product can be measured electrochemically.section{Results} The fluorescent biosensor is able to detect 4-hydroxyphenylacetic acid (4-HPA and 2-phenylacetic acid (PAA concentrations from 15 µM to 3 mM in a dose-responsive manner. The pyocyanin producing biosensor is able to detect 4-HPA concentrations from 1.9 µM to 15.625 µM and PAA concentrations from 15.625 µM to 125 µM, both in a dose-responsive manner.ConclusionsA fluorescent whole cell auxin biosensor and an electrochemical whole cell auxin biosensor have been constructed and tested. Both are able to detect 4-HPA and PAA concentrations that are environmentally relevant in respect to plant growth.

  16. Dynamized Preparations in Cell Culture

    Directory of Open Access Journals (Sweden)

    Ellanzhiyil Surendran Sunila

    2009-01-01

    Full Text Available Although reports on the efficacy of homeopathic medicines in animal models are limited, there are even fewer reports on the in vitro action of these dynamized preparations. We have evaluated the cytotoxic activity of 30C and 200C potencies of ten dynamized medicines against Dalton's Lymphoma Ascites, Ehrlich's Ascites Carcinoma, lung fibroblast (L929 and Chinese Hamster Ovary (CHO cell lines and compared activity with their mother tinctures during short-term and long-term cell culture. The effect of dynamized medicines to induce apoptosis was also evaluated and we studied how dynamized medicines affected genes expressed during apoptosis. Mother tinctures as well as some dynamized medicines showed significant cytotoxicity to cells during short and long-term incubation. Potentiated alcohol control did not produce any cytotoxicity at concentrations studied. The dynamized medicines were found to inhibit CHO cell colony formation and thymidine uptake in L929 cells and those of Thuja, Hydrastis and Carcinosinum were found to induce apoptosis in DLA cells. Moreover, dynamized Carcinosinum was found to induce the expression of p53 while dynamized Thuja produced characteristic laddering pattern in agarose gel electrophoresis of DNA. These results indicate that dynamized medicines possess cytotoxic as well as apoptosis-inducing properties.

  17. Expanding intestinal stem cells in culture

    NARCIS (Netherlands)

    Heo, Inha; Clevers, Hans

    2015-01-01

    Culturing intestinal stem cells into 3D organoids results in heterogeneous cell populations, reflecting the in vivo cell type diversity. In a recent paper published in Nature, Wang et al. established a culture condition for a highly homogeneous population of intestinal stem cells.

  18. Effects of cysteamine on pituitary, MTTW15 tumor, and serum prolactin levels measured by rat lymphoma cell bioassay and radioimmunoassay

    International Nuclear Information System (INIS)

    Cysteamine (CSH), a sulfhydryl compound, reduces both serum and anterior pituitary (AP) PRL measured by RIA. We have used the Nb2 lymphoma cell bioassay (BIO) for PRL to evaluate possible CSH-related changes in PRL levels in sera and tissues of male and MtTW15 mammosomatotropic tumor-bearing female rats. Experimental animals received a single sc injection of CSH (300 mg/kg), and samples were collected 0.5-24 h later. Since CSH and serum from CSH rats were toxic in BIO, samples were dialyzed before assay. All samples were evaluated for PRL and GH by RIA as well. A significant decrease (P less than 0.05) in BIO serum PRL was evident in male rats 0.5 h after CSH; levels remained low for 24 h. Serum PRL by RIA was significantly depressed at 4 h but not at 0.5 h or 24 h. PRL in AP extracts was decreased (60-90%) at all times by BIO and RIA. Significant decreases of BIO- and RIA-detectable PRL were recorded in serum and tissues (AP and tumors) at 4 h in tumor rats. Sequentially bled (0.5-4 h) CSH-treated tumor-bearing rats showed 50% and 80% reductions in serum PRL at 1 and 4 h by both BIO and RIA. CSH had no effect on GH levels in sera and tissues of any animal studied at any time interval. Our results substantiate earlier reports on CSH-induced decreases in RIA-detectable PRL. They show that such changes cannot be attributed to assay effects alone, as significant decreases in circulating and stored PRL (both AP and tumor) were evident by BIO. Results with tissue extracts were the most dramatic. They suggest an action of CSH or a metabolic intermediate with stored PRL which reduces both extractable PRL and hormone release. Such an effect of CSH on PRL extraction has been suggested by others. Whatever the mechanism, it appears to be relatively specific, since GH cells were not affected

  19. Cell Culture as an Alternative in Education.

    Science.gov (United States)

    Nardone, Roland M.

    1990-01-01

    Programs that are intended to inform and provide "hands-on" experience for students and to facilitate the introduction of cell culture-based laboratory exercises into the high school and college laboratory are examined. The components of the CellServ Program and the Cell Culture Toxicology Training Programs are described. (KR)

  20. In vitro culture of human thyroid cells

    International Nuclear Information System (INIS)

    Procedures for establishing primary cultures of human thyroid tissue are described. Tissues removed surgically from patients with papillary carcinoma (PC), follicular adenoma (FA), or hyperthyroidism were grown in culture. In addition, normal cells were separated from the margins of excised tumors and were also cultured. For each gram of thyroid tissue cultured, more than 1 x 105 cells attached to culture dishes. A mixture of 2.5 % fetal bovine serum supplemented with insulin, hydrocortisone, transferrin, glycl-1-histidyl-L-lysine acetate, somatostatin and epidermal growth factor was added to nutrient media containing equal parts of Ham's F-12 and minimum essential medium (αMEM). Complete medium selectively supported epithelial cell growth while restricting fibroblast cell growth, especially during the first two weeks of the primary culture. Cells were stimulated with thyroid stimulating hormone (TSH) and produced raised levels of cAMP and thyroid hormone (T3). Culture conditions that affected the response of cells to X-rays were identified. During the culture period, first and second passage cells were compared for differences in their radiosensitivities. In all cases, cells showed differences in their responses to radiation depending on the cell passage number. However, results of replicate experiments of first passage cells that were exposed to X-rays showed good agreement between experiments. This technique makes it possible to quantitate the effects of chemical and physical cytotoxic agents on proliferating human thyroid epithelial cells. (author)

  1. Determining Resistance of Toxoplasma gondii Oocysts to UV Disinfection Using Cell Culture and a Mouse Bioassay

    Science.gov (United States)

    The effect of UV exposure on Toxoplasma gondii oocysts has not been completely defined for use in water disinfection. This study evaluated irradiated oocysts by three assays: a SCID mouse biassay, an in vitro T. gondii oocyst plaque assay (TOP-assay), and a quantitative reverse-transcriptase real-t...

  2. Culture of Cells from Amphibian Embryos.

    Science.gov (United States)

    Stanisstreet, Martin

    1983-01-01

    Describes a method for in vitro culturing of cells from amphibian early embryos. Such cells can be used to demonstrate such properties of eukaryote cells as cell motility, adhesion, differentiation, and cell sorting into tissues. The technique may be extended to investigate other factors. (Author/JN)

  3. Advantages of embryogenic cell cultures of Gramineae

    International Nuclear Information System (INIS)

    Immature embryos and/or explants from very young leaves and inflorescences of 13 species and over 75 cultivars of Gramineae - including wheat, maize, rye, pearl millet, sugar-cane, Napier grass, Guinea grass, etc. - were used to initiate callus cultures. The cultures are white to yellowish white in colour, compact and contain small and thin-walled meristematic cells which are richly cytoplasmic, non-vacuolated and contain prominent starch grains. These embryogenic tissue cultures provide a long-term, highly reliable and efficient means of rapid mass clonal propagation by the formation of somatic embryos that arise from single cells. The cultures consist largely of cytologically normal diploid cells. During the process of plant regeneration via somatic embryogenesis, there is strong selection in favour of normal cells, so that plants recovered from such cultures neither exhibit any morphological abnormalities nor show any evidence of cytological changes in the number or structure of chromosomes. Embryogenic callus cultures have been used successfully to establish highly dispersed and friable cell-suspension cultures. These fast-growing cultures comprise groups of 2-6 embryogenic cells, which adhere together to form larger unorganized aggregates of up to about 75 cells, but do not contain any organized meristems or callus tissues. Plants were regenerated by somatic embryogenesis from embryogenic cell-suspension cultures of pearl millet, Guinea grass, sugar-cane and maize. Finally, embryogenic cell-suspension cultures are the only current source of totipotent protoplasts in Gramineae. Protoplasts isolated from such cultures have been successfully cultured to produce somatic embryos and plants in pearl millet, Guinea grass, Napier grass and sugar-cane. (author)

  4. Cell Suspension Culture of Neem Tree

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The establishment of suspension culture system for neem (Azadirachta indica A. Juss) cells and the suspension culture condition was studied. It shows that the neem cell suspension culture system was best in B5 liquid medium, 2.0~4.0mg/L NAA with direct spill method. Based on the integrated analysis of cell biomass, Azadirachtin content and productivity, the optimum culture conditions were B5 liquid medium, 2.0-4.0 mg/L NAA, 3% sucrose at 25 ℃. The optimum rotating speed of the shaker and broth content d...

  5. 9 CFR 101.6 - Cell cultures.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Cell cultures. 101.6 Section 101.6 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference...

  6. Callus and cell suspension cultures of carnation

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1972-01-01

    . Cell suspension cultures worked best in media containing 2,4-D in which they had a doubling time of about 2 days. Filtered suspensions were successfully plated on agar in petri dishes, but division was never observed in single cells. The cultures initiated roots at higher concentrations of IAA or NAA...

  7. Bioassays for the determination of nitrification inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Grunditz, Camilla

    1999-07-01

    Requirements for nitrogen reduction in wastewater treatment plants were introduced in Sweden in the early 1990's. This was a governmental move to reduce the nitrogen discharges to the Baltic and Kattegat in order to prevent eutrophication. The nitrification process in wastewater treatment plants is performed by nitrifying bacteria. These are susceptible to inhibition and it is of great importance that the influent water does not contain toxic compounds. Therefore, there is a need for assays for the determination of nitrification inhibition. This thesis describes the development and applications of such bioassays. Pure cultures of Nitrosomonas sp. and Nitrobacter sp. were isolated from activated sludge of a wastewater treatment plant. These cultures were used as test organisms in the development of bioassays for nitrification inhibition measurements. The assays are based on two different principles; cell suspensions of the bacteria, performed in test tubes, and mediated amperometric biosensors with the bacteria immobilised. Ammonia oxidation and nitrite oxidation are studied separately without interference from other organisms, which makes it easier to interpret the results. The cell suspension assays were applied to samples of industrial and municipal wastewater. The Nitrosomonas and Nitrobacter assays showed to have different inhibition patterns. A large percentage of the Swedish municipal wastewater treatment plants were found to receive inhibitory influent water, but the inhibition level was generally low. Compared to an assay based on activated sludge, the screening method, the pure culture assays found more samples of influent water strongly inhibitory or stimulating. The highest correlation was found between the screening method and the Nitrosomonas assay. The Nitrobacter assay was found to be the most sensitive method. Assessment of toxicity of a number of chemical substances was studied using the biosensors, together with the cell suspension assays

  8. Plant cell cultures and their biotechnological potential

    Energy Technology Data Exchange (ETDEWEB)

    Barz, W.; Ellis, B.E.

    1981-01-01

    The potential of plant cell suspension cultures for the biotechnological production of high-cost, plant-specific compounds is critically evaluated. The basic roles of nutrient media and phytohormones are described followed by a description of the recent progress in mass cultivation of plant cell cultures as measured by biomass and doubling time. The accumulation of secondary constituents in cell cultures is reviewed and methods for the selection of high-producing strains are described. The essential features of the selection strategy are the establishment of cell cultures from high-producing plants and a sensitive assay (e.g. radio-immunoassay) for the screening of microcolonies grown on petri dishes. The accumulation of biosynthetic intermediates of secondary constituents in cell culture strains will possibly lead to the isolation of novel compounds.

  9. Studying cell-cell communication in co-culture

    OpenAIRE

    Bogdanowicz, Danielle R.; Lu, Helen H.

    2013-01-01

    Heterotypic and homotypic cellular interactions are essential for biological function, and co-culture models are versatile tools for investigating these cellular interactions in vitro. Physiologically relevant co-culture models have been used to elucidate the effects of cell-cell physical contact and/or secreted factors, as well as the influence of substrate geometry and interaction scale on cell response. Identifying the relative contribution of each cell population to co-culture is often ex...

  10. Cell Culture for Production of Insecticidal Viruses.

    Science.gov (United States)

    Reid, Steven; Chan, Leslie C L; Matindoost, Leila; Pushparajan, Charlotte; Visnovsky, Gabriel

    2016-01-01

    While large-scale culture of insect cells will need to be conducted using bioreactors up to 10,000 l scale, many of the main challenges for cell culture-based production of insecticidal viruses can be studied using small-scale (20-500 ml) shaker/spinner flasks, either in free suspension or using microcarrier-based systems. These challenges still relate to the development of appropriate cell lines, stability of virus strains in culture, enhancing virus yields per cell, and the development of serum-free media and feeds for the desired production systems. Hence this chapter presents mainly the methods required to work with and analyze effectively insect cell systems using small-scale cultures. Outlined are procedures for quantifying cells and virus and for establishing frozen cells and virus stocks. The approach for maintaining cell cultures and the multiplicity of infection (MOI) and time of infection (TOI) parameters that should be considered for conducting infections are discussed.The methods described relate, in particular, to the suspension culture of Helicoverpa zea and Spodoptera frugiperda cell lines to produce the baculoviruses Helicoverpa armigera nucleopolyhedrovirus, HearNPV, and Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, AgMNPV, respectively, and the production of the nonoccluded Oryctes nudivirus, OrNV, using an adherent coleopteran cell line. PMID:27565495

  11. Emulsions Containing Perfluorocarbon Support Cell Cultures

    Science.gov (United States)

    Ju, Lu-Kwang; Lee, Jaw Fang; Armiger, William B.

    1990-01-01

    Addition of emulsion containing perfluorocarbon liquid to aqueous cell-culture medium increases capacity of medium to support mammalian cells. FC-40 Fluorinert (or equivalent) - increases average density of medium so approximately equal to that of cells. Cells stay suspended in medium without mechanical stirring, which damages them. Increases density enough to prevent cells from setting, and increases viscosity of medium so oxygen bubbled through it and nutrients stirred in with less damage to delicate cells.

  12. Constructing a High Density Cell Culture System

    Science.gov (United States)

    Spaulding, Glenn F. (Inventor)

    1996-01-01

    An annular culture vessel for growing mammalian cells is constructed in a one piece integral and annular configuration with an open end which is closed by an endcap. The culture vessel is rotatable about a horizontal axis by use of conventional roller systems commonly used in culture laboratories. The end wall of the endcap has tapered access ports to frictionally and sealingly receive the ends of hypodermic syringes. The syringes permit the introduction of fresh nutrient and withdrawal of spent nutrients. The walls are made of conventional polymeric cell culture material and are subjected to neutron bombardment to form minute gas permeable perforations in the walls.

  13. A cell-based fascin bioassay identifies compounds with potential anti-metastasis or cognition-enhancing functions

    Directory of Open Access Journals (Sweden)

    Robert Kraft

    2013-01-01

    The actin-bundling protein fascin is a key mediator of tumor invasion and metastasis and its activity drives filopodia formation, cell-shape changes and cell migration. Small-molecule inhibitors of fascin block tumor metastasis in animal models. Conversely, fascin deficiency might underlie the pathogenesis of some developmental brain disorders. To identify fascin-pathway modulators we devised a cell-based assay for fascin function and used it in a bidirectional drug screen. The screen utilized cultured fascin-deficient mutant Drosophila neurons, whose neurite arbors manifest the ‘filagree’ phenotype. Taking a repurposing approach, we screened a library of 1040 known compounds, many of them FDA-approved drugs, for filagree modifiers. Based on scaffold distribution, molecular-fingerprint similarities, and chemical-space distribution, this library has high structural diversity, supporting its utility as a screening tool. We identified 34 fascin-pathway blockers (with potential anti-metastasis activity and 48 fascin-pathway enhancers (with potential cognitive-enhancer activity. The structural diversity of the active compounds suggests multiple molecular targets. Comparisons of active and inactive compounds provided preliminary structure-activity relationship information. The screen also revealed diverse neurotoxic effects of other drugs, notably the ‘beads-on-a-string’ defect, which is induced solely by statins. Statin-induced neurotoxicity is enhanced by fascin deficiency. In summary, we provide evidence that primary neuron culture using a genetic model organism can be valuable for early-stage drug discovery and developmental neurotoxicity testing. Furthermore, we propose that, given an appropriate assay for target-pathway function, bidirectional screening for brain-development disorders and invasive cancers represents an efficient, multipurpose strategy for drug discovery.

  14. 3D Cell Culture in Alginate Hydrogels

    Directory of Open Access Journals (Sweden)

    Therese Andersen

    2015-03-01

    Full Text Available This review compiles information regarding the use of alginate, and in particular alginate hydrogels, in culturing cells in 3D. Knowledge of alginate chemical structure and functionality are shown to be important parameters in design of alginate-based matrices for cell culture. Gel elasticity as well as hydrogel stability can be impacted by the type of alginate used, its concentration, the choice of gelation technique (ionic or covalent, and divalent cation chosen as the gel inducing ion. The use of peptide-coupled alginate can control cell–matrix interactions. Gelation of alginate with concomitant immobilization of cells can take various forms. Droplets or beads have been utilized since the 1980s for immobilizing cells. Newer matrices such as macroporous scaffolds are now entering the 3D cell culture product market. Finally, delayed gelling, injectable, alginate systems show utility in the translation of in vitro cell culture to in vivo tissue engineering applications. Alginate has a history and a future in 3D cell culture. Historically, cells were encapsulated in alginate droplets cross-linked with calcium for the development of artificial organs. Now, several commercial products based on alginate are being used as 3D cell culture systems that also demonstrate the possibility of replacing or regenerating tissue.

  15. Human hematopoietic cell culture, transduction, and analyses

    DEFF Research Database (Denmark)

    Bonde, Jesper; Wirthlin, Louisa; Kohn, Donald B;

    2008-01-01

    This unit provides methods for introducing genes into human hematopoietic progenitor cells. The Basic Protocol describes isolation of CD34(+) cells, transduction of these cells with a retroviral vector on fibronectin-coated plates, assaying the efficiency of transduction, and establishing long......-term cultures. Support protocols describe methods for maintenance of vector-producing fibroblasts (VPF) and supernatant collection from these cells, screening medium components for the ability to support hematopoietic cell growth, and establishing colonies from long-term cultures. Other protocols provide PCR...

  16. Methods for Maintaining Insect Cell Cultures

    OpenAIRE

    Dwight E. Lynn

    2002-01-01

    Insect cell cultures are now commonly used in insect physiology, developmental biology, pathology, and molecular biology. As the field has advanced from methods development to a standard procedure, so has the diversity of scientists using the technique. This paper describes methods that are effective for maintaining various insect cell lines. The procedures are differentiated between loosely or non-attached cell strains, attached cell strains, and strongly adherent cell strains.

  17. Callus and cell suspension cultures of carnation

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1972-01-01

    Callus cultures of carnation, Dianthus caryophyllus L. ev. G. J. Sim, were grown on a synthetic medium of half strength Murashige and Skoog salts, 3 % sucrose, 100 mg/l of myo-inositol, 0.5 mg/l each of thiamin, HCl, pyridoxin, HCl and nicotinic acid and 10 g/l agar. Optimal concentrations of....... Cell suspension cultures worked best in media containing 2,4-D in which they had a doubling time of about 2 days. Filtered suspensions were successfully plated on agar in petri dishes, but division was never observed in single cells. The cultures initiated roots at higher concentrations of IAA or NAA...

  18. Cell culture models for study of differentiated adipose cells

    OpenAIRE

    Clynes, Martin

    2014-01-01

    Adipose cells are an important source of mesenchymal stem cells and are important for direct use in research on lipid metabolism and obesity. In addition to use of primary cultures, there is increasing interest in other sources of larger numbers of cells, using approaches including induced pluripotent stem cell differentiation and viral immortalisation.

  19. Human cell culture in a space bioreactor

    Science.gov (United States)

    Morrison, Dennis R.

    1988-01-01

    Microgravity offers new ways of handling fluids, gases, and growing mammalian cells in efficient suspension cultures. In 1976 bioreactor engineers designed a system using a cylindrical reactor vessel in which the cells and medium are slowly mixed. The reaction chamber is interchangeable and can be used for several types of cell cultures. NASA has methodically developed unique suspension type cell and recovery apparatus culture systems for bioprocess technology experiments and production of biological products in microgravity. The first Space Bioreactor was designed for microprocessor control, no gaseous headspace, circulation and resupply of culture medium, and slow mixing in very low shear regimes. Various ground based bioreactors are being used to test reactor vessel design, on-line sensors, effects of shear, nutrient supply, and waste removal from continuous culture of human cells attached to microcarriers. The small Bioreactor is being constructed for flight experiments in the Shuttle Middeck to verify systems operation under microgravity conditions and to measure the efficiencies of mass transport, gas transfer, oxygen consumption and control of low shear stress on cells.

  20. Melphalan metabolism in cultured cells

    International Nuclear Information System (INIS)

    Procedures are presented for the adaptation of reversed-phase-HPLC methods to accomplish separation and isolation of the cancer therapeutic drug melphalan (L-phenylalanine mustard) and its metabolic products from whole cells. Five major degradation products of melphalan were observed following its hydrolysis in phosphate buffer in vitro. The two most polar of these products (or modifications of them) were also found in the cytosol of Chinese hamster CHO cells. The amounts of these two polar products (shown not to be mono- or dihydroxymelphalan) were significantly changed by the pretreatment of cells with ZnC12, one being increased in amount while the other was reduced to an insignificant level. In ZnC12-treated cells, there was also an increased binding of melphalan (or its derivatives) to one protein fraction resolved by gel filtration-HPLC. These observations suggest that changes in polar melphalan products, and perhaps their interaction with a protein, may by involved in the reduction of melphalan cytotoxicity observed in ZnC12-treated cells. While ZnC12 is also known to increase the level of glutathione in cells, no significant amounts of glutathione-melphalan derivatives of the type formed non-enzymatically in vitro could be detected in ZnC12-treated or untreated cells. Formation of derivatives of melphalan with glutathione catabolic products in ZnC12-treated cells has not yet been eliminated, however. 17 refs., 5 figs., 1 tab

  1. Increasing cell culture population doublings for long-term growth of finite life span human cell cultures

    Science.gov (United States)

    Stampfer, Martha R; Garbe, James C

    2015-02-24

    Cell culture media formulations for culturing human epithelial cells are herein described. Also described are methods of increasing population doublings in a cell culture of finite life span human epithelial cells and prolonging the life span of human cell cultures. Using the cell culture media disclosed alone and in combination with addition to the cell culture of a compound associated with anti-stress activity achieves extended growth of pre-stasis cells and increased population doublings and life span in human epithelial cell cultures.

  2. Increasing cell culture population doublings for long-term growth of finite life span human cell cultures

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R.; Garbe, James C.

    2016-06-28

    Cell culture media formulations for culturing human epithelial cells are herein described. Also described are methods of increasing population doublings in a cell culture of finite life span human epithelial cells and prolonging the life span of human cell cultures. Using the cell culture media disclosed alone and in combination with addition to the cell culture of a compound associated with anti-stress activity achieves extended growth of pre-stasis cells and increased population doublings and life span in human epithelial cell cultures.

  3. [CO-CULTURE OF BOAR SPERMATOGONIAL CELLS WITH SERTOLI CELLS].

    Science.gov (United States)

    Savchenkova, I P; Vasil'eva, S A

    2016-01-01

    In the present study, we developed in vitro culture conditions using co-culture of boar spermatogonial cells with Sertoli cells. Testes from 60-day-old crossbred boar were used. A spermatogonia-enriched culture was achieved by enzymatic digestion method and purification by density gradient centrifugation using a discontinuous Percoll gradient and differentiated adherence technique. Lipid drops were detected in isolated Sertoli cells by Oil Red O staining. We have found that the cultivation of boar spermatogonia in the presence of Sertoli cells (up to 35 days) leads to their differentiation as well as in vivo in testis. Association of cells in groups, formation of chains and suspension clusters of the spermatogenic cells were observed on the 10th day. Spermatogonial cellular colonies were noted at the same time. These cellular colonies were analyzed for the expression of genes: Nanog and Plzf in RT PCR. The expression of the Nanog gene in the experimental cellular clones obtained by short-term culture of spermatogonial cells in the presence of Sertoli cells was 200 times higher than the expression of this gene in the freshly isolated spermatogonial cells expression was found in freshly isolated germ cells and in cellular clones derived in vitro. We have found that, in the case of longer cultivation of these cells on Sertoli cells, in vitro process of differentiation of germ cells and formation of single mobile boar spermatozoa occurs at 30-33 days. Cellular population is heterogeneous at this stage. Spermatogenic differentiation in vitro without Sertoli cells stays on the 7th day of cultivation. The results show that co-culture of boar spermatogonia-enriched cells with Sertoli cells can induce their differentiation into spermatozoa in vitro and facilitate obtaining of porcine germ cell culture. PMID:27228660

  4. Cell culture experiments planned for the space bioreactor

    Science.gov (United States)

    Morrison, Dennis R.; Cross, John H.

    1987-01-01

    Culturing of cells in a pilot-scale bioreactor remains to be done in microgravity. An approach is presented based on several studies of cell culture systems. Previous and current cell culture research in microgravity which is specifically directed towards development of a space bioprocess is described. Cell culture experiments planned for a microgravity sciences mission are described in abstract form.

  5. Bioassay of Estrogenic Activity of Effluent and Influent in a Farm Wastewater Treatment Plant Using an in vitro Recombinant Assay with Yeast Cells

    Institute of Scientific and Technical Information of China (English)

    XIANG-MING LI; FANG-NI LUO; GuI-XIA LIU; PING-TING ZHU

    2008-01-01

    Objective Environmental estrogens at an elevated concentration are known to produce adverse effects on human and animal life. However, the majority of researches have been focused on ndustrial discharges, while the impact of livestock wastes as a source of endocrine disrupters in aquatic environments has been rarely elucidated. In order to investigate the contribution of environmental estrogens from livestock, the estrogenic activity in water samples from a farm wastewater treatment plant was analyzed by a recombinant yeast screening method. Methods The extracts prepared from 15 selected water samples from the farm wastewater treatment plant, among which 6 samples were from pre-treatment section (influents) and 9 from post-treatment section (effluents), were analyzed for estrogenic activity by cellar bioassay. Yeast cells transfected with the expression plasmid of human estrogen receptor and the Lac Z reporter plasmid encoding β-galactossidase, were used to measure the estrogen-like compounds in the farm wastewater treatment plant. Results The wastewater samples from influents showed a higher estrogenic potency than the effluent samples showing a low induction of β-galactossidase relative to solvent control condition. By comparison with a standard curve for 1713-estradiol (E2), estrogenic potency in water samples from the influents was calculated as E2-equivalent and ranged from 0.1 to 150 pM E2-equivalent. The estrogenic potency in water samples from the effluents was significantly lower than that in the influents, and 7 water samples had less detectable limit in the total of 9 samples. Conclusion Yeast bioassay of estrogenic activity in most of the samples from the farm wastewater after disposal by traditional sewage treatment showed negative results.

  6. A biocompatible micro cell culture chamber (mu CCC) for the culturing and on-line monitoring of eukaryote cells

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Petronis, Sarunas; Jørgensen, Anders Michael;

    2006-01-01

    We have previously shown that a polymeric (PMMA) chip with medium perfusion and integrated heat regulation provides sufficiently precise heat regulation, pH-control and medium exchange to support cell growth for weeks. However, it was unclear how closely the cells cultured in the chip resembled...... cells cultured in the culture flask. In the current study, gene expression profiles of cells cultured in the chip were compared with gene expression profiles of cells cultured in culture flasks. The results showed that there were only two genes that were differently expressed in cells grown in the cell...... culture chip compared to cell culture flasks. The cell culture chip could without further modification support cell growth of two other cell lines. Light coming from the microscope lamp during optical recordings of the cells was the only external factor identified, that could have a negative effect...

  7. A biocompatible micro cell culture chamber (microCCC) for the culturing and on-line monitoring of eukaryote cells

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Petronis, Sarunas; Jørgensen, A M;

    2006-01-01

    We have previously shown that a polymeric (PMMA) chip with medium perfusion and integrated heat regulation provides sufficiently precise heat regulation, pH-control and medium exchange to support cell growth for weeks. However, it was unclear how closely the cells cultured in the chip resembled...... cells cultured in the culture flask. In the current study, gene expression profiles of cells cultured in the chip were compared with gene expression profiles of cells cultured in culture flasks. The results showed that there were only two genes that were differently expressed in cells grown in the cell...... culture chip compared to cell culture flasks. The cell culture chip could without further modification support cell growth of two other cell lines. Light coming from the microscope lamp during optical recordings of the cells was the only external factor identified, that could have a negative effect...

  8. Propagation of prions causing synucleinopathies in cultured cells

    OpenAIRE

    Woerman, AL; StÖhr, J.; Aoyagi, A; Rampersaud, R; Krejciova, Z; Watts, JC; T. Ohyama; Patel, S; Widjaja, K; Oehler, A; Sanders, DW; Diamond, MI; Seeley, WW; Middleton, LT; Gentleman, SM

    2015-01-01

    © 2015, National Academy of Sciences. All rights reserved. Increasingly, evidence argues that many neurodegenerative diseases, including progressive supranuclear palsy (PSP), are caused by prions, which are alternatively folded proteins undergoing self-propagation. In earlier studies, PSP prions were detected by infecting human embryonic kidney (HEK) cells expressing a tau fragment [TauRD(LM)] fused to yellow fluorescent protein (YFP). Here, we report on an improved bioassay using selective p...

  9. An animal cell culture: Advance technology for modern research

    OpenAIRE

    Sarita Khare; Rajeev Nema

    2012-01-01

    At the present time animal cell culture is more significant and multifarious application tool for current research streams. A lot of field assorted from animal cell culture such: stem cell biology, IVF technology, cancer cell biology, monoclonal antibody production, recombinant protein production, gene therapy, vaccine manufacturing, novel drug selection and improvement. In this review conclude animal cell culture as well as its requirements

  10. Wnt-Dependent Control of Cell Polarity in Cultured Cells.

    Science.gov (United States)

    Runkle, Kristin B; Witze, Eric S

    2016-01-01

    The secreted ligand Wnt5a regulates cell polarity and polarized cell movement during development by signaling through the poorly defined noncanonical Wnt pathway. Cell polarity regulates most aspects of cell behavior including the organization of apical/basolateral membrane domains of epithelial cells, polarized cell divisions along a directional plane, and front rear polarity during cell migration. These characteristics of cell polarity allow coordinated cell movements required for tissue formation and organogenesis during embryonic development. Genetic model organisms have been used to identify multiple signaling pathways including Wnt5a that are required to establish cell polarity and regulate polarized cell behavior. However, the downstream signaling events that regulate these complex cellular processes are still poorly understood. The methods below describe assays to study Wnt5a-induced cell polarity in cultured cells, which may facilitate our understanding of these complex signaling pathways. PMID:27590152

  11. Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor

    Science.gov (United States)

    Parks, Kelsey

    2009-01-01

    Space flight has been shown to alter the astronauts immune systems. Because immune performance is complex and reflects the influence of multiple organ systems within the host, scientists sought to understand the potential impact of microgravity alone on the cellular mechanisms critical to immunity. Lymphocytes and their differentiated immature form, lymphoblasts, play an important and integral role in the body's defense system. T cells, one of the three major types of lymphocytes, play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors. Reported studies have shown that spaceflight can affect the expression of cell surface markers. Cell surface markers play an important role in the ability of cells to interact and to pass signals between different cells of the same phenotype and cells of different phenotypes. Recent evidence suggests that cell-cycle regulators are essential for T-cell function. To trigger an effective immune response, lymphocytes must proliferate. The objective of this project is to investigate the changes in growth of human cells cultured in rotating bioreactors and to measure the growth rate and the cell cycle distribution for different human cell types. Human lymphocytes and lymphoblasts will be cultured in a bioreactor to simulate aspects of microgravity. The bioreactor is a cylindrical culture vessel that incorporates the aspects of clinostatic rotation of a solid fluid body around a horizontal axis at a constant speed, and compensates gravity by rotation and places cells within the fluid body into a sustained free-fall. Cell cycle progression and cell proliferation of the lymphocytes will be measured for a number of days. In addition, RNA from the cells will be isolated for expression of genes related in cell cycle regulations.

  12. Integrated biosensors for cell culture monitoring

    OpenAIRE

    De Micheli, Giovanni; Boero, Cristina; Olivo, Jacopo; Carrara, Sandro

    2014-01-01

    Biosensors for endogenous compounds, such as glucose and lactate, are applied to monitor cell cultures. Cells can be cultivated for several purposes, such as understanding and modeling some biological mechanisms, the development of new drugs and therapies, and in the field of regenerative medicine. We have realized a self-contained monitoring system with remote readout. Metabolite detection is based on oxidases immobilized onto carbon nanotubes. We calibrate the system for glucose and lactate...

  13. Cell culture from sponges: pluripotency and immortality

    NARCIS (Netherlands)

    Caralt Bosch, de S.; Uriz, M.J.; Wijffels, R.H.

    2007-01-01

    Sponges are a source of compounds with potential pharmaceutical applications. In this article, methods of sponge cell culture for production of these bioactive compounds are reviewed, and new approaches for overcoming the problem of metabolite supply are examined. The use of embryos is proposed as a

  14. THE METHODS FOR MAINTAINING INSECT CELL CULTURES

    Science.gov (United States)

    Insect cell cultures are now commonly used in insect physiology, developmental biology, pathology, and molecular biology. As the field has advanced from a methods development to a standard procedure, so has the diversity of scientists using the technique. This paper describes techniques that are e...

  15. ANTHOCYANIN (ACN) STABILITY IN CELL CULTURE MEDIA

    Science.gov (United States)

    Anthocyanins (ACNs) are potential oxygen radical scavengers that have coronary vasoactive and vasoprotective properties. Cell or tissue culture systems have been used to examine the bioactivity and mechanisms of action of ACNs on the vascular system. However, due to their unique chemical structure, ...

  16. Cell Culture Microfluidic Biochips: Experimental Throughput Maximization

    DEFF Research Database (Denmark)

    Minhass, Wajid Hassan; Pop, Paul; Madsen, Jan;

    2011-01-01

    Microfluidic biochips offer a promising alternative to a conventional biochemical laboratory, integrating all necessary functionalities on-chip in order to perform biochemical applications. Researchers have started to propose computer-aided design tools for the synthesis of such biochips. Our focus...... Annealing metaheuristic for experimental design generation for the cell culture microfluidic biochips, and we have evaluated our approach using multiple experimental setups....

  17. 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

  18. Cell culture models using rat primary alveolar type I cells.

    Science.gov (United States)

    Downs, Charles A; Montgomery, David W; Merkle, Carrie J

    2011-10-01

    There is a lack of cell culture models using primary alveolar type I (AT I) cells. The purpose of this study was to develop cell culture models using rat AT I cells and microvascular endothelial cells from the lung (MVECL). Two types of model systems were developed: single and co-culture systems; additionally a 3-dimensional model system was developed. Pure AT I cell (96.3 ± 2.7%) and MVECL (97.9 ± 1.1%) preparations were used. AT I cell morphology, mitochondrial number and distribution, actin filament arrangement and number of apoptotic cells at confluence, and telomere attrition were characterized. AT I cells maintained their morphometric characteristics through at least population doubling (PD) 35, while demonstrating telomere attrition through at least PD 100. Furthermore, AT I cells maintained the expression of their specific markers, T1α and AQ-5, through PD 42. For the co-cultures, AT I cells were grown on the top and MVECL were grown on the bottom of fibronectin-coated 24-well Transwell Fluroblok™ filter inserts. Neither cell type transmigrated the 1 μm pores. Additionally, AT I cells were grown in a thick layer of Matrigel(®) to create a 3-dimensional model in which primary AT I cells form ring-like structures that resemble an alveolus. The development of these model systems offers the opportunities to investigate AT I cells and their interactions with MVECL in response to pharmacological interventions and in the processes of disease, repair and regeneration. PMID:21624488

  19. Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity

    Science.gov (United States)

    Hatton, J. P.; Lewis, M. L.; Roquefeuil, S. B.; Chaput, D.; Cazenave, J. P.; Schmitt, D. A.

    1998-01-01

    The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European 'Biorack' provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the 'Biorack' facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatant in-flight), injection port, and supernatant collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatant, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground

  20. Dynamic cell culture system (7-IML-1)

    Science.gov (United States)

    Cogoli, Augusto

    1992-01-01

    This experiment is one of the Biorack experiments being flown on the International Microgravity Laboratory 1 (MIL-1) mission as part of an investigation studying cell proliferation and performance in space. One of the objectives of this investigation is to assess the potential benefits of bioprocessing in space with the ultimate goal of developing a bioreactor for continuous cell cultures in space. This experiment will test the operation of an automated culture chamber that was designed for use in a Bioreactor in space. The device to be tested is called the Dynamic Cell Culture System (DCCS). It is a simple device in which media are renewed or chemicals are injected automatically, by means of osmotic pumps. This experiment uses four Type I/O experiment containers. One DCCS unit, which contains a culture chamber with renewal of medium and a second chamber without a medium supply fits in each container. Two DCCS units are maintained under zero gravity conditions during the on-orbit period. The other two units are maintained under 1 gh conditions in a 1 g centrifuge. The schedule for incubator transfer is given.

  1. Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices

    OpenAIRE

    Halldórsson, Skarphédinn; Lucumi Moreno, Edinson; Gómez-Sjöberg, Rafael; Fleming, Ronan MT

    2015-01-01

    Culture of cells using various microfluidic devices is becoming more common within experimental cell biology. At the same time, a technological radiation of microfluidic cell culture device designs is currently in progress. Ultimately, the utility of microfluidic cell culture will be determined by its capacity to permit new insights into cellular function. Especially insights that would otherwise be difficult or impossible to obtain with macroscopic cell culture in traditional polystyrene dis...

  2. In vitro bioassays for anticancer drug screening: effects of cell concentration and other assay parameters on growth inhibitory activity.

    Science.gov (United States)

    Lieberman, M M; Patterson, G M; Moore, R E

    2001-11-01

    In vitro growth inhibition assays were performed using human cancer cell lines at various concentrations with experimental anticancer drugs such as the cryptophycins and other cytotoxins. The effect of variations in assay parameters on the observed growth inhibition of these anticancer therapeutic agents was determined. The results demonstrated that the observed inhibitory activity of these compounds varied inversely with the cell concentrations used. The observed differences in activity between different cytotoxins were not necessarily proportionate. Thus, the relative activities of two toxins also varied with cell concentration. Furthermore, the sensitivity of these cell lines to the cytostatic purine analog, 6-mercaptopurine (used as a control), varied with cell concentration as well. The activity of this compound was dependent on the medium used for cell growth, yielding good activity in Eagle's minimum essential medium, but not in Ham's F-12 (Kaigin) medium. Moreover, growth inhibition by cryptophycin as well as 6-mercaptopurine was also dependent on the serum concentration in the medium. Finally, the sensitivity of the cancer cell lines to various organic solvents commonly used as drug vehicles for in vitro testing, such as ethanol, dimethylformamide, and dimethylsulfoxide, was likewise found to vary inversely with cell concentration. PMID:11578805

  3. Prevention and Detection of Mycoplasma Contamination in Cell Culture

    Directory of Open Access Journals (Sweden)

    Parvaneh Farzaneh

    2012-01-01

    Full Text Available One of the main problems in cell culture is mycoplasma infection. It can extensively affectcell physiology and metabolism. As the applications of cell culture increase in research,industrial production and cell therapy, more concerns about mycoplasma contaminationand detection will arise. This review will provide valuable information about: 1. the waysin which cells are contaminated and the frequency and source of mycoplasma species incell culture; 2. the ways to prevent mycoplasma contamination in cell culture; 3. the importanceof mycoplasma tests in cell culture; 4. different methods to identify mycoplasmacontamination; 5. the consequences of mycoplasma contamination in cell culture and 6.available methods to eliminate mycoplasma contamination. Awareness about the sourcesof mycoplasma and pursuing aseptic techniques in cell culture along with reliable detectionmethods of mycoplasma contamination can provide an appropriate situation to preventmycoplasma contamination in cell culture.

  4. Cell culture: Progenitor cells from human brain after death

    Science.gov (United States)

    Palmer, Theo D.; Schwartz, Philip H.; Taupin, Philippe; Kaspar, Brian; Stein, Stuart A.; Gage, Fred H.

    2001-05-01

    Culturing neural progenitor cells from the adult rodent brain has become routine and is also possible from human fetal tissue, but expansion of these cells from postnatal and adult human tissue, although preferred for ethical reasons, has encountered problems. Here we describe the isolation and successful propagation of neural progenitor cells from human postmortem tissues and surgical specimens. Although the relative therapeutic merits of adult and fetal progenitor cells still need to be assessed, our results may extend the application of these progenitor cells in the treatment of neurodegenerative diseases.

  5. Mouse cell culture - Methods and protocols

    Directory of Open Access Journals (Sweden)

    CarloAlberto Redi

    2010-12-01

    Full Text Available The mouse is, out of any doubt, the experimental animal par excellence for many many colleagues within the scientific community, notably for those working in mammalian biology (in a broad sense, from basic genetic to modeling human diseases, starting at least from 1664 Robert Hooke experiments on air’s propertyn. Not surprising then that mouse cell cultures is a well established field of research itself and that there are several handbooks devoted to this discipline. Here, Andrew Ward and David Tosh provide a necessary update of the protocols currently needed. In fact, nearly half of the book is devoted to stem cells culture protocols, mainly embryonic, from a list of several organs (kidney, lung, oesophagus and intestine, pancreas and liver to mention some........

  6. Differentiation of mammalian skeletal muscle cells cultured on microcarrier beads in a rotating cell culture system

    Science.gov (United States)

    Torgan, C. E.; Burge, S. S.; Collinsworth, A. M.; Truskey, G. A.; Kraus, W. E.

    2000-01-01

    The growth and repair of adult skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts. These cells are responsive to a variety of environmental cues, including mechanical stimuli. The overall goal of the research is to examine the role of mechanical signalling mechanisms in muscle growth and plasticity through utilisation of cell culture systems where other potential signalling pathways (i.e. chemical and electrical stimuli) are controlled. To explore the effects of decreased mechanical loading on muscle differentiation, mammalian myoblasts are cultured in a bioreactor (rotating cell culture system), a model that has been utilised to simulate microgravity. C2C12 murine myoblasts are cultured on microcarrier beads in a bioreactor and followed throughout differentiation as they form a network of multinucleated myotubes. In comparison with three-dimensional control cultures that consist of myoblasts cultured on microcarrier beads in teflon bags, myoblasts cultured in the bioreactor exhibit an attenuation in differentiation. This is demonstrated by reduced immunohistochemical staining for myogenin and alpha-actinin. Western analysis shows a decrease, in bioreactor cultures compared with control cultures, in levels of the contractile proteins myosin (47% decrease, p < 0.01) and tropomyosin (63% decrease, p < 0.01). Hydrodynamic measurements indicate that the decrease in differentiation may be due, at least in part, to fluid stresses acting on the myotubes. In addition, constraints on aggregate size imposed by the action of fluid forces in the bioreactor affect differentiation. These results may have implications for muscle growth and repair during spaceflight.

  7. Darwinian Evolution of Prions in Cell Culture*

    OpenAIRE

    Li, Jiali; Browning, Shawn; Mahal, Sukhvir P.; Oelschlegel, Anja M.; Weissmann, Charles

    2009-01-01

    Prions are infectious proteins consisting mainly of PrPSc, a β sheet-rich conformer of the normal host protein PrPC, and occur in different strains. Strain identity is thought to be encoded by PrPSc conformation. We found that biologically cloned prion populations gradually became heterogeneous by accumulating “mutants”, and selective pressures resulted in the emergence of different mutants as major constituents of the evolving population. Thus, when transferred from brain to cultured cells, ...

  8. Bioactive sugar surfaces for hepatocyte cell culture

    OpenAIRE

    Ambury, Rachael

    2010-01-01

    The primary objective of this study was to identify, develop and characterise a novel bioactive surface capable of binding hepatocytes and enabling the retention of hepatocyte-specific cell function during in-vitro culture. The materials were designed to exploit a unique characteristic of hepatocyte biology, with β-galactose moieties displayed to allow cellular adhesion via the specific asialoglycoprotein receptors (ASGP-R) found on hepatocytes. Hydrogels were created by modifying a commercia...

  9. Degradation of TNT by plant cell cultures

    Czech Academy of Sciences Publication Activity Database

    Podlipná, Radka; Nepovím, Aleš; Zeman, S.; Vágner, Martin; Vaněk, Tomáš

    Smolenice, 2003, s. 78-79. [Xenobiochemické sympózium /22./. Smolenice (SK), 09.06.2003-11.06.2003] R&D Projects: GA ČR GP206/02/P065; GA MŠk OC 837.10 Institutional research plan: CEZ:AV0Z5038910; CEZ:AV0Z4055905 Keywords : degradation * plant cell cultures Subject RIV: DK - Soil Contamination ; De-contamination incl. Pesticides

  10. Prevention and Detection of Mycoplasma Contamination in Cell Culture

    OpenAIRE

    Parvaneh Farzaneh; Laleh Nikfarjam

    2011-01-01

    One of the main problems in cell culture is mycoplasma infection. It can extensively affect cell physiology and metabolism. As the applications of cell culture increase in research, industrial production and cell therapy, more concerns about mycoplasma contamination and detection will arise. This review will provide valuable information about: 1. the ways in which cells are contaminated and the frequency and source of mycoplasma species in cell culture; 2. the ways to prevent mycoplasma conta...

  11. A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids

    Science.gov (United States)

    Madeddu, Denise; Cerino, Giulia; Falco, Angela; Frati, Caterina; Gallo, Diego; Deriu, Marco A.; Falvo D’Urso Labate, Giuseppe; Quaini, Federico; Audenino, Alberto; Morbiducci, Umberto

    2016-01-01

    A versatile bioreactor suitable for dynamic suspension cell culture under tunable shear stress conditions has been developed and preliminarily tested culturing cancer cell spheroids. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits the laminar hydrodynamics establishing within the culture chamber enabling dynamic cell suspension in an environment favourable to mass transport, under a wide range of tunable shear stress conditions. The design phase of the device has been supported by multiphysics modelling and has provided a comprehensive analysis of the operating principles of the bioreactor. Moreover, an explanatory example is herein presented with multiphysics simulations used to set the proper bioreactor operating conditions for preliminary in vitro biological tests on a human lung carcinoma cell line. The biological results demonstrate that the ultralow shear dynamic suspension provided by the device is beneficial for culturing cancer cell spheroids. In comparison to the static suspension control, dynamic cell suspension preserves morphological features, promotes intercellular connection, increases spheroid size (2.4-fold increase) and number of cycling cells (1.58-fold increase), and reduces double strand DNA damage (1.5-fold reduction). It is envisioned that the versatility of this bioreactor could allow investigation and expansion of different cell types in the future. PMID:27144306

  12. A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids.

    Science.gov (United States)

    Massai, Diana; Isu, Giuseppe; Madeddu, Denise; Cerino, Giulia; Falco, Angela; Frati, Caterina; Gallo, Diego; Deriu, Marco A; Falvo D'Urso Labate, Giuseppe; Quaini, Federico; Audenino, Alberto; Morbiducci, Umberto

    2016-01-01

    A versatile bioreactor suitable for dynamic suspension cell culture under tunable shear stress conditions has been developed and preliminarily tested culturing cancer cell spheroids. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits the laminar hydrodynamics establishing within the culture chamber enabling dynamic cell suspension in an environment favourable to mass transport, under a wide range of tunable shear stress conditions. The design phase of the device has been supported by multiphysics modelling and has provided a comprehensive analysis of the operating principles of the bioreactor. Moreover, an explanatory example is herein presented with multiphysics simulations used to set the proper bioreactor operating conditions for preliminary in vitro biological tests on a human lung carcinoma cell line. The biological results demonstrate that the ultralow shear dynamic suspension provided by the device is beneficial for culturing cancer cell spheroids. In comparison to the static suspension control, dynamic cell suspension preserves morphological features, promotes intercellular connection, increases spheroid size (2.4-fold increase) and number of cycling cells (1.58-fold increase), and reduces double strand DNA damage (1.5-fold reduction). It is envisioned that the versatility of this bioreactor could allow investigation and expansion of different cell types in the future. PMID:27144306

  13. Reversible gelling culture media for in-vitro cell culture in three-dimensional matrices

    Science.gov (United States)

    An, Yuehuei H.; Mironov, Vladimir A.; Gutowska, Anna

    2000-01-01

    A gelling cell culture medium useful for forming a three dimensional matrix for cell culture in vitro is prepared by copolymerizing an acrylamide derivative with a hydrophilic comonomer to form a reversible (preferably thermally reversible) gelling linear random copolymer in the form of a plurality of linear chains having a plurality of molecular weights greater than or equal to a minimum gelling molecular weight cutoff, mixing the copolymer with an aqueous solvent to form a reversible gelling solution and adding a cell culture medium to the gelling solution to form the gelling cell culture medium. Cells such as chondrocytes or hepatocytes are added to the culture medium to form a seeded culture medium, and temperature of the medium is raised to gel the seeded culture medium and form a three dimensional matrix containing the cells. After propagating the cells in the matrix, the cells may be recovered by lowering the temperature to dissolve the matrix and centrifuging.

  14. An Introductory Undergraduate Course Covering Animal Cell Culture Techniques

    Science.gov (United States)

    Mozdziak, Paul E.; Petitte, James N.; Carson, Susan D.

    2004-01-01

    Animal cell culture is a core laboratory technique in many molecular biology, developmental biology, and biotechnology laboratories. Cell culture is a relatively old technique that has been sparingly taught at the undergraduate level. The traditional methodology for acquiring cell culture training has been through trial and error, instruction when…

  15. Sodium 22+ washout from cultured rat cells

    International Nuclear Information System (INIS)

    The washout of Na+ isotopes from tissues and cells is quite complex and not well defined. To further gain insight into this process, we have studied 22Na+ washout from cultured Wistar rat skin fibroblasts and vascular smooth muscle cells (VSMCs). In these preparations, 22Na+ washout is described by a general three-exponential function. The exponential factor of the fastest component (k1) and the initial exchange rate constant (kie) of cultured fibroblasts decrease in magnitude in response to incubation in K+-deficient medium or in the presence of ouabain and increase in magnitude when the cells are incubated in a Ca++-deficient medium. As the magnitude of the kie declines (in the presence of ouabain) to the level of the exponential factor of the middle component (k2), 22Na+ washout is adequately described by a two-exponential function. When the kie is further diminished (in the presence of both ouabain and phloretin) to the range of the exponential factor of the slowest component (k3), the washout of 22Na+ is apparently monoexponential. Calculations of the cellular Na+ concentrations, based on the 22Na+ activity in the cells at the initiation of the washout experiments, and the medium specific activity agree with atomic absorption spectrometry measurements of the cellular concentration of this ion. Thus, all three components of 22Na+ washout from cultured rat cells are of cellular origin. Using the exponential parameters, compartmental analyses of two models (in parallel and in series) with three cellular Na+ pools were performed. The results indicate that, independent of the model chosen, the relative size of the largest Na+ pool is 92-93% in fibroblasts and approximately 96% in VSMCs. This pool is most likely to represent the cytosol

  16. Cell response of Chlamydomonas actinochloris culture to repeated microwave irradiation

    Directory of Open Access Journals (Sweden)

    OLESIA O. GRYGORIEVA

    2015-05-01

    Full Text Available Abstract. Grygorieva OO, Berezovsjka MA, Dacenko OI. 2015. Cell response of Chlamydomonas actinochloris culture to repeated microwave irradiation. Nusantara Bioscience 7: 38-42. Two cultures of Chlamydomonas actinochloris Deason et Bold in the lag-phase were exposed to the microwave irradiation. One of them (culture 1 was not treated beforehand, whereas the other (culture 2 was irradiated by microwaves 2 years earlier. The measurement of cell quantity as well as measurement of change of intensities and spectra of cultures photoluminescence (PL in the range of chlorophyll a emission was regularly conducted during the cell cultures development. Cell concentration of culture 1 exposed to the microwave irradiation for the first time has quickly restored while cell concentration of culture 2 which was irradiated repeatedly has fallen significantly. The following increasing of cell concentration of culture 2 is negligible. Cell concentration reaches the steady-state level that is about a half of the cell concentration of control culture. Initially the PL efficiency of cells of both cultures decreases noticeable as a result of irradiation. Then there is the monotonic increase to the values which are significantly higher than the corresponding values in the control cultures. The ratio of the intensities at the maxima of the main emission bands of chlorophyll for control samples of both cultures remained approximately at the same level. At the same time effect of irradiation on the cell PL spectrum appears as a temporary reduction of this magnitude.

  17. The cell-surface proteome of cultured adipose stromal cells.

    Science.gov (United States)

    Donnenberg, Albert D; Meyer, E Michael; Rubin, J Peter; Donnenberg, Vera S

    2015-07-01

    In this technical note we describe a method to evaluate the cell surface proteome of human primary cell cultures and cell lines. The method utilizes the BD Biosciences lyoplate, a system covering 242 surface proteins, glycoproteins, and glycosphingolipids plus relevant isotype controls, automated plate-based flow cytometry, conventional file-level analysis and unsupervised K-means clustering of markers on the basis of percent of positive events and mean fluorescence intensity of positive and total clean events. As an example, we determined the cell surface proteome of cultured adipose stromal cells (ASC) derived from 5 independent clinical isolates. Between-sample agreement of very strongly expressed (n = 32) and strongly expressed (n =16) markers was excellent, constituting a reliable profile for ASC identification and determination of functional properties. Known mesenchymal markers (CD29, CD44, CD73, CD90, CD105) were among the identified strongly expressed determinants. Among other strongly expressed markers are several that are potentially immunomodulatory including three proteins that protect from complement mediated effects (CD46, CD55, and CD59), two that regulate apoptosis (CD77 and CD95) and several with ectoenzymatic (CD10, CD26, CD13, CD73, and CD143) or receptor tyrosine kinase (CD140b (PDGFR), CD340 (Her-2), EGFR) activity, suggesting mechanisms for the anti-inflammatory and tissue remodeling properties of ASC. Because variables are standardized for K-means clustering, results generated using this methodology should be comparable between instrumentation platforms. It is widely generalizable to human primary explant cultures and cells lines and will prove useful to determine how cell passage, culture interventions, and gene expression and silencing affect the cell-surface proteome. PMID:25929697

  18. Development of Scalable Culture Systems for Human Embryonic Stem Cells

    OpenAIRE

    Azarin, Samira M.; Palecek, Sean P.

    2010-01-01

    The use of human pluripotent stem cells, including embryonic and induced pluripotent stem cells, in therapeutic applications will require the development of robust, scalable culture technologies for undifferentiated cells. Advances made in large-scale cultures of other mammalian cells will facilitate expansion of undifferentiated human embryonic stem cells (hESCs), but challenges specific to hESCs will also have to be addressed, including development of defined, humanized culture media and su...

  19. Rotating bio-reactor cell culture apparatus

    Science.gov (United States)

    Schwarz, Ray P. (Inventor); Wolf, David A. (Inventor)

    1991-01-01

    A bioreactor system is described in which a tubular housing contains an internal circularly disposed set of blade members and a central tubular filter all mounted for rotation about a common horizontal axis and each having independent rotational support and rotational drive mechanisms. The housing, blade members and filter preferably are driven at a constant slow speed for placing a fluid culture medium with discrete microbeads and cell cultures in a discrete spatial suspension in the housing. Replacement fluid medium is symmetrically input and fluid medium is symmetrically output from the housing where the input and the output are part of a loop providing a constant or intermittent flow of fluid medium in a closed loop.

  20. PHYTOCHEMICAL STUDY OF CELL CULTURE JATROPHA CURCAS

    Directory of Open Access Journals (Sweden)

    KOMAR RUSLAN

    2011-01-01

    Full Text Available Jatropha curcas belongs to the Euphorbiaceae family which has potential economically. This plant has been reported to contain toxic compounds such as curcin and phorbol ester and its derivatives. These compounds may become a problem if J. curcas will be explored as a source of biofuel. In order to provide safety plants, the research on the study of phytochemical and initiation of cell and organ culture have been carried out. J curcas which has been collected from different regions in Indonesia showed to contain relatively the same profile of chemical contents. Dominant compounds that were detected by GCMS are hidrocarbon such as 2-heptenal, decadienal, hexsadecane, pentadecane, cyclooctane etc, fatty acid such as oktadecanoate acid, etthyl linoleate, ethyl stearate, heksadecanoate acid and steroid such as stigmasterol, fucosterol, sitosterol. No phorbol ester and its derivatives have been detected yet by the GCMS method. Callus and suspension cultures of J. curcas have been established to be used for further investigation.

  1. Renotropic stimulation in rat kidney cell culture

    International Nuclear Information System (INIS)

    A circulating renotropic factor specific for renal cells has been described in rats. The addition of sera obtained from unilaterally nephrectomized (uni) rats 24h after operation compared to sham-operated (sham) rats augments 3H-thymidine incorporation into the DNA of incubating kidney slices approximately 10% - 30%. Attempting to amplify the sensitivity of the assay for this renotropic agent, the authors replaced slices with primary rat kidney cultures. The assay system was based on one previously used for rabbits. The cultured cells were synchronized in their growth phase by a period of protein-free starvation. Compared to sera from sham rats, sera from uni rats showed significant stimulation of thymidine incorporation into DNA, 35.5% +/- 9.3 (SEM), p < .0001, at 16 h; 63.3% +/- 10.0 (SEM), p < .001, at 24 h; and 19.5% +/- 6.5 (SEM), p < .01, at 48 h post operation. Accordingly, the maximal stimulation at 24 h was greater than that previously found using the kidney slice assay. Measurable renotropic activity occurred earlier and over a shorter duration than in rabbits. Stimulation was similar when a D-valine medium, relatively specific for renal epithelial cells, replaced DME medium

  2. Optical Oxygen Sensors for Applications in Microfluidic Cell Culture

    OpenAIRE

    Grist, Samantha M.; Lukas Chrostowski; Cheung, Karen C.

    2010-01-01

    The presence and concentration of oxygen in biological systems has a large impact on the behavior and viability of many types of cells, including the differentiation of stem cells or the growth of tumor cells. As a result, the integration of oxygen sensors within cell culture environments presents a powerful tool for quantifying the effects of oxygen concentrations on cell behavior, cell viability, and drug effectiveness. Because microfluidic cell culture environments are a promising alternat...

  3. Cardiac Cells Beating in Culture: A Laboratory Exercise

    Science.gov (United States)

    Weaver, Debora

    2007-01-01

    This article describes how to establish a primary tissue culture, where cells are taken directly from an organ of a living animal. Cardiac cells are taken from chick embryos and transferred to culture dishes. These cells are not transformed and therefore have a limited life span. However, the unique characteristics of cardiac cells are maintained…

  4. Growth of cultured porcine retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Wiencke, A.K.; Kiilgaard, Jens Folke; Nicolini, Jair;

    2003-01-01

    To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation.......To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation....

  5. Equipment for large-scale mammalian cell culture.

    Science.gov (United States)

    Ozturk, Sadettin S

    2014-01-01

    This chapter provides information on commonly used equipment in industrial mammalian cell culture, with an emphasis on bioreactors. The actual equipment used in the cell culture process can vary from one company to another, but the main steps remain the same. The process involves expansion of cells in seed train and inoculation train processes followed by cultivation of cells in a production bioreactor. Process and equipment options for each stage of the cell culture process are introduced and examples are provided. Finally, the use of disposables during seed train and cell culture production is discussed. PMID:24429549

  6. Ethanol Extracts of Selected Cyathea Species Decreased Cell Viability and Inhibited Growth in MCF 7 Cell Line Cultures.

    Science.gov (United States)

    Janakiraman, Narayanan; Johnson, Marimuthu

    2016-06-01

    Cancer is the cause of more than 6 million deaths worldwide every year. For centuries, medicinal plants have been used in the treatment of cancer. Chemotherapy, radiotherapy, surgery and acupuncture point stimulation are also used to treat cancer. The present study was intended to reveal the cytotoxic and anticancer potential of selected Cyathea species and to highlight their importance in the pharmaceutical industry for the development of cost-effective drugs. Cytotoxic studies using brine shrimp lethality bioassays and MCF 7 cell line cultures were carried out. Compared to petroleum ether, chloroform and acetone extracts, the ethanol extracts of selected Cyathea species were found to be more effective against brine shrimps. The ethanol extracts were further subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assays. A decrease in cell viability and an increase in growth inhibition were observed for the MCF 7 cell line. The maximum percentage of cell inhibition was observed in Cyathea crinit, followed by Cyathea nilgirensis and Cyathea gigantea. The results of the present study suggest that Cyathea species are an effective source of cytotoxic compounds. PMID:27342889

  7. Development of primary cell culture from Scylla serrata: Primary cell cultures from Scylla serrata

    OpenAIRE

    Sashikumar, Anu; Desai, P. V.

    2008-01-01

    This paper reports for the first time, the Primary cell culture of hepatopancreas from edible crab Scylla serrata using crab saline, L-15 (Leibovitz), 1 × L-15 + crab saline, 2 × L-15 + crab saline, 3 × L-15 and citrate buffer without any serum. We could isolate and maintain E (Embryonalzellen), F (Fibrenzellen), B (Blasenzellen), R (Restzellen) and G (Granular cells). Upon seeding the hepatopancreatic E, F, B, and R cells showed different survival pattern over time than granular cells. A mod...

  8. Phosphatidylinositol species of suspension cultured plant cells

    Energy Technology Data Exchange (ETDEWEB)

    Heim, S.; Wagner, K.G.

    Suspension cultured Nicotiana tabacum and Catharanthus roseus cells were labeled with (/sup 3/H)inositol, the phospholipid fraction extracted and separated by thin layer chromatography. Three different solvent systems and reference compounds were used to assign the different /sup 3/H-labeled species by autoradiography. The ratio of (/sup 3/H)inositol incorporation into PI, PIP and PIP/sub 2/ was found to be 95:4:1; with some preparations a lyso-PI band was obtained which incorporated about a tenth of the label of the PIP band. With Catharanthus roseus cells a very faint band between PI and lyso-PI was detected which could not be assigned to a reference compound.

  9. Metabolic flux rewiring in mammalian cell cultures.

    Science.gov (United States)

    Young, Jamey D

    2013-12-01

    Continuous cell lines (CCLs) engage in 'wasteful' glucose and glutamine metabolism that leads to accumulation of inhibitory byproducts, primarily lactate and ammonium. Advances in techniques for mapping intracellular carbon fluxes and profiling global changes in enzyme expression have led to a deeper understanding of the molecular drivers underlying these metabolic alterations. However, recent studies have revealed that CCLs are not necessarily entrenched in a glycolytic or glutaminolytic phenotype, but instead can shift their metabolism toward increased oxidative metabolism as nutrients become depleted and/or growth rate slows. Progress to understand dynamic flux regulation in CCLs has enabled the development of novel strategies to force cultures into desirable metabolic phenotypes, by combining fed-batch feeding strategies with direct metabolic engineering of host cells. PMID:23726154

  10. Evaluation of osteogenic cell culture and osteogenic/peripheral blood mononuclear human cell co-culture on modified titanium surfaces

    International Nuclear Information System (INIS)

    This study aimed to determine the effect of a bioactive ceramic coating on titanium in the nanothickness range on human osteogenic cells, peripheral blood mononuclear cells (PBMC) and on osteogenic cells co-cultured with PBMC without exogenous stimuli. Cell viability, proliferation, adhesion, cytokine release (IL1β, TGFβ1, IL10 and IL17) and intracellular stain for osteopontin and alkaline phosphatase were assessed. Morphologic evaluation showed smaller and less spread cell aspects in co-culture relative to osteogenic cell culture. Cell viability, proliferation and adhesion kinetics were differently influenced by surface texture/chemistry in culture versus co-culture. Cytokine release was also influenced by the interaction between mononuclear and osteogenic cells (mediators released by mononuclear cells acted on osteogenic cells and vice versa). In general, ‘multi-cell type’ interactions played a more remarkable role than the surface roughness or chemistry utilized on the in vitro cellular events related to initial stages of bone formation. (paper)

  11. Recombinant Protein Production and Insect Cell Culture and Process

    Science.gov (United States)

    Spaulding, Glenn F. (Inventor); Goodwin, Thomas J. (Inventor); OConnor, Kim C. (Inventor); Francis, Karen M. (Inventor); Andrews, Angela D. (Inventor); Prewett, Tracey L. (Inventor)

    1997-01-01

    A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using virtually infected or stably transformed insect cells containing a gene encoding the described polypeptide. The insect cells can also be a host for viral production.

  12. System-level modeling and simulation of the cell culture microfluidic biochip ProCell

    DEFF Research Database (Denmark)

    Minhass, Wajid Hassan; Pop, Paul; Madsen, Jan;

    2010-01-01

    -defined micro-channels using valves and pumps. We present an approach to the system-level modeling and simulation of a cell culture microfluidic biochip called ProCell, Programmable Cell Culture Chip. ProCell contains a cell culture chamber, which is envisioned to run 256 simultaneous experiments (viewed as a...

  13. Protection of cultured mammalian cells by rebamipide

    Energy Technology Data Exchange (ETDEWEB)

    Antoku, Shigetoshi; Aramaki, Ryoji [Kyushu Univ., Fukuoka (Japan). Faculty of Medicine; Tanaka, Hisashi; Kusumoto, Naotoshi

    1997-06-01

    Rebamipide which is used as a drug for gastritis and stomach ulcer has large capability for OH radical scavenging. It is expected that rebamipide has protective effect against ionizing radiations. The present paper deals with protective effect of rebamipide for cultured mammalian cells exposed to ionizing radiations. As rebamipide is insoluble in water, three solvents were used to dissolve. Rebamipide dissolved in dimethyl sulfoxide (DMSO), dimethyl formamide (DMFA) and 0.02 N NaOH was added to the cells in Eagle`s minimum essential medium (MEM) supplemented with 10% fetal calf serum and the cells were irradiated with X-rays. After irradiation, the cells were trypsinized, plated in MEM with 10% fetal calf serum and incubated for 7 days in a CO{sub 2} incubator to form colonies. Rebamipide dissolved in 0.02 N NaOH exhibited the protective effect expected its OH radical scavenging capability. However, the protective effect of rebamipide dissolved in DMSO was about half of that expected by its radical scavenging capability and that of rebamipide dissolved in DMFA was not observed. Uptake of rebamipide labeled with {sup 14}C increased with increasing contact time with rebamipide. These rebamipide mainly distributed in nucleus rather than cytoplasm. (author)

  14. Bioefficacy of crude extract of Cyperus aromaticus (Family:Cyperaceae) cultured cells, against Aedes aegypti and Aedes albopictus mosquitoes

    Institute of Scientific and Technical Information of China (English)

    Fatemeh Kamiabi; Zairi Jaal; Chan Lai Keng

    2013-01-01

    Objective:To evaluate the growth inhibition activity of the crude extract of Cyperus aromaticus (C. aromaticus) cultured cells against the 3rd instar larvae of Aedes aegypti (Linn.) and Aedes albopictus Skuse (Ae. albopictus) under laboratory conditions, and determine the sublethal effects (EI50) of the crude extract of C. aromaticus cultured cells on some biological and morphological parameters of both Aedes mosquito species during two generations as well. Methods:The cell suspension cultures of C. aromaticus were activated from five callus lines (P4, Pa, Z1, Z6 and Ml) derived from the root explants of in vitro plantlets. The cultured cells were extracted in chloroform and used as plant material for the present study. For detection of juvenile hormone III, the crude extracts were analyzed by HPLC. Then the crude extracts of the three C. aromaticus cultured cell lines which contained varied amounts of juvenile hormone III [high level (P4 cell line), medium level (Z1 cell line) and low level (Ml cell line)] were tested against Aedes mosquito species. Laboratory evaluation was performed against late third instar larvae of the Vector Control Research Unit strains of Ae. aegypti and Ae. albopictus using the standard WHO method. The effects of EI50 of the C. aromaticus cultured P4 cells on fecundity, fertility, growth period, sex ratio, adult size and longevity of Aedes mosquitoes were assessed. Results:Bioassay tests presented the remarkable growth inhibition activity of the crude extracts of C. aromaticus cultured cells against the two Aedes mosquitoes. Between the two mosquito species, Ae. albopictus was more susceptible to the crude extracts with lower EI50 values. EI50 of the crude extract of C. aromaticus cultured cells (P4) increased the sterility indices in the parental generation females in both Aedes mosquito species. A significant delay in the pupal formation and adult emergence were observed in the parental generation of the both mosquito species. The sex

  15. A rapid bioassay to monitor murine leukemia virus infection in mice using cellular gp71 binding

    International Nuclear Information System (INIS)

    A rapid and sensitive bioassay based on the availability of cell surface receptors for the binding of purified envelope glycoprotein, gp71, of Rauscher murine leukemia virus (R-MuLV) was developed to serially monitor viral-induced leukemogenesis in individual BALB/cAnN mice. The specificity of the bioassay was demonstrated by the competition of [125I]gp71 cellular binding with murine ecotropic viruses, purified unlabelled R-MuLV envelope glycoprotein and by antiserum to R-MuLV gp71. In contrast, there was no effect on the [125I]gp71 binding level with the addition of murine xenotropic viruses, R-MuLV p30, or several other proteins. The [125I]gp71 binding level of circulating leukocytes was significantly (P < 0.05) reduced in mice after R-MuLV infection. The reduction of cellular gp71 binding developed in two stages and the latter stage was highly dependent (P < 0.05) on circulating infections virus titer. Using this technique, the gp71 cellular binding levels of 48-60 individual mice can be assayed in a 4 h period. The advantages of this bioassay compared to standard immunological and tissue culture techniques used in studying retrovirus expression and viral-cell interactions are discussed. (Auth.)

  16. Determining Cell Number During Cell Culture using the Scepter Cell Counter

    OpenAIRE

    Ongena, Kathleen; Das, Chandreyee; Smith, Janet L.; Gil, Sónia; Johnston, Grace

    2010-01-01

    Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measuremen...

  17. Three-Dimensional Cell Culture: A Breakthrough in Vivo

    Directory of Open Access Journals (Sweden)

    Delphine Antoni

    2015-03-01

    Full Text Available Cell culture is an important tool for biological research. Two-dimensional cell culture has been used for some time now, but growing cells in flat layers on plastic surfaces does not accurately model the in vivo state. As compared to the two-dimensional case, the three-dimensional (3D cell culture allows biological cells to grow or interact with their surroundings in all three dimensions thanks to an artificial environment. Cells grown in a 3D model have proven to be more physiologically relevant and showed improvements in several studies of biological mechanisms like: cell number monitoring, viability, morphology, proliferation, differentiation, response to stimuli, migration and invasion of tumor cells into surrounding tissues, angiogenesis stimulation and immune system evasion, drug metabolism, gene expression and protein synthesis, general cell function and in vivo relevance. 3D culture models succeed thanks to technological advances, including materials science, cell biology and bioreactor design.

  18. Organ culture-cell culture system for studying multistage carcinogenesis in respiratory epithelium. [Mice

    Energy Technology Data Exchange (ETDEWEB)

    Steele, Vernon E.; Marchok, Ann C.; Nettesheim, Paul

    1977-01-01

    An organ culture-cell culture system was used to demonstrate carcinogen dose-dependent transformation of tracheal epithelial cells in vitro. Tracheal explants were exposed to MNNG (N-methyl-N/sup 1/-nitro-N-nitrosoguanidine) in organ culture. Outgrowths from these explants provided epithelial cell cultures. The numbers of long term epithelial cell cultures and cell lines that were established per explant increased as MNNG exposure concentration increased. At the present time, more cell lines derived from explants exposed to the highest MNNG concentration have produced palpable tumors than cell lines derived from explants exposed to lower MNNG concentrations. No cell lines were established from primaries derived from control explants. TPA (12-0-tetradecanoyl-phorbol-13-acetate), stimulates DNA synthesis in tracheal epithelium in organ culture in a manner simular to that described for mouse skin. Short exposures to TPA not only stimulated DNA synthesis earlier, but the stimulation was greater than that obtained with continuous exposure. At the present time, exposure of tracheal organ cultures to MNNG followed by TPA has resulted in an enhanced production of morphologically altered cells in primary epithelial cell cultures, than exposure to either agent alone.

  19. Electrospinning of microbial polyester for cell culture

    International Nuclear Information System (INIS)

    Biodegradable and biocompatible poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a copolymer of microbial polyester, was fabricated as a nanofibrous mat by electrospinning. The specific surface area and the porosity of electrospun PHBV nanofibrous mat were determined. When the mechanical properties of flat film and electrospun PHBV nanofibrous mats were investigated, both the tensile modulus and strength of electrospun PHBV were less than those of cast PHBV film. However, the elongation ratio of nanofiber mat was higher than that of the cast film. The structure of electrospun nanofibers using PHBV-trifluoroethanol solutions depended on the solution concentrations. When x-ray diffraction patterns of bulk PHBV before and after electrospinning were compared, the crystallinity of PHBV was not significantly affected by the electrospinning process. Chondrocytes adhered and grew on the electrospun PHBV nanofibrous mat better than on the cast PHBV film. Therefore, the electrospun PHBV was considered to be suitable for cell culture

  20. Particle Trajectories in Rotating Wall Cell Culture Devices

    Science.gov (United States)

    Ramachandran N.; Downey, J. P.

    1999-01-01

    Cell cultures are extremely important to the medical community since such cultures provide an opportunity to perform research on human tissue without the concerns inherent in experiments on individual humans. Development of cells in cultures has been found to be greatly influenced by the conditions of the culture. Much work has focused on the effect of the motions of cells in the culture relative to the solution. Recently rotating wall vessels have been used with success in achieving improved cellular cultures. Speculation and limited research have focused on the low shear environment and the ability of rotating vessels to keep cells suspended in solution rather than floating or sedimenting as the primary reasons for the improved cellular cultures using these devices. It is widely believed that the cultures obtained using a rotating wall vessel simulates to some degree the effect of microgravity on cultures. It has also been speculated that the microgravity environment may provide the ideal acceleration environment for culturing of cellular tissues due to the nearly negligible levels of sedimentation and shear possible. This work predicts particle trajectories of cells in rotating wall vessels of cylindrical and annular design consistent with the estimated properties of typical cellular cultures. Estimates of the shear encountered by cells in solution and the interactions with walls are studied. Comparisons of potential experiments in ground and microgravity environments are performed.

  1. HAIR CELL-LIKE CELL GENERATION INDUCED BY NATURE CULTURE OF ADULT RAT AUDITORY EPITHELIUM

    Institute of Scientific and Technical Information of China (English)

    Liu Hui; Zhu Hongliang; Li Shengli; Yao Xiaobao; Wang Xiaoxia

    2006-01-01

    Objective To establish adult rat auditory epithelial cell culture and try to find precursor cells of auditory hair cells in vitro. Methods With refinement of culture media and techniques, cochlear sensory epithelial cells of adult rat were cultured. Immunocytochemistry and Bromodeoxyuridine (BrdU)labeling were used to detect properties and mitotic status of cultured cells. Results The cultured auditory epithelial cells showed a large, flat epithelial morphotype and expressed F-actin and cytokeratin, a subset of cells generated from auditory epithelium were labeled by calretinin, a specific marker of early hair cell. Conclusion Adult rat auditory epithelium can be induced to generate hair cell-like cells by nature culture, this phenomenon suggests that progenitor cells may exist in rat cochlea and they may give birth to new hair cells. Whether these progenitor cells are tissue specific stem cells is still need more study.

  2. Usability and Applicability of Microfluidic Cell Culture Systems

    DEFF Research Database (Denmark)

    Hemmingsen, Mette

    devices still lack general implementation into biological research laboratories. In this project, the usability and applicability of microfluidic cell culture systems have been investigated. The tested systems display good properties regarding optics and compatibility with standard laboratory equipment...... possibilities for, for example, precise control of the chemical environment, 3D cultures, controlled co-culture of different cell types or automated, individual control of up to 96 cell culture chambers in one integrated system. Despite the great new opportunities to perform novel experimental designs, these...... shown to be needed. This is possibly one of the reasons for the lack of implementation of microfluidic cell culture systems into biological research laboratories. Procedures to perform long-term microfluidic perfusion cell culture experiments have been established. Furthermore, successful application of...

  3. THE ALKALOID CYTISINE IN THE CELL CULTURE

    Directory of Open Access Journals (Sweden)

    Gazaliev A.M.

    2012-08-01

    Full Text Available Alkaloids are vegetative establishments of complex and original structure with nitrous heterocycles in the basis. For a long time they drew researchers’ attention because of their unique and specific physiological effect on alive organisms. Not all the representatives of the globe’s flora contain these unique substances. Alkaloid cytisine is to be found mainly in the plants of the fabaceous family - Fabaceae. For the cytisine production the seeds of Thermopsis lanceolata R.Br (T. lanceolata R.Br and Cytisus laburnum (C. laburnum are used as a raw material. The object of the research is T. lanceolata cell culture. Sterile sprouts are used at the first stage of the experiment. Callus genesis is accompanied with dedifferentiation. It leads to the cellular organization simplification. Based on an important property of a plant cell, such as totipotency, there appears the formation of the “de novo” biosynthetic device. The cultivation algorithm consists of two basic stages: (i the cultivation conditions optimization of callus with a high level of the primary metabolites biosynthesis (Aspartat – lysine; (ii the research of cultivation chemical and physical factors influence on the secondary metabolite (cytisine biosynthesis and accumulation. During the cultivation the Murashige and Skoog classical recipe of nutrient medium will be used. Optimization of the cultivation conditions will concern the phytohormones, macro- and micronutrients content, as the purpose of optimization is the production of the determined high-level competence embriogenical callus. The main problem is genetic heterogeneity of a cellular population and instability of morpho-physiological processes. The correct management of higher plants cells population is possible at the synchronization of a cellular cycle phases. The references analysis has shown that it is almost impossible to synchronize cellular cycles in the culture of plant tissue. The application of chemical

  4. Using Tissue Culture To Investigate Plant Cell Differentiation and Dedifferentiation.

    Science.gov (United States)

    Bozzone, Donna M.

    1997-01-01

    Describes an experimental project that uses plant tissue culture techniques to examine cell differentiation in the carrot. Allows students to gain experience in some important techniques and to explore fundamental questions about cell differentiation. (DDR)

  5. Systems Biology for Organotypic Cell Cultures

    Energy Technology Data Exchange (ETDEWEB)

    Grego, Sonia [RTI International, Research Triangle Park, NC (United States); Dougherty, Edward R. [Texas A & M Univ., College Station, TX (United States); Alexander, Francis J. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Auerbach, Scott S. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Berridge, Brian R. [GlaxoSmithKline, Research Triangle Park, NC (United States); Bittner, Michael L. [Translational Genomics Research Inst., Phoenix, AZ (United States); Casey, Warren [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Cooley, Philip C. [RTI International, Research Triangle Park, NC (United States); Dash, Ajit [HemoShear Therapeutics, Charlottesville, VA (United States); Ferguson, Stephen S. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Fennell, Timothy R. [RTI International, Research Triangle Park, NC (United States); Hawkins, Brian T. [RTI International, Research Triangle Park, NC (United States); Hickey, Anthony J. [RTI International, Research Triangle Park, NC (United States); Kleensang, Andre [Johns Hopkins Univ., Baltimore, MD (United States). Center for Alternatives to Animal Testing; Liebman, Michael N. [IPQ Analytics, Kennett Square, PA (United States); Martin, Florian [Phillip Morris International, Neuchatel (Switzerland); Maull, Elizabeth A. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Paragas, Jason [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Qiao, Guilin [Defense Threat Reduction Agency, Ft. Belvoir, VA (United States); Ramaiahgari, Sreenivasa [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Sumner, Susan J. [RTI International, Research Triangle Park, NC (United States); Yoon, Miyoung [The Hamner Inst. for Health Sciences, Research Triangle Park, NC (United States); ScitoVation, Research Triangle Park, NC (United States)

    2016-08-04

    Translating in vitro biological data into actionable information related to human health holds the potential to improve disease treatment and risk assessment of chemical exposures. While genomics has identified regulatory pathways at the cellular level, translation to the organism level requires a multiscale approach accounting for intra-cellular regulation, inter-cellular interaction, and tissue/organ-level effects. Tissue-level effects can now be probed in vitro thanks to recently developed systems of three-dimensional (3D), multicellular, “organotypic” cell cultures, which mimic functional responses of living tissue. However, there remains a knowledge gap regarding interactions across different biological scales, complicating accurate prediction of health outcomes from molecular/genomic data and tissue responses. Systems biology aims at mathematical modeling of complex, non-linear biological systems. We propose to apply a systems biology approach to achieve a computational representation of tissue-level physiological responses by integrating empirical data derived from organotypic culture systems with computational models of intracellular pathways to better predict human responses. Successful implementation of this integrated approach will provide a powerful tool for faster, more accurate and cost-effective screening of potential toxicants and therapeutics. On September 11, 2015, an interdisciplinary group of scientists, engineers, and clinicians gathered for a workshop in Research Triangle Park, North Carolina, to discuss this ambitious goal. Participants represented laboratory-based and computational modeling approaches to pharmacology and toxicology, as well as the pharmaceutical industry, government, non-profits, and academia. Discussions focused on identifying critical system perturbations to model, the computational tools required, and the experimental approaches best suited to generating key data. This consensus report summarizes the discussions held.

  6. Induced engulfment of Neisseria gonorrhoeae by tissue culture cells.

    OpenAIRE

    Richardson, W P; Sadoff, J C

    1988-01-01

    Engulfment of gonococci by mammalian tissue culture cells was examined as a model of the penetration of host cells in gonorrhea. Engulfment required viable organisms; killing the gonococci with heat or refrigeration abolished the process. Engulfment also required tissue culture cell microtubule- and microfilament-dependent movement; treating the cells with cytochalasin B (0.5 micrograms/ml) or demecolcine (Colcemid; Ciba-Geigy AG, Basel, Switzerland) (10 micrograms/ml) also prevented his proc...

  7. Increased exosome production from tumour cell cultures using the Integra CELLine Culture System.

    Science.gov (United States)

    Mitchell, J Paul; Court, Jacqueline; Mason, Malcolm David; Tabi, Zsuzsanna; Clayton, Aled

    2008-06-01

    Exosomes are nanometer-sized vesicles, secreted from most cell types, with documented immune-modulatory functions. Exosomes can be purified from cultured cells but to do so effectively, requires maintenance of cells at high density in order to obtain sufficient accumulation of exosomes in the culture medium, prior to purification. Whilst high density cultures can be achieved with cells in suspension, this remains difficult with adherent cells, resulting in low quantity of exosomes for subsequent study. We have used the Integra CELLine culture system, originally designed for hybridoma cultures, to achieve a significant increase in obtainable exosomes from adherent and non-adherent tumour cells. Traditional cultures of mesothelioma cells (cultured in 75 cm(2) flasks) gave an average yield of 0.78 microg+/-0.14 microg exosome/ml of conditioned medium. The CELLine Adhere 1000 (CLAD1000) flask, housing the same cell line, increased exosome yield approximately 12 fold to 10.06 microg+/-0.97 microg/ml. The morphology, phenotype and immune function of these exosomes were compared, and found to be identical in all respects. Similarly an 8 fold increase in exosome production was obtained from NKL cells (a suspension cell line) using a CELLine 1000 (CL1000) flask. The CELLine system also incurred ~5.5 fold less cost and reduced labour for cell maintenance. This simple culture system is a cost effective, useful method for significantly increasing the quantity of exosomes available from cultured cells, without detrimental effects. This tool should prove advantageous in future studies of exosome-immune modulation in cancer and other settings. PMID:18423480

  8. High-Aspect-Ratio Rotating Cell-Culture Vessel

    Science.gov (United States)

    Wolf, David A.; Sams, Clarence; Schwarz, Ray P.

    1992-01-01

    Cylindrical rotating cell-culture vessel with thin culture-medium layer of large surface area provides exchange of nutrients and products of metabolism with minimal agitation. Rotation causes averaging of buoyant forces otherwise separating components of different densities. Vessel enables growth of cells in homogeneous distribution with little agitation and little shear stress.

  9. Detecting mycoplasma contamination in cell cultures by polymerase chain reaction.

    Science.gov (United States)

    Uphoff, Cord C; Drexler, Hans G

    2011-01-01

    The detection of mycoplasmas in human and animal cell cultures is mandatory for every cell culture laboratory, because these bacteria are common contaminants, persist unrecognized in cell cultures for many years, and affect research results as well as the purity of cell culture products. The reliability of the mycoplasma detection depends on the sensitivity and specificity of the method and should also be convenient to be included in the basic routine of cell culture quality assessment. Polymerase chain reaction (PCR) detection is one of the acknowledged methodologies to detect mycoplasmas in cell cultures and cell culture products. Although the PCR offers a fast and simple technique to detect mycoplasmas, the method is also susceptible to errors and can produce false positive as well as false-negative results. Thus, the establishment and the routine application of the PCR assay require optimization and the inclusion of the appropriate control reactions. The presented protocol describes sample preparation, DNA extraction, PCR run, the analysis of the PCR products, and speciation of the contaminant. It also provides detailed information on how to avoid artifacts produced by the method. Established properly, PCR is a reliable, fast, and sensitive method and should be applied regularly to monitor the contamination status of cell cultures. PMID:21516400

  10. Growth and Plating of Cell Suspension Cultures of Datura Innoxia

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1974-01-01

    ammonium malate) or on NO3−-N alone. Dry weight yield was proportional to the amount of nitrate-N added (47 mg/mg N). Filtered suspension cultures containing single cells (plating cultures) could be grown in agar in petri dishes when NAA or 2,4-D were used as growth substances. Cells grew at densities...

  11. Batch variation between branchial cell cultures: An analysis of variance

    DEFF Research Database (Denmark)

    Hansen, Heinz Johs. Max; Grosell, M.; Kristensen, L.

    2003-01-01

    We present in detail how a statistical analysis of variance (ANOVA) is used to sort out the effect of an unexpected batch-to-batch variation between cell cultures. Two separate cultures of rainbow trout branchial cells were grown on permeable filtersupports ("inserts"). They were supposed to be...

  12. 21 CFR 864.2280 - Cultured animal and human cells.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cultured animal and human cells. 864.2280 Section 864.2280 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 §...

  13. Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

    OpenAIRE

    Akopian, Veronika; Andrews, Peter W.; Beil, Stephen; Benvenisty, Nissim; Brehm, Jennifer; Christie, Megan; Ford, Angela; Fox, Victoria; Gokhale, Paul J; Healy, Lyn; Holm, Frida; Hovatta, Outi; Knowles, Barbara B; Ludwig, Tenneille E; Ronald D G McKay

    2010-01-01

    There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with pr...

  14. Autophagic response to cell culture stress in pluripotent stem cells.

    Science.gov (United States)

    Gregory, Siân; Swamy, Sushma; Hewitt, Zoe; Wood, Andrew; Weightman, Richard; Moore, Harry

    2016-05-01

    Autophagy is an important conserved cellular process, both constitutively as a recycling pathway for long lived proteins and as an upregulated stress response. Recent findings suggest a fundamental role for autophagic processes in the maintenance of pluripotent stem cell function. In human embryonic stem cells (hESCS), autophagy was investigated by transfection of LC3-GFP to visualize autophagosomes and with an antibody to LC3B protein. The presence of the primary cilium (PC) in hESCs as the site of recruitment of autophagy-related proteins was also assessed. HESCs (mShef11) in vitro displayed basal autophagy which was upregulated in response to deprivation of culture medium replacement. Significantly higher levels of autophagy were exhibited on spontaneous differentiation of hESCs in vitro. The PC was confirmed to be present in hESCs and therefore may serve to coordinate autophagy function. PMID:26385182

  15. Horizontally rotated cell culture system with a coaxial tubular oxygenator

    Science.gov (United States)

    Wolf, David A. (Inventor); Schwarz, Ray P. (Inventor); Trinh, Tinh T. (Inventor)

    1991-01-01

    The present invention relates to a horizontally rotating bioreactor useful for carrying out cell and tissue culture. For processing of mammalian cells, the system is sterilized and fresh fluid medium, microcarrier beads, and cells are admitted to completely fill the cell culture vessel. An oxygen containing gas is admitted to the interior of the permeable membrane which prevents air bubbles from being introduced into the medium. The cylinder is rotated at a low speed within an incubator so that the circular motion of the fluid medium uniformly suspends the microbeads throughout the cylinder during the cell growth period. The unique design of this cell and tissue culture device was initially driven by two requirements imposed by its intended use for feasibility studies for three dimensional culture of living cells and tissues in space by JSC. They were compatible with microgravity and simulation of microgravity in one G. The vessels are designed to approximate the extremely quiescent low shear environment obtainable in space.

  16. Biona-C Cell Culture pH Monitoring System

    Science.gov (United States)

    Friedericks, C.

    1999-01-01

    Sensors 2000! is developing a system to demonstrate the ability to perform accurate, real-time measurements of pH and CO2 in a cell culture media in Space. The BIONA-C Cell Culture pH Monitoring System consists of S2K! developed ion selective sensors and control electronics integrated with the fluidics of a cell culture system. The integrated system comprises a "rail" in the Cell Culture Module (CCM) of WRAIR (Space Biosciences of Walter Read Army Institute of Research). The CCM is a Space Shuttle mid-deck locker experiment payload. The BIONA-C is displayed along with associated graphics and text explanations. The presentation will stimulate interest in development of sensor technology for real-time cell culture measurements. The transfer of this technology to other applications will also be of interest. Additional information is contained in the original document.

  17. PECULIARITIES OF SECONDARY METABOLITES BIOSYNTHESIS IN PLANT CELL CULTURES

    Directory of Open Access Journals (Sweden)

    A.M. NOSOV

    2014-06-01

    Full Text Available metabolites formation in plant cell cultures of Panax spp., (ginsenosides; Dioscorea deltoidea (steroid glycosides; Ajuga reptans, Serratula coronata, Rhaponticum carthamoides (ecdisteroids; Polyscias spp., (triterpene glycosides, Taxus spp. (taxoids, Stevia rebaudiana (diterpene steviol-glycosides, Stephania glabra (alkaloids. They are some regular trends of secondary metabolites synthesis in the plant cell culture:It can be noted the stable synthesis of the compound promoting cell proliferation. Indeed, cell cultures of Dioscorea deltoidea were demonstrated to accumulate only furostanol glycosides, which promoted cell division. Furostanol glycoside content of Dioscorea strain DM-0.5 was up to 6 - 12% by dry biomass.Panax ginseng and P. japonicus plant cell cultures synthesize as minimum seven triterpene glycosides (ginsenosides, the productivity of these compounds was up to 6.0 - 8.0% on dry biomass.By contrast, the detectable synthesis of diterpene steviol-glycosides in cultivated cells of Stevia rebaudiana initiated in the mixotrophic cultures during chloroplast formation only.Despite these differences, or mainly due to them, plant cell cultures have become an attractive source of phytochemicals in alternative to collecting wild plants. It provides a guideline to bioreactor-based production of isoprenoids using undifferentiated plant cell cultures

  18. Morphological and Immunohistochemical Characterization of Canine Osteosarcoma Spheroid Cell Cultures.

    Science.gov (United States)

    Gebhard, C; Gabriel, C; Walter, I

    2016-06-01

    Spheroid cell culture emerges as powerful in vitro tool for experimental tumour research. In this study, we established a scaffold-free three-dimensional spheroid system built from canine osteosarcoma (OS) cells (D17). Spheroids (7, 14 and 19 days of cultivation) and monolayer cultures (2 and 7 days of cultivation) were evaluated and compared on light and electron microscopy. Monolayer and spheroid cultures were tested for vimentin, cytokeratin, alkaline phosphatase, osteocalcin and collagen I by means of immunohistochemistry. The spheroid cell culture exhibited a distinct network of collagen I in particular after 19-day cultivation, whereas in monolayer cultures, collagen I was arranged as a lamellar basal structure. Necrotic centres of large spheroids, as observed in 14- and 19-day cultures, were characterized by significant amounts of osteocalcin. Proliferative activity as determined by Ki-67 immunoreactivity showed an even distribution in two-dimensional cultures. In spheroids, proliferation was predominating in the peripheral areas. Metastasis-associated markers ezrin and S100A4 were shown to be continuously expressed in monolayer and spheroid cultures. We conclude that the scaffold-free spheroid system from canine OS cells has the ability to mimic the architecture of the in vivo tumour, in particular cell-cell and cell-matrix interactions. PMID:26287450

  19. Culturing of PC12 Cells, Neuronal Cells, Astrocytes Cultures and Brain Slices in an Open Microfluidic System

    DEFF Research Database (Denmark)

    Al Atraktchi, Fatima Al-Zahraa; Bakmand, Tanya; Rømer Sørensen, Ane;

    culturing PC12 cells, neuronal cells, astrocytes cultures and brain slices. The microfluidic system provides efficient nutrient delivery, waste removal, access to oxygen, fine control over the neurochemical environment and access to modern microscopy. Additionally, the setup consists of an in vitro...

  20. Cytotoxicity of Spent Pot Liner on Allium cepa root tip cells: A comparative analysis in meristematic cell type on toxicity bioassays.

    Science.gov (United States)

    Palmieri, Marcel José; Andrade-Vieira, Larissa Fonseca; Campos, José Marcello Salabert; Dos Santos Gedraite, Leonardo; Davide, Lisete Chamma

    2016-11-01

    Spent Pot Liner (SPL) is a waste generated during the production of aluminum. It is comprised of a mixture of substances most of which, like cyanide, aluminum and fluoride, are toxic. Previous studies indicate the highly toxic nature of SPL. However studies using cells of the differentiation/elongation zone of the root meristem (referred as M2 cells in this study) after a proper recovery period in water were never considered. Using these cells could be useful to further understanding the toxicity mechanisms of SPL. A comparative approach between the effects on M2 cells and meristematic cells of the proximal meristem zone (referred as M1 cells in this study) could lead to understanding how DNA damage caused by SPL behaves on successive generations of cells. Allium cepa cells were exposed to 4 different concentrations of SPL (2.5, 5, 7.5 and 10gL(-1)) mixed with soil and diluted in a CaCl2 0.01M to simulate the ionic forces naturally encountered on the environment. A solution containing only soil diluted on CaCl2 0.01M was used as control. M1 and M2 cells were evaluated separately, taking into account four different parameters: (1) mitotic alterations (MA); (2) presence of condensed nuclei (CN); (3) mitotic index (MI); (4) presence of micronucleus (MCN). Significant differences were observed between M1 and M2 roots tip cells for these four parameters accessed. M1 cells was more prompt to reveal citogenotoxicity through the higher frequency of MA observed. Meanwhile, for M2 cells higher frequencies of MCN and CN was noticed, followed by a reduction of MI. Also, it was possible to detect significant differences between the tested treatments and the control on every case. These results indicate SPL toxic effects carries on to future cells generations. This emphasizes the need to properly manage this waste. Joint evaluation of cells from both M1 and M2 regions was proven valuable for the evaluation of a series of parameters on all toxicity tests. PMID:27517141

  1. Microglial cells in astroglial cultures: a cautionary note

    Directory of Open Access Journals (Sweden)

    Saura Josep

    2007-10-01

    Full Text Available Abstract Primary rodent astroglial-enriched cultures are the most popular model to study astroglial biology in vitro. From the original methods described in the 1970's a great number of minor modifications have been incorporated into these protocols by different laboratories. These protocols result in cultures in which the astrocyte is the predominant cell type, but astrocytes are never 100% of cells in these preparations. The aim of this review is to bring attention to the presence of microglia in astroglial cultures because, in my opinion, the proportion of and the role that microglial cells play in astroglial cultures are often underestimated. The main problem with ignoring microglia in these cultures is that relatively minor amounts of microglia can be responsible for effects observed on cultures in which the astrocyte is the most abundant cell type. If the relative contributions of astrocytes and microglia are not properly assessed an observed effect can be erroneously attributed to the astrocytes. In order to illustrate this point the case of NO production in activated astroglial-enriched cultures is examined. Lipopolysaccharide (LPS induces nitric oxide (NO production in astroglial-enriched cultures and this effect is very often attributed to astrocytes. However, a careful review of the published data suggests that LPS-induced NO production in rodent astroglial-enriched cultures is likely to be mainly microglial in origin. This review considers cell culture protocol factors that can affect the proportion of microglial cells in astroglial cultures, strategies to minimize the proportion of microglia in these cultures, and specific markers that allow the determination of such microglial proportions.

  2. Isolation, Culture, and Maintenance of Mouse Intestinal Stem Cells

    Science.gov (United States)

    O’Rourke, Kevin P.; Ackerman, Sarah; Dow, Lukas E; Lowe, Scott W

    2016-01-01

    In this protocol we describe our modifications to a method to isolate, culture and maintain mouse intestinal stem cells as crypt-villus forming organoids. These cells, isolated either from the small or large intestine, maintain self-renewal and multilineage differentiation potential over time. This provides investigators a tool to culture wild type or transformed intestinal epithelium, and a robust assay for stem cell tissue homeostasis in vitro.

  3. SPONTANEOUS TRANSFORMATION OF CULTURED PORCINE BONE MARROW STROMAL CELLS

    DEFF Research Database (Denmark)

    Zou, Lijin; Zou, Xuenong; Li, Haisheng;

    -term culture are transformed into malignant cells. MATERIAL AND METHODS BMSC from 6 pigs were isolated and propagated continuously. Cell morphology was observed. Transformation properties were evaluated by means of serum dependence assay, Ki- 67 immunostaining, soft agar colony assay, karyotyping, telomerase...... was increased and TGF‚ signaling pathway was upregulated. However, telomerase activity maintained negative during culture. CONCLUSION Porcine BMSC can undergo spontaneous transformation, which provides a useful model to study the mechanisms associated with the tumorigenic potential of adult stem cells....

  4. Effects of methyl isocyanate on rat brain cells in culture.

    Science.gov (United States)

    Anderson, D; Goyle, S; Phillips, B J; Tee, A; Beech, L; Butler, W H

    1990-09-01

    Since the disaster in Bhopal, India, people exposed to methyl isocyanate (MIC) have complained of various disorders including neuromuscular dysfunction. In an attempt to get information about such dysfunction we have previously shown that MIC can affect muscle cells in culture. The present communication reports investigations into the effect of MIC on brain cells in culture. MIC was toxic to brain cells and the response was dose related. The observations were supported by light and electron microscopy. PMID:2207030

  5. Multizone paper platform for 3D cell cultures.

    Directory of Open Access Journals (Sweden)

    Ratmir Derda

    Full Text Available In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other environmental factors (temperature, mechanical stress, etc. The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously ("cells-in-gels-in-paper" or CiGiP, this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D culture, by peeling apart the sheets of paper, "sections" all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures.

  6. Chemotherapy in heterogeneous cultures of cancer cells with interconversion

    International Nuclear Information System (INIS)

    Recently, the interconversion between differentiated and stem-like cancer cells has been observed. Here, we model the in vitro growth of heterogeneous cell cultures in the presence of interconversion from differentiated cancer cells to cancer stem cells (CSCs), showing that, by targeting only CSC with cytotoxic agents, it is not always possible to eradicate cancer. We have determined the kinetic conditions under which cytotoxic agents in in vitro heterogeneous cultures of cancer cells eradicate cancer. In particular, we have shown that the chemotherapeutic elimination of in vitro cultures of heterogeneous cancer cells is effective only if it targets all cancer cell types, and if the induced death rates for the different subpopulations of cancer cell types are large enough. The quantitative results of the model are compared and validated with experimental data. (paper)

  7. Feeding Frequency Affects Cultured Rat Pituitary Cells in Low Gravity

    Science.gov (United States)

    Hymer, W. C.; Grindeland, R. E.; Salada, T.; Cenci, R.; Krishnan, K.; Mukai, C.; Nagaoka, S.

    1996-01-01

    In this report, we describe the results of a rat pituitary cell culture experiment done on STS-65 in which the effect of cell feeding on the release of the six anterior pituitary hormones was studied. We found complex microgravity related interactions between the frequency of cell feeding and the quantity and quality (i.e. biological activity) of some of the six hormones released in flight. Analyses of growth hormone (GH) released from cells into culture media on different mission days using gel filtration and ion exchange chromatography yielded qualitatively similar results between ground and flight samples. Lack of cell feeding resulted in extensive cell clumping in flight (but not ground) cultures. Vigorous fibroblast growth occurred in both ground and flight cultures fed 4 times. These results are interpreted within the context of autocrine and or paracrine feedback interactions. Finally the payload specialist successfully prepared a fresh trypsin solution in microgravity, detached the cells from their surface and reinserted them back into the culture chamber. These cells reattached and continued to release hormone in microgravity. In summary, this experiment shows that pituitary cells are microgravity sensitive and that coupled operations routinely associated with laboratory cel1 culture can also be accomplished in low gravity.

  8. Mammosphere culture of cancer stem cells in a microfluidic device

    Science.gov (United States)

    Saadin, Katayoon; White, Ian M.

    2012-03-01

    It is known that tumor-initiating cells with stem-like properties will form spherical colonies - termed mammospheres - when cultured in serum-free media on low-attachment substrates. Currently this assay is performed in commercially available 96-well trays with low-attachment surfaces. Here we report a novel microsystem that features on-chip mammosphere culture on low attachment surfaces. We have cultured mammospheres in this microsystem from well-studied human breast cancer cell lines. To enable the long-term culture of these unattached cells, we have integrated diffusion-based delivery columns that provide zero-convection delivery of reagents, such as fresh media, staining agents, or drugs. The multi-layer system consists of parallel cell-culture chambers on top of a low-attachment surface, connected vertically with a microfluidic reagent delivery layer. This design incorporates a reagent reservoir, which is necessary to reduce evaporation from the cell culture micro-chambers. The development of this microsystem will lead to the integration of mammosphere culture with other microfluidic functions, including circulating tumor cell recovery and high throughput drug screening. This will enable the cancer research community to achieve a much greater understanding of these tumor initiating cancer stem cells.

  9. UNIFYING SCALER FOR BIOASSAY TESTS

    Science.gov (United States)

    An extensive set of interlaboratory root bioassay data was unified using centroids of individual tests as scalers. It is shown that the dose response obeys a first order differential equation with the constant of the equation related to the sensitivity of the dose response relati...

  10. Epithelial cell detachment by Porphyromonas gingivalis biofilm and planktonic cultures.

    Science.gov (United States)

    Huang, Lijia; van Loveren, Cor; Ling, Junqi; Wei, Xi; Crielaard, Wim; Deng, Dong Mei

    2016-04-01

    Porphyromonas gingivalis is present as a biofilm at the sites of periodontal infections. The detachment of gingival epithelial cells induced by P. gingivalis biofilms was examined using planktonic cultures as a comparison. Exponentially grown planktonic cultures or 40-h biofilms were co-incubated with epithelial cells in a 24-well plate for 4 h. Epithelial cell detachment was assessed using imaging. The activity of arginine-gingipain (Rgp) and gene expression profiles of P. gingivalis cultures were examined using a gingipain assay and quantitative PCR, respectively. P. gingivalis biofilms induced significantly higher cell detachment and displayed higher Rgp activity compared to the planktonic cultures. The genes involved in gingipain post-translational modification, but not rgp genes, were significantly up-regulated in P. gingivalis biofilms. The results underline the importance of including biofilms in the study of bacterial and host cell interactions. PMID:26963862

  11. [Continuous perfusion culture hybridoma cells for production of monoclonal antibody].

    Science.gov (United States)

    Mi, Li; Li, Ling; Feng, Qiang; Yu, Xiao-Ling; Chen, Zhi-Nan

    2002-05-01

    Hybridoma cells were cultured by continuous perfusion in Fibra-Cel of 5L packed-bed bioreactor for 22 days in low serum or serum-free media. The corresponded amino acids were fed and serum concentration was decreased by analyzing glucose concentration, oxygen uptake rate, secretary antibody amount and amino acids concentration in culture supernatant. Comparing with continuous perfusion culture that amino acids were not fed, antibody amount of production was increased about 2-3 times. The inoculated cell density was 2.5 x 10(5) cells/mL, while the final cell density was 8.79 x 10(8) cells/mL. Antibody production was reached 295 mg/L/d at average level, and the highest level was reached 532 mg/L/d. These results provided a primary mode of enlarge culture for monoclonal antibody industralization. PMID:12192875

  12. A practical guide to hydrogels for cell culture.

    Science.gov (United States)

    Caliari, Steven R; Burdick, Jason A

    2016-04-28

    There is growing appreciation of the role that the extracellular environment plays in regulating cell behavior. Mechanical, structural, and compositional cues, either alone or in concert, can drastically alter cell function. Biomaterials, and particularly hydrogels, have been developed and implemented to present defined subsets of these cues for investigating countless cellular processes as a means of understanding morphogenesis, aging, and disease. Although most scientists concede that standard cell culture materials (tissue culture plastic and glass) do a poor job of recapitulating native cellular milieus, there is currently a knowledge barrier for many researchers in regard to the application of hydrogels for cell culture. Here, we introduce hydrogels to those who may be unfamiliar with procedures to culture and study cells with these systems, with a particular focus on commercially available hydrogels. PMID:27123816

  13. THE ULTRASTRUCTURE OF SEPARATED AND CULTURED CELL OF PORPHYRA YEZOENSIS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    There are many reports that cells (protoplasts) separated from the thallus of Porphyra by enzyme can develop to normal leafy thalli in the same way as monospores. But there are few investigations on the subcellular structure of the isolated vegetative cell for comparison with the subcellular structure of monospores. To clarify whether the separated and cultured cells undergo the same or similar ultrastructure changes during culture and germination as monospores undergo in their formation and germination, we observed their ultrastructure, compared them with those of the monospore and found that the ultrastructure of separated and cultured cells did not have the characteristic feature as that of monospore formation, such as production of small and large fibrous vesicles, but was accompanied by vacuolation and starch mobilization like that in monospore germination. The paper also discusses the relations between monospores and separated and cultured cells.

  14. Biomarkers and bioassays as alternative screening methods for the presence and effects of PCDD, PCDF and PCB

    International Nuclear Information System (INIS)

    Polyhalogenated aromatic hydrocarbons (PHAH) are wide spread, highly toxic, environmental contaminants. As such they pose risks for both humans and wildlife. For risk assessment purposes, concentrations are generally analyzed by HRGC-HR/LRMS. With the analytical data, mixture toxicity is calculated using the TEF concept. With this method only the defined congeners are taken into account and additivity for all congeners is assumed, whereas synergistic and antagonistic effects for several PCDD/F in combination with PCB have also been reported. To avoid these problems and high analytical costs, bioassays can be used for screening purposes. Cytochrome P 450 1 A 1 induction and vitamin A and thyroid hormone levels are shown to be useful markers for PHAH exposure. When bioassays based on cytochrome P 450 1 A 1 induction, in cultured cells, in multi-well culturing plates, are used, 2,3,7,8-TCDD detection limits <0.2 pg are possible. As such these bioassays are highly sensitive, cost effective and time saving. This application can be used as a pre-screening method to determine total ''dioxin'' content of environmental samples. (orig.)

  15. Surface modified alginate microcapsules for 3D cell culture

    Science.gov (United States)

    Chen, Yi-Wen; Kuo, Chiung Wen; Chueh, Di-Yen; Chen, Peilin

    2016-06-01

    Culture as three dimensional cell aggregates or spheroids can offer an ideal platform for tissue engineering applications and for pharmaceutical screening. Such 3D culture models, however, may suffer from the problems such as immune response and ineffective and cumbersome culture. This paper describes a simple method for producing microcapsules with alginate cores and a thin shell of poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) to encapsulate mouse induced pluripotent stem (miPS) cells, generating a non-fouling surface as an effective immunoisolation barrier. We demonstrated the trapping of the alginate microcapsules in a microwell array for the continuous observation and culture of a large number of encapsulated miPS cells in parallel. miPS cells cultured in the microcapsules survived well and proliferated to form a single cell aggregate. Droplet formation of monodisperse microcapsules with controlled size combined with flow cytometry provided an efficient way to quantitatively analyze the growth of encapsulated cells in a high-throughput manner. The simple and cost-effective coating technique employed to produce the core-shell microcapsules could be used in the emerging field of cell therapy. The microwell array would provide a convenient, user friendly and high-throughput platform for long-term cell culture and monitoring.

  16. Crystal Violet Assay for Determining Viability of Cultured Cells.

    Science.gov (United States)

    Feoktistova, Maria; Geserick, Peter; Leverkus, Martin

    2016-01-01

    Adherent cells detach from cell culture plates during cell death. This characteristic can be used for the indirect quantification of cell death and to determine differences in proliferation upon stimulation with death-inducing agents. One simple method to detect maintained adherence of cells is the staining of attached cells with crystal violet dye, which binds to proteins and DNA. Cells that undergo cell death lose their adherence and are subsequently lost from the population of cells, reducing the amount of crystal violet staining in a culture. This protocol describes a quick and reliable screening method that is suitable for the examination of the impact of chemotherapeutics or other compounds on cell survival and growth inhibition. However, characterization of the cause of reduced crystal violet staining requires additional methods detailed elsewhere. PMID:27037069

  17. 77 FR 14837 - Bioassay at Uranium Mills

    Science.gov (United States)

    2012-03-13

    ... COMMISSION Bioassay at Uranium Mills AGENCY: Nuclear Regulatory Commission. ACTION: Draft regulatory guide... for public comment draft regulatory guide (DG), DG-8051, ``Bioassay at Uranium Mills.'' This guide describes a bioassay program acceptable to the NRC staff for uranium mills and applicable portions...

  18. Controlling the diversity of cell populations in a stem cell culture

    NARCIS (Netherlands)

    Heo, Inha; Clevers, Hans

    2015-01-01

    Culturing intestinal stem cells into 3D organoids results in heterogeneous cell populations, reflecting the in vivo cell type diversity. In a recent paper published in Nature, Wang et al. established a culture condition for a highly homogeneous population of intestinal stem cells.

  19. A Place to Call Home: Bioengineering Pluripotential Stem Cell Cultures

    OpenAIRE

    Mark Weingarten; Nathan Akhavan; Joshua Hanau; Yakov Peter

    2015-01-01

    Pluripotent stem cells (PSCs) have the power to revolutionize the future of cell-based therapies and regenerative medicine. However, stem/progenitor cell use in the clinical arsenal has been hampered by discrepancies resulting from stem cell engineering and expansion, as well as in their (mass) differentiation in culture. Moreover, the manner in which external conditions affect PSC and induced-pluripotent stem cell lineage establishment as well as maturation remains controversial. In this rev...

  20. Air pollutant production by algal cell cultures

    Science.gov (United States)

    Fong, F.; Funkhouser, E. A.

    1982-01-01

    The production of phytotoxic air pollutants by cultures of Chlorella vulgaris and Euglena gracilis is considered. Algal and plant culture systems, a fumigation system, and ethylene, ethane, cyanide, and nitrogen oxides assays are discussed. Bean, tobacco, mustard green, cantaloupe and wheat plants all showed injury when fumigated with algal gases for 4 hours. Only coleus plants showed any resistance to the gases. It is found that a closed or recycled air effluent system does not produce plant injury from algal air pollutants.

  1. Insect cell culture and applications to research and pest management

    Science.gov (United States)

    Building on earlier research, insect cell culture began with the successful establishment of one cell line from pupal ovarian tissue. The field has grown to the extent that now over 500 insect cell lines have been established from many insect species representing numerous insect Orders and from seve...

  2. Detection and Treatment of Mycoplasma Contamination in Cultured Cells

    Directory of Open Access Journals (Sweden)

    Hsuan Jung

    2003-04-01

    Full Text Available Background: Mycoplasmas, the smallest and simplest prokaryotes that reside in endosomesof mammalian cells, are widespread contaminants found in cell cultures.About 30% of all cell cultures, varying from 15 to 80%, are reportedlycontaminated with mycoplasmas. Here, we present our experience in successfullydetecting and treating mycoplasmal infection in various cell lines.Methods: The nested polymerase chain reaction (PCR detection and microscopicexamination, including phase-contrast, fluorescent, as well as differentialinterference contrast, were used for detecting potential mycoplasma contaminationof cell lines used in our laboratory. As soon as mycoplasma was identified,antibiotic treatment was initiated.Results: Mycoplasmal contamination was detected in six of 15 cell lines using thenested PCR amplification of mycoplasma DNA, which was further demonstratedusing 4, 6-Diamidino-2-phenylindole (DAPI staining and fluorescentmicroscopy. Alternate treatment with two antibiotics, macrolide (tiamulinand tetracycline (minocycline, effectively eliminated mycoplasma, whichwas validated by both PCR and microscopic studies.Conclusions: The nested PCR using genomic DNA extracted from cultured cells as templatesis a rapid and sensitive method for detecting mycoplasma contamination.Treatment with combined antibiotics can completely eradicatemycoplasmal infection from cultured cells. For the ease of use, PCR and/orDAPI staining appear suitable for detecting potential mycoplasmal contaminationin laboratories that rely heavily on the cell culture system.

  3. The effects of glucocorticoids on cultured human endothelial cells.

    Science.gov (United States)

    Maca, R D; Fry, G L; Hoak, J C

    1978-04-01

    The effects of hydrocortisone, dexamethasone and prednisone on the morphology, replication, DNA synthesis, cell protein content and protein synthesis of cultured, human endothelial cells were evaluated. After culturing the cells with these glucocorticoids for 24-48 h, the cells covered a greater portion of the culture surface area. The mean surface area of the individual endothelial cell treated with glucocorticoids was 1.53 times greater than that of the untreated control endothelial cell. When compared with controls, the endothelial cover provided by the cells treated with glucocorticoids was more extensive and in many instances covered the entire culture surface. The change in morphology was associated with an increase in protein synthesis and protein content of the cells without an increase in DNA synthesis or cellular replication. Dexamethasone was approximately 10-fold more effective than hydrocortisone, while prednisone was the least effective. Aldosterone, DOCA, testosterone, progesterone, oestradiol and oestriol were ineffective. These studies indicate that glucocorticoids can alter the morphology and biochemistry of cultured endothelial cells and may have implications for the effects of steroids in the treatment of thrombocytopenic states and vascular disorders in man. PMID:646949

  4. Novel culturing platform for brain slices and neuronal cells

    DEFF Research Database (Denmark)

    Svendsen, Winnie Edith; Al Atraktchi, Fatima Al-Zahraa; Bakmand, Tanya;

    2015-01-01

    In this paper we demonstrate a novel culturing system for brain slices and neuronal cells, which can control the concentration of nutrients and the waste removal from the culture by adjusting the fluid flow within the device. The entire system can be placed in an incubator. The system has been te...

  5. Cell/Tissue Culture Radiation Exposure Facility Project

    Data.gov (United States)

    National Aeronautics and Space Administration — We propose to develop a Cell/Tissue Culture Radiation Exposure Facility (CTC-REF) to enable radiobiologists to investigate the real-time radiation effects on...

  6. Convoluted cells as a marker for maternal cell contamination in CVS cultures

    DEFF Research Database (Denmark)

    Hertz, Jens Michael; Jensen, P K; Therkelsen, A J

    1987-01-01

    In order to identify cells of maternal origin in CVS cultures, tissue from 1st trimester abortions were cultivated and the cultures stained in situ for X-chromatin. Convoluted cells and maternal fibroblasts were found to be positive. By chromosome analysis of cultures from 105 diagnostic placenta...... biopsies, obtained by the transabdominal route, metaphases of maternal origin were found in nine cases. In eight of these cases colonies of convoluted cells were observed. We conclude that convoluted cells are of maternal origin and are a reliable marker for maternal cell contamination in CVS cultures....

  7. Effects of UVC-irradiation on cultured mouse embryonic cells

    International Nuclear Information System (INIS)

    Effects of UVC-irradiation on the cultured differentiating mouse embryonic cells were investigated. Embryonic mesenchymal cells, isolated from fore-and hind-limbs or mid brain of Day 11 mouse embryos, and 3T3 cells, a reference mouse fibroblast cell line, were irradiated with UVC at a dose range of 0∼30 J/m2. Dose-dependent inhibition was found for both cellular proliferation and differentiation, dose-dependent induction of DNA cyclobutane pyrimidine dimers and (6-4) photoproducts were found in the embryonic cells. Mesenchymal chondrogenesis was more sensitive to the UVC than proliferation, and the UVC-induced DNA damage and their repair kinetics in the cultured embryonic cells were similar to those in mouse 3T3 cells. No effects of treatments by the fluorescent light pre or post UVC-irradiation were found on the repair kinetics of DNA damage in all of the cells

  8. Exposure to Music Alters Cell Viability and Cell Motility of Human Nonauditory Cells in Culture

    Science.gov (United States)

    Lestard, Nathalia R.

    2016-01-01

    Although music is part of virtually all cultures in the world, little is known about how it affects us. Since the beginning of this century several studies suggested that the response to music, and to sound in general, is complex and might not be exclusively due to emotion, given that cell types other than auditory hair cells can also directly react to audible sound. The present study was designed to better understand the direct effects of acoustic vibrations, in the form of music, in human cells in culture. Our results suggest that the mechanisms of cell growth arrest and/or cell death induced by acoustic vibrations are similar for auditory and nonauditory cells. PMID:27478480

  9. Exposure to Music Alters Cell Viability and Cell Motility of Human Nonauditory Cells in Culture.

    Science.gov (United States)

    Lestard, Nathalia R; Capella, Marcia A M

    2016-01-01

    Although music is part of virtually all cultures in the world, little is known about how it affects us. Since the beginning of this century several studies suggested that the response to music, and to sound in general, is complex and might not be exclusively due to emotion, given that cell types other than auditory hair cells can also directly react to audible sound. The present study was designed to better understand the direct effects of acoustic vibrations, in the form of music, in human cells in culture. Our results suggest that the mechanisms of cell growth arrest and/or cell death induced by acoustic vibrations are similar for auditory and nonauditory cells. PMID:27478480

  10. Qualitative study of three cell culture methods.

    Science.gov (United States)

    Wang, Aiguo; Xia, Tao; Ran, Peng; Chen, Xuemin; Nuessler, Andreas K

    2002-01-01

    Primary rat hepatocytes were cultured using different in vitro models and the enzyme leakage, albumin secretion, and cytochrome P450 1A (CYP 1A) activity were observed. The results showed that the level of LDH was decreased over time in culture. However, on day 5, LDH showed a significant increase in monolayer culture (MC) while after day 8 no LDH was detectable in sandwich culture (SC). The levels of AST and ALT did not change significantly over the investigated time. The CYP 1A activity was gradually decreased in a time-dependent manner in MC and SC. The decline of CYP 1A was faster in MC than in SC. This effect was partially reversed by using cytochrome P450 (CYP450) inducer such as Omeprazol and 3-methylcholanthrene (3-MC) and the CYP 1A induction was always higher in MC than in SC. In bioreactor basic CYP 1A activity was preserved over 2 weeks and the highest albumin production was observed in bioreactor followed by SC and MC. Taken together, it was indicated each investigated model had its advantages and disadvantages. It was also underlined that various in vitro models may address different questions. PMID:12674760

  11. [Effect evaluation of three cell culture models].

    Science.gov (United States)

    Wang, Aiguo; Xia, Tao; Yuan, Jing; Chen, Xuemin

    2003-11-01

    Primary rat hepatocytes were cultured using three kinds of models in vitro and the enzyme leakage, albumin secretion, and cytochrome P450 1A (CYP 1A) activity were observed. The results showed that the level of LDH in the medium decreased over time in the period of culture. However, on 5 days, LDH showed a significant increase in monolayer culture (MC) while after 8 days LDH was not detected in sandwich culture (SC). The levels of AST and ALT in the medium did not change significantly over the investigated time. The basic CYP 1A activity gradually decreased with time in MC and SC. The decline of CYP 1A in rat hepatocytes was faster in MC than that in SC. This effect was partially reversed by using cytochrome P450 (CYP450) inducers such as omeprazol and 3-methylcholanthrene (3-MC) and the CYP 1A induction was always higher in MC than that in SC. Basic CYP 1A activity in bioreactor was keeped over 2 weeks and the highest albumin production was observed in bioreactor, and next were SC and MC. In conclusion, our results clearly indicated that there have some advantages and disadvantages in each of models in which can address different questions in metabolism of toxicants and drugs. PMID:14963896

  12. Hydrofocusing Bioreactor for Three-Dimensional Cell Culture

    Science.gov (United States)

    Gonda, Steve R.; Spaulding, Glenn F.; Tsao, Yow-Min D.; Flechsig, Scott; Jones, Leslie; Soehnge, Holly

    2003-01-01

    The hydrodynamic focusing bioreactor (HFB) is a bioreactor system designed for three-dimensional cell culture and tissue-engineering investigations on orbiting spacecraft and in laboratories on Earth. The HFB offers a unique hydrofocusing capability that enables the creation of a low-shear culture environment simultaneously with the "herding" of suspended cells, tissue assemblies, and air bubbles. Under development for use in the Biotechnology Facility on the International Space Station, the HFB has successfully grown large three-dimensional, tissuelike assemblies from anchorage-dependent cells and grown suspension hybridoma cells to high densities. The HFB, based on the principle of hydrodynamic focusing, provides the capability to control the movement of air bubbles and removes them from the bioreactor without degrading the low-shear culture environment or the suspended three-dimensional tissue assemblies. The HFB also provides unparalleled control over the locations of cells and tissues within its bioreactor vessel during operation and sampling.

  13. Learning about Cells as Dynamic Entities: An Inquiry-Driven Cell Culture Project

    Science.gov (United States)

    Palombi, Peggy Shadduck; Jagger, Kathleen Snell

    2008-01-01

    Using cultured fibroblast cells, undergraduate students explore cell division and the responses of cultured cells to a variety of environmental changes. The students learn new research techniques and carry out a self-designed experiment. Through this project, students enhance their creative approach to scientific inquiry, learn time-management and…

  14. Stability of resazurin in buffers and mammalian cell culture media

    DEFF Research Database (Denmark)

    Rasmussen, Eva; Nicolaisen, G.M.

    1999-01-01

    The utility of a ferricyanide/ferrocyanide system used in the AlamarBlue(TM) (Serotec, Oxford, UK) vital. dye to inhibit the reduction of resazurin by mammalian cell culture media is questioned. Resazurin was found to be relatively stable when dissolved in phosphate-buffered saline (PBS). The use...... of HEPES resulted in a huge immediate dye reduction, which was significantly enhanced by exposure to diffuse light from fluorescent tubes in the laboratory 8 h per day. The reduction of resazurin by various cell culture media was time and temperature dependent, and it was significantly enhanced by......'s nutrient mixture F-10 and F-12. Fetal calf serum (5-20%) slightly decreased resazurin reduction during the first 2 days of incubation. The reduction of resazurin by mammalian cell culture media do not appear to be problematic under normal culture conditions, and it is primarily dependent upon the presence...

  15. Towards dynamic metabolic flux analysis in CHO cell cultures.

    Science.gov (United States)

    Ahn, Woo Suk; Antoniewicz, Maciek R

    2012-01-01

    Chinese hamster ovary (CHO) cells are the most widely used mammalian cell line for biopharmaceutical production, with a total global market approaching $100 billion per year. In the pharmaceutical industry CHO cells are grown in fed-batch culture, where cellular metabolism is characterized by high glucose and glutamine uptake rates combined with high rates of ammonium and lactate secretion. The metabolism of CHO cells changes dramatically during a fed-batch culture as the cells adapt to a changing environment and transition from exponential growth phase to stationary phase. Thus far, it has been challenging to study metabolic flux dynamics in CHO cell cultures using conventional metabolic flux analysis techniques that were developed for systems at metabolic steady state. In this paper we review progress on flux analysis in CHO cells and techniques for dynamic metabolic flux analysis. Application of these new tools may allow identification of intracellular metabolic bottlenecks at specific stages in CHO cell cultures and eventually lead to novel strategies for improving CHO cell metabolism and optimizing biopharmaceutical process performance. PMID:22102428

  16. Detection of multiple mycoplasma infection in cell cultures by PCR

    Directory of Open Access Journals (Sweden)

    J. Timenetsky

    2006-07-01

    Full Text Available A total of 301 cell cultures from 15 laboratories were monitored for mycoplasma (Mollicutes using PCR and culture methodology. The infection was detected in the cell culture collection of 12 laboratories. PCR for Mollicutes detected these bacteria in 93 (30.9% samples. Although the infection was confirmed by culture for 69 (22.9% samples, PCR with generic primers did not detect the infection in five (5.4%. Mycoplasma species were identified with specific primers in 91 (30.2% of the 98 samples (32.6% considered to be infected. Mycoplasma hyorhinis was detected in 63.3% of the infected samples, M. arginini in 59.2%, Acholeplasma laidlawii in 20.4%, M. fermentans in 14.3%, M. orale in 11.2%, and M. salivarium in 8.2%. Sixty (61.2% samples were co-infected with more than one mycoplasma species. M. hyorhinis and M. arginini were the microorganisms most frequently found in combination, having been detected in 30 (30.6% samples and other associations including up to four species were detected in 30 other samples. Failure of the treatments used to eliminate mycoplasmas from cell cultures might be explained by the occurrence of these multiple infections. The present results indicate that the sharing of non-certified cells among laboratories may disseminate mycoplasma in cell cultures.

  17. Integrin VLA-3: ultrastructural localization at cell-cell contact sites of human cell cultures

    OpenAIRE

    1989-01-01

    The integrin VLA-3 is a cell surface receptor, which binds to fibronectin, laminin, collagen type I and VI (Takada, Y., E. A. Wayner, W. G. Carter, and M. E. Hemler. 1988. J. Cell. Biochem. 37:385-393) and is highly expressed in substrate adherent cultures of almost all human cell types. The ligand specificity of VLA-3 and the inhibition of cell adhesion by anti-VLA-3 monoclonal antibodies suggest its involvement in cell-substrate interaction. In normal tissues, VLA-3 is restricted to few cel...

  18. Challenges of culturing human norovirus in three-dimensional organoid intestinal cell culture models.

    Directory of Open Access Journals (Sweden)

    Efstathia Papafragkou

    Full Text Available Human noroviruses are the most common cause of acute gastroenteritis worldwide. Recently, cell culture systems have been described using either human embryonic intestinal epithelial cells (Int-407 or human epithelial colorectal adenocarcinoma cells (Caco-2 growing on collagen-I porous micro carrier beads in a rotating bioreactor under conditions of physiological fluid shear. Here, we describe the efforts from two independent laboratories to implement this three dimensional (3D cell culture system for the replication of norovirus. Int-407 and Caco-2 were grown in a rotating bioreactor for up to 28 days. Prior to infection, cells were screened for the presence of microvilli by electron microscopy and stained for junction proteins (zonula occludens-1, claudin-1, and β-catenin. Differentiated 3D cells were transferred to 24-well plates and infected with bacteria-free filtrates of various norovirus genotypes (GI.1, GI.3, GI.8, GII.2, GII.4, GII.7, and GII.8. At 12 h, 24 h, and 48 h post inoculation, viral RNA from both cells and supernatants were collected and analyzed for norovirus RNA by real-time reverse transcription PCR. Despite observations of high expression of junction proteins and microvilli development in stained thin sections, our data suggest no significant increase in viral titer based on norovirus RNA copy number during the first 48 h after inoculation for the different samples and virus culture conditions tested. Our combined efforts demonstrate that 3D cell culture models using Int-407 or Caco-2 cells do not support norovirus replication and highlight the complexity and difficulty of developing a reproducible in vitro cell culture system for human norovirus.

  19. Metabolism Kinetics of Glucose in Anchorage-dependent Cell Cultures

    Institute of Scientific and Technical Information of China (English)

    孙祥明; 张元兴

    2001-01-01

    The kinetic model of glucose metabolism was established and successfully applied to batchcultures of rCHO and rBHK cells. It was found that a large amount of glucose was utilized for cellmaintenance, and the overwhelming majority of maintenance energy from glucose was by its anaerobicmetabolism in both rBHK and rCHO cell cultures. The overall maintenance coefficients from aerobicmetabolism were 1.9×10-13 mmol/(cell.h) for rCHO cells and 7×10-13 mmol/(cell.h) for rBHK cells. Inaddition, all Go/T and Eo/T gradually increased with the same trend as the cell growth in the culture ofboth rCHO and rBHK cells. The overall molecule yield coefficients of lactate to glucose were 1.61 for rCHO cells and 1.38 for rBHK cells. The yield coefficients of cell to glucose were 4.5×108 cells/mmol for rCHO cells and 1.9 × 108 cells/mmol for rBHK cells, respectively.

  20. A Simple Hydrophilic Treatment of SU-8 Surfaces for Cell Culturing and Cell Patterning

    DEFF Research Database (Denmark)

    Wang, Zhenyu; Stangegaard, Michael; Dufva, Hans Martin;

    2005-01-01

    SU-8, an epoxy-based photoresist, widely used in constitution different mTAS systems, is incompatible with mammalian cell adhesion and culture in its native form. Here, we demonstrate a simple, cheap and robust two-step method to render a SU-8 surface hydrophilic and compatible with cell culture....... The contact angle of SU-8 surface was significantly reduced from 90° to 25° after the surface modification. The treated SU-8 surfaces provided a cell culture environment that was comparable with cell culture flask surface in terms of generation time and morphology....

  1. Replica-moulded polydimethylsiloxane culture vessel lids attenuate osmotic drift in long-term cell cultures

    Indian Academy of Sciences (India)

    Axel Blau; Tanja Neumann; Christiane Ziegler; Fabio Benfenati

    2009-03-01

    An imbalance in medium osmolarity is a determinant that affects cell culture longevity. Even in humidified incubators, evaporation of water leads to a gradual increase in osmolarity overtime. We present a simple replica-moulding strategy for producing self-sealing lids adaptable to standard, small-size cell-culture vessels. They are made of polydimethylsiloxane (PDMS), a flexible, transparent and biocompatible material, which is gas-permeable but largely impermeable to water. Keeping cell cultures in a humidified 5% CO2 incubator at 37°C, medium osmolarity increased by +6.86 mosmol/kg/day in standard 35 mm Petri dishes, while PDMS lids attenuated its rise by a factor of four to changes of +1.72 mosmol/kg/ day. Depending on the lid membrane thickness, pH drifts at ambient CO2 levels were attenuated by a factor of 4 to 9. Comparative evaporation studies at temperatures below 60°C yielded a 10-fold reduced water vapour flux of 1.75 g/day/dm2 through PDMS lids as compared with 18.69 g/day/dm2 with conventional Petri dishes. Using such PDMS lids, about 2/3 of the cell cultures grew longer than 30 days in vitro. Among these, the average survival time was 69 days with the longest survival being 284 days under otherwise conventional cell culture conditions.

  2. Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Met, Özcan;

    2011-01-01

    Standard cell culture plastic was surface modified by passive adsorption or covalent attachment of interleukin (IL)-4 and investigated for its ability to induce differentiation of human monocytes into mature dendritic cells, a process dose-dependently regulated by IL-4. Covalent attachment of IL-4...

  3. Endothelial cell cultures as a tool in biomaterial research

    NARCIS (Netherlands)

    Kirkpatrick, CJ; Otto, M; Kooten, TV; Krump, [No Value; Kriegsmann, J; Bittinger, F

    1999-01-01

    Progress in biocompatibility and tissue engineering would today be inconceivable without the aid of in vitro techniques. Endothelial cell cultures represent a valuable tool not just in haemocompatibility testing, but also in the concept of designing hybrid organs. In the past endothelial cells (EC)

  4. Cell cultures from the symbiotic soft coral Sinularia flexibilis

    NARCIS (Netherlands)

    Khalesi, M.K.; Vera-Jimenez, N.I.; Aanen, D.K.; Beeftink, H.H.; Wijffels, R.H.

    2008-01-01

    The symbiotic octocoral Sinularia flexibilis is a producer of potential pharmaceuticals. Sustainable mass production of these corals as a source of such compounds demands innovative approaches, including coral cell culture. We studied various cell dissociation methodologies and the feasibility of cu

  5. Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Met, Ozcan;

    2011-01-01

    Standard cell culture plastic was surface modified by passive adsorption or covalent attachment of interleukin (IL)-4 and investigated for its ability to induce differentiation of human monocytes into mature dendritic cells, a process dose-dependently regulated by IL-4. Covalent attachment of IL-...

  6. Duchenne muscular dystrophy: normal ATP turnover in cultured cells

    International Nuclear Information System (INIS)

    This paper examines ATP metabolism in cultured muscle cells and fibroblasts from patients with Duchenne dystrophy. ATP and ADP levels were the same in cultured cells from normal subjects and patients and there was no difference in ATP synthesis or degradation. The ATP synthesis was measured by the incorporation of C 14-U-adenine into aTP and ADP. although there was a significant decrease in radioactively labelled ATP after incubation with deoxyglucose in Duchenne muscle cells, there was no difference in ATP concentration of ADP metabolism

  7. Schwann cell cultures from human fetal dorsal root ganglia

    Institute of Scientific and Technical Information of China (English)

    Yaping Feng; Hui Zhu; Jiang Hao; Xinmin Wang; Shengping Wu; Li Bai; Xiangming Li; Yun Zha

    2009-01-01

    BACKGROUND:Previous studies have used many methods for in vitro Schwann cells (SCs) cul-tures and purification,such as single cell suspension and cytosine arabinoside.However,it has been difficult to obtain sufficient cellular density,and the procedures have been quite tedious.OBJECTIVE:To investigate the feasibility of culturing high-density SCs using fetal human dorsal root ganglion tissue explants.DESIGN,TIME AND SETTING:Cell culture and immunohistochemistry were performed at the Cen-tral Laboratory of Kunming General Hospital of Chinese PLA between March 2001 and October 2008.MATERIALS:Culture media containing 10% fetal bovine serum,as well as 0.2% collagenase and 0.25% trypsin were purchased from Gibco,USA;mouse anti-human S-100 monoclonal antibody and goat anti-mouse IgG labeled with horseradish peroxidase were provided by Beijing Institute of Bi-ological Products,China.METHODS:Primarily cultured SCs were dissociated from dorsal root ganglia of human aborted fe-tuses at 4-6 months pregnancy.Following removal of the dorsal root ganglion perineurium,the gan-glia were dissected into tiny pieces and digested with 0.2% collagenase and 0.25% trypsin (volume ratio 1:1),then explanted and cultured.SC purification was performed with 5 mL 10% fetal bovine serum added to the culture media,followed by differential adhesion.MAIN OUTCOME MEASURES:SCs morphology was observed under inverted phase contrast light microscopy.SC purity was evaluated according to percentage of S-100 immunostained cells.RESULTS:SCs were primarily cultured for 5-6 days and then subcultured for 4-5 passages.The highly enriched SC population reached > 95% purity and presented with normal morphology.CONCLUSION:A high purity of SCs was obtained with culture methods using human fetal dorsal root ganglion tissue explants.

  8. Miniature Bioreactor System for Long-Term Cell Culture

    Science.gov (United States)

    Gonda, Steve R.; Kleis, Stanley J.; Geffert, Sandara K.

    2010-01-01

    A prototype miniature bioreactor system is designed to serve as a laboratory benchtop cell-culturing system that minimizes the need for relatively expensive equipment and reagents and can be operated under computer control, thereby reducing the time and effort required of human investigators and reducing uncertainty in results. The system includes a bioreactor, a fluid-handling subsystem, a chamber wherein the bioreactor is maintained in a controlled atmosphere at a controlled temperature, and associated control subsystems. The system can be used to culture both anchorage-dependent and suspension cells, which can be either prokaryotic or eukaryotic. Cells can be cultured for extended periods of time in this system, and samples of cells can be extracted and analyzed at specified intervals. By integrating this system with one or more microanalytical instrument(s), one can construct a complete automated analytical system that can be tailored to perform one or more of a large variety of assays.

  9. Hydrodynamic effects on cells in agitated tissue culture reactors

    Science.gov (United States)

    Cherry, R. S.; Papoutsakis, E. T.

    1986-01-01

    The mechanisms by which hydrodynamic forces can affect cells grown on microcarrier beads in agitated cell culture reactors were investigated by analyzing the motion of microcarriers relative to the surrounding fluid, to each other, and to moving or stationary solid surfaces. It was found that harmful effects on cell cultures that have been previously attributed to shear can be better explained as the effects of turbulence (of a size scale comparable to the microcarriers or the spacing between them) or collisions. The primary mechanisms of cell damage involve direct interaction between microcarriers and turbulent eddies, collisions between microcarriers in turbulent flow, and collisions against the impeller or other solid surfaces. The implications of these analytical results for the design of tissue culture reactors are discussed.

  10. Culture and immortalization of pancreatic ductal epithelial cells.

    Science.gov (United States)

    Lawson, Terence; Ouellette, Michel; Kolar, Carol; Hollingsworth, Michael

    2005-01-01

    Some populations of the epithelial cells from the duct and ductular network of the mammalian pancreas have been isolated and maintained in vitro for up to 3 mo. These cells express many of the surface factors that are unique to them in vivo. They also retain significant drug- and carcinogen-metabolizing capacity in vitro. In this chapter we review the progression of the methods for the isolation, culture and maintenance in vitro for these cells from the earliest when only duct/ductular fragments were obtainable to the current ones which provide epithelial cells. The critical steps in the isolation process are identified and strategies are provided to facilitate these steps. These include the selection of tissue digestive enzymes, the importance of extensive mincing before culture and the importance of roles of some co-factors used in the culture medium. PMID:15542901

  11. Microfluidic bioreactors for culture of non-adherent cells

    DEFF Research Database (Denmark)

    Shah, Pranjul Jaykumar; Vedarethinam, Indumathi; Kwasny, Dorota;

    2011-01-01

    Microfluidic bioreactors (μBR) are becoming increasingly popular for cell culture, sample preparation and analysis in case of routine genetic and clinical diagnostics. We present a novel μBR for non-adherent cells designed to mimic in vivo perfusion of cells based on diffusion of media through a...... sandwiched membrane. The culture chamber and perfusion chamber are separated by a sandwiched membrane and each chamber has separate inlet/outlets for easy loading/unloading of cells and perfusion of the media. The perfusion of media and exchange of nutrients occur through the sandwiched membrane, which was...... of CFSE staining and subsequent counting in a flow cytometer. To conclude on the applicability of μBR for genetic diagnostics, we prepare chromosome spreads on glass slides from the cultured samples, which is the primary step for metaphase FISH analysis....

  12. Dose verification by OSLDs in the irradiation of cell cultures

    International Nuclear Information System (INIS)

    The determination of value of irradiation dose presents difficulties when targets are irradiated located in regions where electronic equilibrium of charged particle is not reached, as in the case of irradiation -in vitro- of cell lines monolayer-cultured, in culture dishes or flasks covered with culture medium. The present study aimed to implement a methodology for dose verification in irradiation of cells in culture media by optically stimulated luminescence dosimetry (OSLD). For the determination of the absorbed dose in terms of cell proliferation OSL dosimeters of aluminum oxide doped with carbon (Al2O3:C) were used, which were calibrated to the irradiation conditions of culture medium and at doses that ranged from 0.1 to 15 Gy obtained with a linear accelerator of 6 MV photons. Intercomparison measurements were performed with an ionization chamber of 6 cm3. Different geometries were evaluated by varying the thicknesses of solid water, air and cell culture medium. The results showed deviations below 2.2% when compared with the obtained doses of OSLDs and planning system used. Also deviations were observed below 3.4% by eccentric points of the irradiation plane, finding homogeneous dose distribution. Uncertainty in the readings was less than 2%. The proposed methodology contributes a contribution in the dose verification in this type of irradiations, eliminating from the calculation uncertainties, potential errors in settling irradiation or possible equipment failure with which is radiating. It also provides certainty about the survival curves to be plotted with the experimental data. (Author)

  13. Radiosensitivity of cultured insect cells: I. Lepidoptera

    International Nuclear Information System (INIS)

    The radiosensitivity of five lepidopteran insect cell lines representing five different genera has been investigated. These lines are: (1) TN-368, Trichoplusia ni; (2) IPLB-SF-1254, Spodoptera frugiperda; (3) IPLB-1075, Heliothis zea; (4) MRRL-CHl, clone GVl, Manduca sexta; and (5) IAL-PID2, Plodia interpunctella. The cell lines grew at different rates and had population doubling times that ranged from 19 to 52 hr. All of the lines are highly heteroploid and have approximate chromosome numbers near or above 100. The chromosomes are very small. All of the lines are extremely radioresistant; cell populations are able to recover from 260 kVp X-ray exposures up to and including 400 Gy, the highest dose examined. Cell survival curves were obtainable for only the TN-368 and IPLB-SF-1254 lines. The TN-368 cells displayed a biphasic survival response with D0, d/sub q/, and n values of 65.7 and 130.2 Gy, 9.0 and -36.1 Gy, and 1.2 and 0.8, respectively, for the steep and shallow portions of the curve. The IPLB-SF-1254 cells had a D0 of 63.9 Gy. D/sub q/ of 19.0 Gy, and n value of 1.4. These studies provide definitive evidence of the radioresistance of lepidopteran cells, and suggest that this radioresistance is a characteristic of lepidopteran insects

  14. Establishing a stem cell culture laboratory for clinical trials

    Directory of Open Access Journals (Sweden)

    Elíseo Joji Sekiya

    2012-01-01

    Full Text Available Adult stem/progenitor cells are found in different human tissues. An in vitro cell culture is needed for their isolation or for their expansion when they are not available in a sufficient quantity to regenerate damaged organs and tissues. The level of complexity of these new technologies requires adequate facilities, qualified personnel with experience in cell culture techniques, assessment of quality and clear protocols for cell production. The rules for the implementation of cell therapy centers involve national and international standards of good manufacturing practices. However, such standards are not uniform, reflecting the diversity of technical and scientific development. Here standards from the United States, the European Union and Brazil are analyzed. Moreover, practical solutions encountered for the implementation of a cell therapy center appropriate for the preparation and supply of cultured cells for clinical studies are described. Development stages involved the planning and preparation of the project, the construction of the facility, standardization of laboratory procedures and development of systems to prevent cross contamination. Combining the theoretical knowledge of research centers involved in the study of cells with the practical experience of blood therapy services that manage structures for cell transplantation is presented as the best potential for synergy to meet the demands to implement cell therapy centers.

  15. The Chemopreventive Effect of Tanacetum Polycephalum Against LA7-Induced Breast Cancer in Rats and the Apoptotic Effect of a Cytotoxic Sesquiterpene Lactone in MCF7 Cells: A Bioassay-Guided Approach

    Directory of Open Access Journals (Sweden)

    Hamed Karimian

    2015-06-01

    Full Text Available Background: Tanacetum polycephalum L. Schultz-Bip is a member of the Asteraceae family. This study evaluated the chemopreventive effect of a T. polycephalum hexane extract (TPHE using in in vivo and in vitro models. Methods and Results: Five groups of rats: normal control, cancer control, TPHE low dose, TPHE high dose and positive control (tamoxifen were used for the in vivo study. Histopathological examination showed that TPHE significantly suppressed the carcinogenic effect of LA7 tumour cells. The tumour sections from TPHE-treated rats demonstrated significantly reduced expression of Ki67 and PCNA compared to the cancer control group. Using a bioassay-guided approach, the cytotoxic compound of TPHE was identified as a tricyclic sesquiterpene lactone, namely, 8β- hydroxyl- 4β, 15- dihydrozaluzanin C (HDZC. Signs of early and late apoptosis were observed in MCF7 cells treated with HDZC and were attributed to the mitochondrial intrinsic pathway based on the up-regulation of Bax and the down-regulation of Bcl-2. HDZC induced cell cycle arrest in MCF7 cells and increased the expression of p21 and p27 at the mRNA and protein levels. Conclusion: This results of this study substantiate the anticancer effect of TPHE and highlight the involvement of HDZC as one of the contributing compounds that act by initiating mitochondrial-mediated apoptosis.

  16. The replacement of serum by hormones in cell culture media.

    Science.gov (United States)

    Sato, G; Hayashi, I

    1976-12-01

    The replacement of serum by hormones in cell culture media. (Reemplazo del suero por hormonas en el medio de cultivo de células). Arch. Biol. Med. Exper. 10: 120-121, 1976. The serum used in cell culture media can be replaced by a mixture of hormones and some accesory blood factors. The pituitary cell line GH3 can be grown in a medium in which serum is replaced by triiodothyronine, transferrin, parathormone, tyrotrophin releasing hormone and somatomedins. Hela and BHK cell strains can also be grown in serum free medium supplemented with hormones. Each cell type appears to have different hormonal requirements yet it may found that some hormones are required for most cell types. PMID:1026199

  17. Continuous culture of immobilized streptomyces cells for kasugamycin production.

    Science.gov (United States)

    Kim, C J; Chang, Y K; Chun, G T; Jeong, Y H; Lee, S J

    2001-01-01

    Continuous cultures of immobilized Streptomyces kasugaensis, a kasugamycin producer, were carried out on Celite beads. When using a prototype separator for immobilized-cell separation and recycling, the continuous operation could not be sustained for an extended period as a result of an excessive loss of immobilized cells caused by the poor performance of the separator. Accordingly, the immobilized-cell separator was revised to provide better immobilized-cell settling and thus recycling into the reactor. In a subsequent culture using the revised separator, a stable operation was maintained for over 820 h with a high kasugamycin productivity. The kasugamycin productivity ranged from 9.8 to 16.1 mg/L/h, which was about 14- to 23-fold higher than that in a batch suspended-cell culture. When the original feeding medium concentration was doubled at the end of the continuous culture, the productivity became severely impaired for several reasons, which will be discussed. An excessive formation of free cells and loss of immobilized cells through the separator were also observed. PMID:11386865

  18. Isolation, culture and characterization of primary mouse RPE cells.

    Science.gov (United States)

    Fernandez-Godino, Rosario; Garland, Donita L; Pierce, Eric A

    2016-07-01

    Mouse models are powerful tools for the study of ocular diseases. Alterations in the morphology and function of the retinal pigment epithelium (RPE) are common features shared by many ocular disorders. We report a detailed protocol to collect, seed, culture and characterize RPE cells from mice. We describe a reproducible method that we previously developed to collect and culture murine RPE cells on Transwells as functional polarized monolayers. The collection of RPE cells takes ∼3 h, and the cultures mimic in vivo RPE cell features within 1 week. This protocol also describes methods to characterize the cells on Transwells within 1-2 weeks by transmission and scanning electron microscopy (TEM and SEM, respectively), immunostaining of vibratome sections and flat mounts, and measurement of transepithelial electrical resistance. The RPE cell cultures are suitable to study the biology of the RPE from wild-type and genetically modified strains of mice between the ages of 10 d and 12 months. The RPE cells can also be manipulated to investigate molecular mechanisms underlying the RPE pathology in the numerous mouse models of ocular disorders. Furthermore, modeling the RPE pathology in vitro represents a new approach to testing drugs that will help accelerate the development of therapies for vision-threatening disorders such as macular degeneration (MD). PMID:27281648

  19. Differential heat shock response of primary human cell cultures and established cell lines

    DEFF Research Database (Denmark)

    Richter, W W; Issinger, O G

    1986-01-01

    degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized a...

  20. Formation and action of oxygen activated species in cell cultures

    International Nuclear Information System (INIS)

    The differences of hydrogen peroxide sensibility of mammal cell lineages (man, mouse, chinese hamster) in culture are studied. The cellular survival and the frequency of DNA induced breaks by hydrogen peroxide are analysed. The efficiency of elimination of DNA breaks by cells is determined. The possible relation between the cell capacity of repair and its survival to hydrogen peroxide action is also discussed. (M.A.)

  1. Polyphosphoinositides are present in plant tissue culture cells

    International Nuclear Information System (INIS)

    Polyphosphoinositides have been isolated from wild carrot cells grown in suspension culture. This is the first report of polyphosphoinositides in plant cells. The phospholipids were identified by comigration with known standards on thin-layer plates. After overnight labeling of the cells with myo-[2-3H] inositol, the phosphoinositides as percent recovered inositol were 93% phosphatidylinositol., 3.7% lysophosphatidylinositol, 1.7% phosphatidylinositol monophosphate, 0.8% phosphatidylinositol bisphosphate

  2. Culture of Neural Stem Cells in Calcium-alginate Microbeads

    Institute of Scientific and Technical Information of China (English)

    Li-Song YAO; Tian-Qing LIU; Dan GE; Xue-Hu MA; Zhan-Feng CUI

    2005-01-01

    @@ 1 Introduction Recent research shows that neural stem cells may play an important role in the nerve injury reparation and nerve disease treatment. The shortage of the source and the number of NSCs, however, is the main challenge for its clinic application. In this situation, expansion of NSCs in large scale and culture in three dimensional environment are very worth of exploration. Notablely, the shear stress existed in bioreactors can cause serious cell injury especially for the shear sensitive cells like NSCs.

  3. Culture of Neural Stem Cells in Calcium-alginate Microbeads

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    1 Introduction Recent research shows that neural stem cells may play an important role in the nerve injury reparation and nerve disease treatment. The shortage of the source and the number of NSCs, however, is the main challenge for its clinic application. In this situation, expansion of NSCs in large scale and culture in three dimensional environment are very worth of exploration. Notablely, the shear stress existed in bioreactors can cause serious cell injury especially for the shear sensitive cells like ...

  4. Epithelial morphogenesis in three-dimensional cell culture system

    OpenAIRE

    Liu, Mengfei; 刘梦菲

    2014-01-01

    In human body, the most common structures formed by epithelial cells are hollow cysts or tubules. The key feature of the cysts and tubules is the central lumen, which is lined by epithelial cell sheets. The central lumen allows material exchange, thus it is indispensable for the proper function of the epithelial tissue. In order to understand the way that the epithelial cells form highly specialized structure, an in vitro three-dimensional (3D) culture system was established. The Caco-2 c...

  5. Carbon Nanotubes-Based Electrochemical Sensing for Cell Culture Monitoring

    OpenAIRE

    Boero, Cristina; Carrara, Sandro; Del Vecchio, Giovanna; Albini, Giuseppe D.; Calzà, Laura; De Micheli, Giovanni

    2010-01-01

    Monitoring of metabolic compounds, such as glucose and lactate, is extensively reported in literature, especially for clinical purposes. Instead, the application of such technologies for monitoring metabolites in cell cultures has not been explored. From one side, such devices can provide information to the current state-of-the-art of cell lines, particularly those which are not fully known, as stem and embryonic cells. On the other hand, those systems can pave the way to fully automation for...

  6. Optimization of Seeding Density in Microencapsulated Recombinant CHO Cell Culture

    OpenAIRE

    Zhang, Ying; Zhou, Jing; Zhang, Xulang; Yu, Weiting; Guo, Xin; Wang, Wei; Ma, Xiaojun

    2008-01-01

    Microencapsulation technology is an alternative large-scale mammalian cell culture method. The semi-permeable membrane of the microcapsule allows free diffusion of nutrients, oxygen and toxic metabolites to support cell growth, and the microcapsule membrane can protect the cells from the mechanical damage of shear forces associated with agitation and aeration. Many polymers have been used to make microcapsules, such as chitosan, polyacrylates, alginate, polyamino acids, and polyamides. One of...

  7. Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

    Science.gov (United States)

    Stover, Alexander E.; Herculian, Siranush; Banuelos, Maria G.; Navarro, Samantha L.; Jenkins, Michael P.; Schwartz, Philip H.

    2016-01-01

    This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy. PMID:27341536

  8. Specimen Sample Preservation for Cell and Tissue Cultures

    Science.gov (United States)

    Meeker, Gabrielle; Ronzana, Karolyn; Schibner, Karen; Evans, Robert

    1996-01-01

    The era of the International Space Station with its longer duration missions will pose unique challenges to microgravity life sciences research. The Space Station Biological Research Project (SSBRP) is responsible for addressing these challenges and defining the science requirements necessary to conduct life science research on-board the International Space Station. Space Station will support a wide range of cell and tissue culture experiments for durations of 1 to 30 days. Space Shuttle flights to bring experimental samples back to Earth for analyses will only occur every 90 days. Therefore, samples may have to be retained for periods up to 60 days. This presents a new challenge in fresh specimen sample storage for cell biology. Fresh specimen samples are defined as samples that are preserved by means other than fixation and cryopreservation. The challenge of long-term storage of fresh specimen samples includes the need to suspend or inhibit proliferation and metabolism pending return to Earth-based laboratories. With this challenge being unique to space research, there have not been any ground based studies performed to address this issue. It was decided hy SSBRP that experiment support studies to address the following issues were needed: Fixative Solution Management; Media Storage Conditions; Fresh Specimen Sample Storage of Mammalian Cell/Tissue Cultures; Fresh Specimen Sample Storage of Plant Cell/Tissue Cultures; Fresh Specimen Sample Storage of Aquatic Cell/Tissue Cultures; and Fresh Specimen Sample Storage of Microbial Cell/Tissue Cultures. The objective of these studies was to derive a set of conditions and recommendations that can be used in a long duration microgravity environment such as Space Station that will permit extended storage of cell and tissue culture specimens in a state consistent with zero or minimal growth, while at the same time maintaining their stability and viability.

  9. Expression of CD44 in Cultured Human Trabecular Meshwork Cells

    Institute of Scientific and Technical Information of China (English)

    Zhongguo Li; Hong Zhang

    2004-01-01

    Purpose:To determine whether cultured human trabecular meshwork cells express CD44 and to discuss their possible relationship with primary open angle glaucoma.Methods:Human trabecular meshwork cells were cultured in DMEM/F12 media. Total RNAs from the cells were extracted with Trizol reagent. Messenger RNA expression of CD44 in human trabecular meshwork cells was examined by using reverse transcriptasepolymerase chain reaction ( RT-PCR ) analysis. Expression of CD44 was confirmed by Western-blotting and immunofiuorescent microscopy. Effect of CD44-specific antisense oligonucleotide on adhesion of trabecular meshwork cells to hyaluronate was determined by MTT assay.Results:A single RT-PCR product whose size was 471bp was obtained.A band about 80kD was stained by Western-blot. Immunofiuorescent examination of expression of CD44 on the cell surface was positive and reactions were mainly localized in cell membranes.Adhesion of trabecular meshwork cells to hyaluronate was inhibited by CD44-specific antisense oligonucleotide.Conclusions: Cultured human trabecular meshwork cells express CD44. CD44 may play a role in pathogenesis of primary open angle glaucoma. Eye Science 2004;20:52-56.

  10. Preparation of 14C-catechins by tea cell culture

    International Nuclear Information System (INIS)

    The preparation of 14C labelled catechins was studied by feeding 14C labelled precursor to tea cultured cells. Two labelled precursors were tested and their effects were compared. The dynamics of absorption and transformation of fed precursors were analyzed and the effects of pre-culture as well as UV light pretreatment on product labelling rate were evaluated. Product analysis was also made by HSCCC and HPLC techniques

  11. Enhanced casein kinase II activity in human tumour cell cultures

    DEFF Research Database (Denmark)

    Prowald, K; Fischer, H; Issinger, O G

    1984-01-01

    Casein kinase II (CKII) activity is enhanced as much as 2-3 fold in established and 4-5-fold in transformed human cell lines when compared to that of fibroblasts and primary human tumour cell cultures where CKII activity never exceeded a basic level. The high activity of CKII in transformed cells...... and in established cell lines was reduced to about the same basic level after treatment with heparin, a highly specific inhibitor of CKII activity. The activity of the cAMP-dependent protein kinase was virtually the same in fibroblasts and various human tumour cell lines investigated....

  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...

  13. Hollow fiber clinostat for simulating microgravity in cell culture

    Science.gov (United States)

    Rhodes, Percy H. (Inventor); Miller, Teresa Y. (Inventor); Snyder, Robert S. (Inventor)

    1992-01-01

    A clinostat for simulating microgravity on cell systems carried in a fiber fixedly mounted in a rotatable culture vessel is disclosed. The clinostat is rotated horizontally along its longitudinal axis to simulate microgravity or vertically as a control response. Cells are injected into the fiber and the ends of the fiber are sealed and secured to spaced end pieces of a fiber holder assembly which consists of the end pieces, a hollow fiber, a culture vessel, and a tension spring with three alignment pins. The tension spring is positioned around the culture vessel with its ends abutting the end pieces for alignment of the spring. After the fiber is secured, the spring is decompressed to maintain tension on the fiber while it is being rotated. This assures that the fiber remains aligned along the axis of rotation. The fiber assembly is placed in the culture vessel and culture medium is added. The culture vessel is then inserted into the rotatable portion of the clinostat and subjected to rotate at selected rpms. The internal diameter of the hollow fiber determines the distance the cells are from the axis of rotation.

  14. Virtual screening of bioassay data

    Directory of Open Access Journals (Sweden)

    Schierz Amanda C

    2009-12-01

    Full Text Available Abstract Background There are three main problems associated with the virtual screening of bioassay data. The first is access to freely-available curated data, the second is the number of false positives that occur in the physical primary screening process, and finally the data is highly-imbalanced with a low ratio of Active compounds to Inactive compounds. This paper first discusses these three problems and then a selection of Weka cost-sensitive classifiers (Naive Bayes, SVM, C4.5 and Random Forest are applied to a variety of bioassay datasets. Results Pharmaceutical bioassay data is not readily available to the academic community. The data held at PubChem is not curated and there is a lack of detailed cross-referencing between Primary and Confirmatory screening assays. With regard to the number of false positives that occur in the primary screening process, the analysis carried out has been shallow due to the lack of cross-referencing mentioned above. In six cases found, the average percentage of false positives from the High-Throughput Primary screen is quite high at 64%. For the cost-sensitive classification, Weka's implementations of the Support Vector Machine and C4.5 decision tree learner have performed relatively well. It was also found, that the setting of the Weka cost matrix is dependent on the base classifier used and not solely on the ratio of class imbalance. Conclusions Understandably, pharmaceutical data is hard to obtain. However, it would be beneficial to both the pharmaceutical industry and to academics for curated primary screening and corresponding confirmatory data to be provided. Two benefits could be gained by employing virtual screening techniques to bioassay data. First, by reducing the search space of compounds to be screened and secondly, by analysing the false positives that occur in the primary screening process, the technology may be improved. The number of false positives arising from primary screening leads to

  15. A microwell cell culture platform for the aggregation of pancreatic β-cells.

    Science.gov (United States)

    Bernard, Abigail B; Lin, Chien-Chi; Anseth, Kristi S

    2012-08-01

    Cell-cell contact between pancreatic β-cells is important for maintaining survival and normal insulin secretion. Various techniques have been developed to promote cell-cell contact between β-cells, but a simple yet robust method that affords precise control over three-dimensional (3D) β-cell cluster size has not been demonstrated. To address this need, we developed a poly(ethylene glycol) (PEG) hydrogel microwell platform using photolithography. This microwell cell-culture platform promotes the formation of 3D β-cell aggregates of defined sizes from 25 to 210 μm in diameter. Using this platform, mouse insulinoma 6 (MIN6) β-cells formed aggregates with cell-cell adherin junctions. These naturally formed cell aggregates with controllable sizes can be removed from the microwells for macroencapsulation, implantation, or other biological assays. When removed and subsequently encapsulated in PEG hydrogels, the aggregated cell clusters demonstrated improved cellular viability (>90%) over 7 days in culture, while the β-cells encapsulated as single cells maintained only 20% viability. Aggregated MIN6 cells also exhibited more than fourfold higher insulin secretion in response to a glucose challenge compared with encapsulated single β-cells. Further, the cell aggregates stained positively for E-cadherin, indicative of the formation of cell junctions. Using this hydrogel microwell cell-culture method, viable and functional β-cell aggregates of specific sizes were created, providing a platform from which other biologically relevant questions may be answered. PMID:22320435

  16. Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions

    OpenAIRE

    Saha, Krishanu; Mei, Ying; Reisterer, Colin M.; Pyzocha, Neena Kenton; Yang, Jing; Muffat, Julien; Davies, Martyn C.; Alexander, Morgan R.; Langer, Robert; Anderson, Daniel G.; Jaenisch, Rudolf

    2011-01-01

    The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here, we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells, as predicted from a numerical model of cell...

  17. Lingual Epithelial Stem Cells and Organoid Culture of Them

    Directory of Open Access Journals (Sweden)

    Hiroko Hisha

    2016-01-01

    Full Text Available As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP, were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine.

  18. Cancer Stem Cells: Cell Culture, Markers and Targets for New Therapies

    OpenAIRE

    Gilbert, Candace A.; Ross, Alonzo H.

    2009-01-01

    A cancer stem cell is defined as an undifferentiated cell with the ability to self-renew, differentiate to multiple lineages and initiate tumors that mimic the parent tumor. In this review, we focus on glioblastomas, describing recent progress and problems in characterizing these cells. There have been advances in cancer stem cell culture, but tumor cell heterogeneity has made purification of cancer stem cells difficult. Indeed, it may be that cancer stem cells significantly vary from tumor t...

  19. Metabolism of quercetin in cell suspension culture of Nicotiana tabacum

    International Nuclear Information System (INIS)

    Quercetin was oxidized on a rotating glassy-carbon electrode in phosphate-methanol buffer. The half-wave potentials for several pH were determined. Oxidation of quercetin by one of Nicotiana tabacum cell suspension cultures was carried out in vitro and final products were characterized by means of UV spectra and mass-spectrophotometry. Each product of oxidation was assayed for oxygen consumption inhibiting activity, using a 3 days old cell suspension culture of Nicotiana tabacum. Dimers and polymers showed strong inhibiting activity in O-2 consumption

  20. Dynamic cell culture system: a new cell cultivation instrument for biological experiments in space

    Science.gov (United States)

    Gmunder, F. K.; Nordau, C. G.; Tschopp, A.; Huber, B.; Cogoli, A.

    1988-01-01

    The prototype of a miniaturized cell cultivation instrument for animal cell culture experiments aboard Spacelab is presented (Dynamic cell culture system: DCCS). The cell chamber is completely filled and has a working volume of 200 microliters. Medium exchange is achieved with a self-powered osmotic pump (flowrate 1 microliter h-1). The reservoir volume of culture medium is 230 microliters. The system is neither mechanically stirred nor equipped with sensors. Hamster kidney (Hak) cells growing on Cytodex 3 microcarriers were used to test the biological performance of the DCCS. Growth characteristics in the DCCS, as judged by maximal cell density, glucose consumption, lactic acid secretion and pH, were similar to those in cell culture tubes.

  1. Cell sources for in vitro human liver cell culture models.

    Science.gov (United States)

    Zeilinger, Katrin; Freyer, Nora; Damm, Georg; Seehofer, Daniel; Knöspel, Fanny

    2016-09-01

    In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described. PMID:27385595

  2. Pedigree analysis of proliferation kinetics in cultured mammalian cells

    International Nuclear Information System (INIS)

    Quantitative analysis of proliferation kinetics in cultured mammalian cells was given an outline by extremely low speed photography (pedigree method). Photographing method of this analysis, camera used in this analysis, cultivation method and apparatus for cultivation, and film analysis were explained. As to changes of generation time by this analysis, relationship of each stage of cell cycle to colony formation or to proliferation capacity were explained in non-irradiated cells and irradiated cells. On abnormal cell division in time of large dose irradiation, a condition from cell fusion to cell death via multipolar division was explained. Mechanisms of proliferation death and interphase death were explained by analysis of pedigree data on radiation injuries in time of division and by mentioning division probability. Some information about inhibition of cell proliferation by radiation and lethal effect of radiation was described. (Kanao, N.)

  3. Metabolic flux rewiring in mammalian cell cultures

    OpenAIRE

    Young, Jamey D.

    2013-01-01

    Continuous cell lines (CCLs) engage in “wasteful” glucose and glutamine metabolism that leads to accumulation of inhibitory byproducts, primarily lactate and ammonium. Advances in techniques for mapping intracellular carbon fluxes and profiling global changes in enzyme expression have led to a deeper understanding of the molecular drivers underlying these metabolic alterations. However, recent studies have revealed that CCLs are not necessarily entrenched in a glycolytic or glutaminolytic phe...

  4. Differential Effect of Culture Temperature and Specific Growth Rate on CHO Cell Behavior in Chemostat Culture

    OpenAIRE

    Vergara, Mauricio; Becerra, Silvana; Berrios, Julio; Osses, Nelson; Reyes, Juan; Rodríguez-Moyá, María; Gonzalez, Ramon; Altamirano, Claudia

    2014-01-01

    Mild hypothermia condition in mammalian cell culture technology has been one of the main focuses of research for the development of breeding strategies to maximize productivity of these production systems. Despite the large number of studies that show positive effects of mild hypothermia on specific productivity of r-proteins, no experimental approach has addressed the indirect effect of lower temperatures on specific cell growth rate, nor how this condition possibly affects less specific pro...

  5. Culture and characterization of rat hair follicle stem cells.

    Science.gov (United States)

    Quan, Renfu; Zheng, Xuan; Ni, Yueming; Xie, Shangju; Li, Changming

    2016-08-01

    The purpose of this study was to establish methods for isolation, culture, expansion, and characterization of rat hair follicle stem cells (rHFSCs). Hair follicles were harvested from 1-week-old Sprague-Dawley rats and digested with dispase and collagenase IV. The bulge of the hair follicle was dissected under a microscope and cultured in Dulbecco's modified Eagle's medium/F12 supplemented with KnockOut™ Serum Replacement serum substitute, penicillin-streptomycin, L-glutamine, non-essential amino acids, epidermal growth factor, basic fibroblast growth factor, polyhydric alcohol, and hydrocortisone. The rHFSCs were purified using adhesion to collagen IV. Cells were characterized by detecting marker genes with immunofluorescent staining and real-time polymerase chain reaction (PCR). The proliferation and vitality of rHFSCs at different passages were evaluated. The cultured rHFSCs showed typical cobblestone morphology with good adhesion and colony-forming ability. Expression of keratin 15, integrin α6, and integrin β1 were shown by immunocytochemistry staining. On day 1-2, the cells were in the latent phase. On day 5-6, the cells were in the logarithmic phase. Cell vitality gradually decreased from the 7th passage. Real-time PCR showed that the purified rHFSCs had good vitality and proliferative capacity and contained no keratinocytes. Highly purified rHFSCs can be obtained using tissue culture and adhesion to collagen IV. The cultured cells had good proliferative capacity and could therefore be a useful cell source for tissue-engineered hair follicles, vessels, and skin. PMID:25407732

  6. Cell culture systems for the hepatitis C virus

    Institute of Scientific and Technical Information of China (English)

    Gilles Duverlie; Czeslaw Wychowski

    2007-01-01

    Since the discovery of HCV in 1989, the lack of a cell culture system has hampered research progress on this important human pathogen. No robust system has been obtained by empiric approaches, and HCV cell culture remained hypothetical until 2005. The construction of functional molecular clones has served as a starting point to reconstitute a consensus infectious cDNA that was able to transcribe infectious HCV RNAs as shown by intrahepatic inoculation in a chimpanzee. Other consensus clones have been selected and established in a human hepatoma cell line as replicons, i.e. self-replicating subgenomic or genomic viral RNAs. However, these replicons did not support production of infectious virus. Interestingly, some full-length replicons could be established without adaptive mutations and one of them was able to replicate at very high levels and to release virus particles that are infectious in cell culture and in vivo. This new cell culture system represents a major breakthrough in the HCV field and should enable a broad range of basic and applied studies to be achieved.

  7. Immunological assays for chemokine detection in in-vitro culture of CNS cells

    OpenAIRE

    Mahajan Supriya D.; Schwartz Stanley A; Nair Madhavan P.N.

    2003-01-01

    Herein we review the various methods currently in use for determining the expression of chemokines by CNS cells in vitro. Chemokine detection assays are used in conjuction with one another to provide a comprehensive, biologically relevant assessment of the chemokines which is necessary for correct data interpretation of a specific observed biological effect. The methods described include bioassays for soluble chemokine receptors, RNA extraction, RT-PCR, Real - time quantitative PCR, gene arra...

  8. Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Ping, E-mail: fanpinggoodluck@163.com [Department of Rheumatism and Immunity, The First Affiliated Hospital Xi' an Jiaotong University School of Medicine, Xi' an, Shaanxi 710061 (China); He, Lan; Pu, Dan; Lv, Xiaohong; Zhou, Wenxu; Sun, Yining; Hu, Nan [Department of Rheumatism and Immunity, The First Affiliated Hospital Xi' an Jiaotong University School of Medicine, Xi' an, Shaanxi 710061 (China)

    2011-01-21

    Research highlights: {yields} The proliferation of dramatic increased by co-cultured with Sertoli cells. {yields} VEGF receptor-2 expression of ECs was up-regulated by co-cultured with Sertoli cells. {yields} The MHC expression of ECs induced by INF-{gamma} and IL-6, IL-8 and sICAM induced by TNF-{alpha} decreased respectively after co-cultured with Sertoli cells. {yields} ECs co-cultured with Sertoli cells also didn't increase the stimulation index of spleen lymphocytes. -- Abstract: The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertoli cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 x 10{sup 3}, 1 x 10{sup 4} or 1 x 10{sup 5} cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-{gamma} and TNF-{alpha} were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 x 10{sup 4} cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P < 0.05). Western blotting showed that 1 x 10{sup 4} cell/ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli

  9. Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells

    International Nuclear Information System (INIS)

    Research highlights: → The proliferation of dramatic increased by co-cultured with Sertoli cells. → VEGF receptor-2 expression of ECs was up-regulated by co-cultured with Sertoli cells. → The MHC expression of ECs induced by INF-γ and IL-6, IL-8 and sICAM induced by TNF-α decreased respectively after co-cultured with Sertoli cells. → ECs co-cultured with Sertoli cells also didn't increase the stimulation index of spleen lymphocytes. -- Abstract: The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertoli cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 x 103, 1 x 104 or 1 x 105 cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-γ and TNF-α were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 x 104 cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P 4 cell/ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli cells can effectively suppress INF-γ-induced MHC II antigen expression in co-cultured ECs compared with single culture

  10. Enhancement effect of shikonin in cell suspension culture and transfermanant culture by radiation application

    International Nuclear Information System (INIS)

    The cell lines 679, 679-29 and 622-46 of L. erythrorhizon could be selected on LS agar medium for the production shikonin in cell suspension culture. The shikonin was increased moderately in suspension culture of cell line 622-46 in LS liquid medium containing BA 2 mg·L-1 and IAA 0.2 mg·L-1 in the dark, and was increased by adding 1 μM Cu2+ and 100 μM methyl jasmonate The accumulation of shikonin in the liquid medium was increased significantly by 2 Gy irradiation to callus of cell line 622-46 and culture in LS liquid medium containing BA 2 mg·L-1 and IAA 0.2 mg·L-1 in the dark and shikonin in cell debris was higher by 16 Gy irradiation. The activity of p-hydroxybenzoate geranyltransferase was increased by irradiation of 2 Gy and 16 Gy of γ radiation. Seedling hypocotyles of L. erythrorhizon were infected with Agrogacterium rhizogenes strain 15834 harboring a binary vector with an intron bearing the GUS (β-glucuronidase) gene driven by cauliflower mosaic virus (CaMV) 35S promotor as well as the HPT (hygromycin phosphotransferase) gene as the selection marker. Hairy roots isolated were hygromycin resistant and had integrated GUS gene in DNA. The root tip grown on M-9 medium showed normal pigment production pattern in border cells and root hairs

  11. Expansion and properties of human bone marrow stromal cells cultured in different culture media

    Czech Academy of Sciences Publication Activity Database

    Syrová, Zdeňka; Glogarová, Kateřina; Jendelová, Pavla; Syková, Eva

    2006-01-01

    Roč. 8, Supplement 1 (2006), s. 238-238. ISSN 1465-3249. [ ISCT 2006. 04.05.2006-07.05.2006, Berlin] R&D Projects: GA MZd(CZ) NR8339; GA MŠk 1M0538 Institutional research plan: CEZ:AV0Z50390512 Keywords : Cells cultured Subject RIV: FH - Neurology

  12. A novel bioassay for the activity determination of therapeutic human brain natriuretic peptide (BNP.

    Directory of Open Access Journals (Sweden)

    Lei Yu

    Full Text Available BACKGROUND: Recombinant human brain natriuretic peptide (rhBNP is an important peptide-based therapeutic drug indicated for the treatment of acute heart failure. Accurate determination of the potency of therapeutic rhBNP is crucial for the safety and efficacy of the drug. The current bioassay involves use of rabbit aortic strips, with experiments being complicated and time-consuming and markedly variable in results. Animal-less methods with better precision and accuracy should be explored. We have therefore developed an alternative cell-based assay, which relies on the ability of BNP to induce cGMP production in HEK293 cells expressing BNP receptor guanylyl cyclase-A. METHODOLOGY/PRINCIPAL FINDINGS: An alternative assay based on the measurement of BNP-induced cGMP production was developed. Specifically, the bioassay employs cells engineered to express BNP receptor guanylyl cyclase-A (GCA. Upon rhBNP stimulation, the levels of the second messager cGMP in these cells drastically increased and subsequently secreted into culture supernatants. The quantity of cGMP, which corresponds to the rhBNP activity, was determined using a competitive ELISA developed by us. Compared with the traditional assay, the novel cell-based assay demonstrated better reproducibility and precision. CONCLUSION/SIGNIFICANCE: The optimized cell-based assay is much simpler, more rapid and precise compared with the traditional assay using animal tissues. To our knowledge, this is the first report on a novel and viable alternative assay for rhBNP potency analysis.

  13. Arsenic exposure induces the Warburg effect in cultured human cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Fei; Severson, Paul; Pacheco, Samantha; Futscher, Bernard W.; Klimecki, Walter T., E-mail: klimecki@pharmacy.arizona.edu

    2013-08-15

    Understanding how arsenic exacts its diverse, global disease burden is hampered by a limited understanding of the particular biological pathways that are disrupted by arsenic and underlie pathogenesis. A reductionist view would predict that a small number of basic pathways are generally perturbed by arsenic, and manifest as diverse diseases. Following an initial observation that arsenite-exposed cells in culture acidify their media more rapidly than control cells, the report here shows that low level exposure to arsenite (75 ppb) is sufficient to induce aerobic glycolysis (the Warburg effect) as a generalized phenomenon in cultured human primary cells and cell lines. Expanded studies in one such cell line, the non-malignant pulmonary epithelial line, BEAS-2B, established that the arsenite-induced Warburg effect was associated with increased accumulation of intracellular and extracellular lactate, an increased rate of extracellular acidification, and inhibition by the non-metabolized glucose analog, 2-deoxy-D-glucose. Associated with the induction of aerobic glycolysis was a pathway-wide induction of glycolysis gene expression, as well as protein accumulation of an established glycolysis master-regulator, hypoxia-inducible factor 1A. Arsenite-induced alteration of energy production in human cells represents the type of fundamental perturbation that could extend to many tissue targets and diseases. - Highlights: • Chronic arsenite exposure induces aerobic glycolysis, dubbed the “Warburg effect”. • Arsenite-induced Warburg effect is a general phenomenon in cultured human cells. • HIF-1A may mediate arsenite induced Warburg effect.

  14. Testing of serum atherogenicity in cell cultures: questionable data published

    Directory of Open Access Journals (Sweden)

    Sergei V. Jargin

    2012-01-01

    Full Text Available In a large series of studies was reported that culturing of smooth muscle cells with serum from atherosclerosis patients caused intracellular lipid accumulation, while serum from healthy controls had no such effect. Cultures were used for evaluation of antiatherogenic drugs. Numerous substances were reported to lower serum atherogenicity: statins, trapidil, calcium antagonists, garlic derivatives etc. On the contrary, beta-blockers, phenothiazines and oral hypoglycemics were reported to be pro-atherogenic. Known antiatherogenic agents can influence lipid metabolism and cholesterol synthesis, intestinal absorption or endothelium-related mechanisms. All these targets are absent in cell monocultures. Inflammatory factors, addressed by some antiatherogenic drugs, are also not reproduced. In vivo, relationship between cholesterol uptake by cells and atherogenesis must be inverse rather than direct: in familial hypercholesterolemia, inefficient clearance of LDL-cholesterol by cells predisposes to atherosclerosis. Accordingly, if a pharmacological agent reduces cholesterol uptake by cells in vitro, it should be expected to elevate cholesterol in vivo. Validity of clinical recommendations, based on serum atherogenicity testing in cell monocultures, is therefore questionable. These considerations pertain also to the drugs developed on the basis of the cell culture experiments.

  15. Arsenic exposure induces the Warburg effect in cultured human cells

    International Nuclear Information System (INIS)

    Understanding how arsenic exacts its diverse, global disease burden is hampered by a limited understanding of the particular biological pathways that are disrupted by arsenic and underlie pathogenesis. A reductionist view would predict that a small number of basic pathways are generally perturbed by arsenic, and manifest as diverse diseases. Following an initial observation that arsenite-exposed cells in culture acidify their media more rapidly than control cells, the report here shows that low level exposure to arsenite (75 ppb) is sufficient to induce aerobic glycolysis (the Warburg effect) as a generalized phenomenon in cultured human primary cells and cell lines. Expanded studies in one such cell line, the non-malignant pulmonary epithelial line, BEAS-2B, established that the arsenite-induced Warburg effect was associated with increased accumulation of intracellular and extracellular lactate, an increased rate of extracellular acidification, and inhibition by the non-metabolized glucose analog, 2-deoxy-D-glucose. Associated with the induction of aerobic glycolysis was a pathway-wide induction of glycolysis gene expression, as well as protein accumulation of an established glycolysis master-regulator, hypoxia-inducible factor 1A. Arsenite-induced alteration of energy production in human cells represents the type of fundamental perturbation that could extend to many tissue targets and diseases. - Highlights: • Chronic arsenite exposure induces aerobic glycolysis, dubbed the “Warburg effect”. • Arsenite-induced Warburg effect is a general phenomenon in cultured human cells. • HIF-1A may mediate arsenite induced Warburg effect

  16. Adenosine formation in contracting primary rat skeletal muscle cells and endothelial cells in culture

    DEFF Research Database (Denmark)

    Hellsten, Ylva; Frandsen, Ulrik

    1997-01-01

    1. The present study examined the capacity for adenosine formation, uptake and metabolism in contracting primary rat muscle cells and in microvascular endothelial cells in culture. 2. Strong and moderate electrical simulation of skeletal muscle cells led to a significantly greater increase in the...... extracellular adenosine concentration (421 +/- 91 and 235 +/- 30 nmol (g protein)-1, respectively; P < 0.05) compared with non-stimulated muscle cells (161 +/- 20 nmol (g protein)-1). The ATP concentration was lower (18%; P < 0.05) in the intensely contracted, but not in the moderately contracted muscle cells....... 3. Addition of microvascular endothelial cells to the cultured skeletal muscle cells enhanced the contraction-induced accumulation of extracellular adenosine (P < 0.05), whereas endothelial cells in culture alone did not cause extracellular accumulation of adenosine. 4. Skeletal muscle cells were...

  17. PDMS/glass microfluidic cell culture system for cytotoxicity tests and cells passage

    DEFF Research Database (Denmark)

    Ziolkowska, K.; Jedrych, E.; Kwapiszewski, R.; Lopacinska, Joanna M.; Skolimowski, Maciej; Chudy, M.

    2010-01-01

    In this paper, hybrid (PDMS/glass) microfluidic cell culture system (MCCS) integrated with the concentration gradient generator (CGG) is presented. PDMS gas permeability enabled cells' respiration in the fabricated microdevices and excellent glass hydrophilicity allowed successful cells' seeding....... The human lung carcinoma cells (A549) were cultured in the microdevice for several days. The growth and proliferation of cells was monitored using an inverted fluorescence microscope. After the cells' confluence was achieved in the microchambers, the novel method of cells' passaging in the designed...... microdevice was developed and successfully tested. The MCCS microdevice is fully reusable, i.e. it can be used several times for various cell culture and cytotoxic experiments. The suitability of designed MCCS for cell-based cytotoxicity assay application was verified using 1,4-dioxane as a model toxic agent...

  18. Effects of Visible Light on Cultured Bovine Trabecular Cells

    Institute of Scientific and Technical Information of China (English)

    姜发纲; 郝风芹; 魏厚仁; 许德胜

    2004-01-01

    To explore the biological effects of light on trabecular cells, cultured bovine trabecular cells were exposed to visible light of different wavelength with different energy. Cellular morphology, structure, proliferation, and phagocytosis were observed. The cells showed no remarkable changes when the energy was low. When the exposure energy reached 1. 12 mW/cm2 , the cytoplasm showed a rough appearance, and cell proliferation and phagocytosis decreased. This phototoxicity was strong with white light (compound chromatic light), moderate with violet light or yellow light, and mild with red light.

  19. Kinetics of meta-iodo-benzylguanidine in neuroblastoma cell cultures

    International Nuclear Information System (INIS)

    The continuously cultured human neuroblastoma cell line SK-N-SH possesses an active transport mechanism for uptake of meta-iodobenzylguanidine (MIBG). The uptake rate at an MIBG-concentration of 6,4x10-8 M was 0.35x10-12 mol/minx106 cells, corresponding to values as measured in human pheochromocytoma cell lines. MIBG is released from the cells with a biological half-life of 81,3 h in correspondence to half-life values as measured in vivo in neuroblastoma patients. (orig.)

  20. Hybridoma cell behaviour in continuous culture under hyperosmotic stress.

    Science.gov (United States)

    Cherlet, M; Marc, A

    1999-01-01

    In this paper, we propose an alternative strategy to the ones proposed before (Oh et al., 1993; Øyaas et al., 1994a) to get real increases of global final antibody titer and production at hyperosmotic stress, by reducing the detrimental effect of such a stress on cell growth, and conserving the stimulating effect on antibody production. It consists of cultivating the cells in continuous culture and increasing the osmolality stepwise. In this way, the cells could progressively adapt to the higher osmolality at each step and antibody titers could be nearly doubled at 370 and 400 mOsm kg-1, compared to the standard osmolality of 335 mOsm kg-1. Surprisingly, the stimulation of antibody production was not confirmed for higher osmolalities, 425 and 450 mOsm kg- 1, despite the minor negative effect on cell growth. Intracellular IgG analysis by flow cytometry revealed at these osmolalities a significant population of non-producing cells. However, even when taking into account this non-producing population, a stimulating effect on antibody production could not be shown at these highest osmolalities. It seems to us that osmolality has a significant effect on the appearance of these non-producing cells, since they were not observed in continuous cultures at standard osmolality, of comparable duration and at an even higher dilution rate. The appearance of the non-producing cells coincides furthermore with modifications of the synthesised antibody, as shown by electrophoretic techniques. It is however not really clear if these two observations reflect actually the same phenomenon. Hyperosmolality affects the cell behaviour in continuous culture in multiple ways, independently of the growth rate, counting all at least partially for the observed stimulation of antibody production: acceleration of the amino acid, and in particular the glutamine metabolism, increase of the cell volume, increase of the intracellular pH and accumulation of cells in the G1 cell cycle phase. PMID

  1. Isolation and culture of umbilical vein mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    D.T. Covas

    2003-09-01

    Full Text Available Bone marrow contains a population of stem cells that can support hematopoiesis and can differentiate into different cell lines including adipocytes, osteocytes, chondrocytes, myocytes, astrocytes, and tenocytes. These cells have been denoted mesenchymal stem cells. In the present study we isolated a cell population derived from the endothelium and subendothelium of the umbilical cord vein which possesses morphological, immunophenotypical and cell differentiation characteristics similar to those of mesenchymal stem cells isolated from bone marrow. The cells were isolated from three umbilical cords after treatment of the umbilical vein lumen with collagenase. The cell population isolated consisted of adherent cells with fibroblastoid morphology which, when properly stimulated, gave origin to adipocytes and osteocytes in culture. Immunophenotypically, this cell population was found to be positive for the CD29, CD13, CD44, CD49e, CD54, CD90 and HLA-class 1 markers and negative for CD45, CD14, glycophorin A, HLA-DR, CD51/61, CD106, and CD49d. The characteristics described are the same as those presented by bone marrow mesenchymal stem cells. Taken together, these findings indicate that the umbilical cord obtained from term deliveries is an important source of mesenchymal stem cells that could be used in cell therapy protocols.

  2. Cannabinoids induce incomplete maturation of cultured human leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Murison, G.; Chubb, C.B.H.; Maeda, S.; Gemmell, M.A.; Huberman, E.

    1987-08-01

    Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 ..mu..M ..delta../sup 9/-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody of the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 ..mu..M THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. The THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype. However, treatment of these incompletely matured cells with either phorbol 12-myristate 13-acetate of 1..cap alpha..,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced incomplete cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells.

  3. Culturing intestinal stem cells: applications for colorectal cancer research

    OpenAIRE

    Fujii, Masayuki; Sato, Toshiro

    2014-01-01

    Recent advance of sequencing technology has revealed genetic alterations in colorectal cancer (CRC). The biological function of recurrently mutated genes has been intensively investigated through mouse genetic models and CRC cell lines. Although these experimental models may not fully reflect biological traits of human intestinal epithelium, they provided insights into the understanding of intestinal stem cell self-renewal, leading to the development of novel human intestinal organoid culture...

  4. New Generation of Cell Culture Assay for Smallpox Vaccine Potency

    OpenAIRE

    Leparc-Goffart, Isabelle; Poirier, Bertrand; El Zaouk, Annie; Tissier, Marie-Hélène; Fuchs, Florence

    2003-01-01

    The potency of smallpox vaccines produced in the 1970s was tested by titration onto chorioallantoic membranes of fertilized hen eggs (CAM assay). The potency specification commonly approved for these vaccines was a titer above 108 pock-forming units per milliliter. We developed and validated a cell culture titration assay to have a more reliable potency test. The cell titration assay and the CAM assay were tested in parallel on 34 first-generation smallpox vaccine lots. These allowed us to de...

  5. Radiation transformation in differentiated human cells in culture

    International Nuclear Information System (INIS)

    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)

  6. A Microwell Cell Culture Platform for the Aggregation of Pancreatic β-Cells

    OpenAIRE

    Bernard, Abigail B.; Lin, Chien-Chi; Anseth, Kristi S.

    2012-01-01

    Cell–cell contact between pancreatic β-cells is important for maintaining survival and normal insulin secretion. Various techniques have been developed to promote cell–cell contact between β-cells, but a simple yet robust method that affords precise control over three-dimensional (3D) β-cell cluster size has not been demonstrated. To address this need, we developed a poly(ethylene glycol) (PEG) hydrogel microwell platform using photolithography. This microwell cell-culture platform promotes t...

  7. Mitodepressive and clastogenic effects of aqueous extracts of the lichens Myelochroa lindmanii and Canoparmelia texana (Lecanorales, Parmeliaceae on meristematic cells in plant bioassays

    Directory of Open Access Journals (Sweden)

    José Marcello Salabert de Campos

    2008-01-01

    Full Text Available The cytotoxicity effect of aqueous extracts of the lichens species Myelochroa lindmanii and Canoparmelia texana (Lecanorales, Parmeliaceae were evaluated using meristematic cells of lettuce (Lactuca sativa and maize (Zea mays. The seasonal effect was also evaluated. Extracts of M. lindmanii and C. texana inhibited root growth and/or percentage germination, possibly due to alterations in the cell cycle. The M. lindmanii extract showed anti-mitotic effects and blocked the cell cycle in metaphase so that c-mitosis and cells with chromosome duplication were produced. The C. texana extract appeared to hinder cell division, increasing the number of interphase cells. In addition, both extracts caused an increase in percentage of cell death. Clastogenic effects were also observed, such as sticky chromosomes, bridges, fragments and later segregation. Both lichen species are thus potential sources of biologically active substances with possible applications in biology, medicine and agronomy.

  8. Endocytic activity of Sertoli cells grown in bicameral culture chambers

    International Nuclear Information System (INIS)

    Immature rat Sertoli cells were cultured for 7 to 14 days on Millipore filters impregnated with a reconstituted basement membrane extract in dual-environment (bicameral) culture chambers. Electron microscopy of the cultured cells revealed the presence of rod-shaped mitochondria, Golgi apparatus, rough endoplasmic reticulum, and Sertoli-Sertoli tight junctions, typical of these cells in vivo. The endocytic activity of both the apical and basal surfaces of the Sertoli cells was examined by either adding alpha 2-macroglobulin (alpha 2-M) conjugated to 20 nm gold particles to the apical chamber or by adding 125I labeled alpha 2-M to the basal chamber. During endocytosis from the apical surface of Sertoli cells, the alpha 2-M-gold particles were bound initially to coated pits and then internalized into coated vesicles within 5 minutes. After 10 minutes, the alpha 2-M-gold was found in multi-vesicular bodies (MVBs) and by 30 minutes it was present in the lysosomes. The proportion of alpha 2-M-gold found within endocytic cell organelles after 1 hour of uptake was used to estimate the approximate time that this ligand spent in each type of organelle. The alpha 2-M-gold was present in coated pits, coated vesicles, multivesicular bodies, and lysosomes for approximately 3, 11, 22, and 24 minutes, respectively. This indicates that the initial stages of endocytosis are rapid, whereas MVBs and lysosomes are relatively long-lived

  9. Staurosporine induces different cell death forms in cultured rat astrocytes

    International Nuclear Information System (INIS)

    Astroglial cells are frequently involved in malignant transformation. Besides apoptosis, necroptosis, a different form of regulated cell death, seems to be related with glioblastoma genesis, proliferation, angiogenesis and invasion. In the present work we elucidated mechanisms of necroptosis in cultured astrocytes, and compared them with apoptosis, caused by staurosporine. Cultured rat cortical astrocytes were used for a cell death studies. Cell death was induced by different concentrations of staurosporine, and modified by inhibitors of apoptosis (z-vad-fmk) and necroptosis (nec-1). Different forms of a cell death were detected using flow cytometry. We showed that staurosporine, depending on concentration, induces both, apoptosis as well as necroptosis. Treatment with 10−7 M staurosporine increased apoptosis of astrocytes after the regeneration in a staurosporine free medium. When caspases were inhibited, apoptosis was attenuated, while necroptosis was slightly increased. Treatment with 10−6 M staurosporine induced necroptosis that occurred after the regeneration of astrocytes in a staurosporine free medium, as well as without regeneration period. Necroptosis was significantly attenuated by nec-1 which inhibits RIP1 kinase. On the other hand, the inhibition of caspases had no effect on necroptosis. Furthermore, staurosporine activated RIP1 kinase increased the production of reactive oxygen species, while an antioxidant BHA significantly attenuated necroptosis. Staurosporine can induce apoptosis and/or necroptosis in cultured astrocytes via different signalling pathways. Distinction between different forms of cell death is crucial in the studies of therapy-induced necroptosis

  10. Rapid method for culturing embryonic neuron-glial cell cocultures

    DEFF Research Database (Denmark)

    Svenningsen, Åsa Fex; Shan, Wei-Song; Colman, David R;

    2003-01-01

    enteric neurons is seen after 3 weeks (2 weeks in ascorbic acid), suggesting that basal lamina production is important even for glial ensheathment in the enteric nervous system. No overgrowth of fibroblasts or other nonneuronal cells was noted in any cultures, and myelination of the peripheral nervous...

  11. Plant Cell Cultures as Source of Cosmetic Active Ingredients

    Directory of Open Access Journals (Sweden)

    Ani Barbulova

    2014-04-01

    Full Text Available The last decades witnessed a great demand of natural remedies. As a result, medicinal plants have been increasingly cultivated on a commercial scale, but the yield, the productive quality and the safety have not always been satisfactory. Plant cell cultures provide useful alternatives for the production of active ingredients for biomedical and cosmetic uses, since they represent standardized, contaminant-free and biosustainable systems, which allow the production of desired compounds on an industrial scale. Moreover, thanks to their totipotency, plant cells grown as liquid suspension cultures can be used as “biofactories” for the production of commercially interesting secondary metabolites, which are in many cases synthesized in low amounts in plant tissues and differentially distributed in the plant organs, such as roots, leaves, flowers or fruits. Although it is very widespread in the pharmaceutical industry, plant cell culture technology is not yet very common in the cosmetic field. The aim of the present review is to focus on the successful research accomplishments in the development of plant cell cultures for the production of active ingredients for cosmetic applications.

  12. Biodegradable Mg corrosion and osteoblast cell culture studies

    International Nuclear Information System (INIS)

    Magnesium (Mg) is a biodegradable metal that has significant potential advantages as an implant material. In this paper, corrosion and cell culture experiments were performed to evaluate the biocompatibility of Mg. The corrosion current and potential of a Mg disk were measured in different physiological solutions including deionized (DI) water, phosphate-buffered saline (PBS), and McCoy's 5A culture medium. The corrosion currents in the PBS and in the McCoy's 5A-5% FBS media were found to be higher than in DI water, which is expected because corrosion of Mg occurs faster in a chloride solution. Weight loss, open-circuit potential, and electrochemical impedance spectroscopy measurements were also performed. The Mg specimens were also characterized using an environmental scanning electron microscope and energy-dispersive X-ray analysis (EDAX). The X-ray analysis showed that in the cell culture media a passive interfacial layer containing oxygen, chloride, phosphate, and potassium formed on the samples. U2OS cells were then co-cultured with a Mg specimen for up to one week. Cytotoxicity results of magnesium using MTT assay and visual observation through cell staining were not significantly altered by the presence of the corroding Mg sample. Further, bone tissue formation study using von Kossa and alkaline phosphatase staining indicates that Mg may be suitable as a biodegradable implant material.

  13. Test chambers for cell culture in static magnetic field

    International Nuclear Information System (INIS)

    Article presents a test chamber intended to be used for in vitro cell culture in homogenous constant magnetic field with parametrically variable magnitude. We constructed test chambers with constant parameters of control homeostasis of cell culture for the different parameters of static magnetic field. The next step was the computer calculation of 2D and 3D simulation of the static magnetic field distribution in the chamber. The analysis of 2D and 3D calculations of magnetic induction in the cells' exposition plane reveals, in comparison to the detection results, the greater accuracy of 2D calculations (Figs. 9 and 10). The divergence in 2D method was 2–4% and 8 to 10% in 3D method (reaching 10% only out of the cells′ cultures margins). -- Highlights: ► We present test chamber to be used for in vitro cell culture in static magnetic field. ► The technical data of the chamber construction was presented. ► 2D versus 3D simulation of static magnetic field distribution in chamber was reported. ► We report the accuracy of 2D calculation than 3D

  14. System-level modeling and simulation of the cell culture microfluidic biochip ProCell

    OpenAIRE

    Minhass, Wajid Hassan; Pop, Paul; Madsen, Jan; Hemmingsen, Mette; Dufva, Martin

    2010-01-01

    Microfluidic biochips offer a promising alternative to a conventional biochemical laboratory. There are two technologies for the microfluidic biochips: droplet-based and flow-based. In this paper we are interested in flow-based microfluidic biochips, where the liquid flows continuously through pre-defined micro-channels using valves and pumps. We present an approach to the system-level modeling and simulation of a cell culture microfluidic biochip called ProCell, Programmable Cell Culture Chi...

  15. Fabrication of a thermoresponsive cell culture dish: a key technology for cell sheet tissue engineering

    OpenAIRE

    Jun Kobayashi and Teruo Okano

    2010-01-01

    This article reviews the properties and characterization of an intelligent thermoresponsive surface, which is a key technology for cell sheet-based tissue engineering. Intelligent thermoresponsive surfaces grafted with poly(N-isopropylacrylamide) exhibit hydrophilic/hydrophobic alteration in response to temperature change. Cultured cells are harvested on thermoresponsive cell culture dishes by decreasing the temperature without the use of digestive enzymes or chelating agents. Our group has d...

  16. INCREASED OSMOLARITY AND CELL CLUSTERING PRESERVES CANINE NOTOCHORDAL CELL PHENOTYPE IN CULTURE

    OpenAIRE

    Spillekom, S; Smolders, L.A.; Grinwis, G.C.M.; Arkesteijn, I.; Ito, K.; Meij, B. P.; Tryfonidou, M.A.

    2013-01-01

    Abstract Degeneration of the intervertebral disc (IVD) is associated with a loss of notochordal cells (NCs) from the nucleus pulposus (NP) and their replacement by chondrocyte-like cells. NCs are known to maintain extracellular matrix quality and stimulate the chondrocyte-like NP cells, making NCs attractive for designing new tissue engineering approaches for IVD regeneration. However, optimal conditions, such as osmolarity and other characteristics of the culture media, for long-term culture...

  17. Neural differentiation of human placenta-derived mesenchymal stem cells following neural cell co-culture

    Institute of Scientific and Technical Information of China (English)

    Nailong Yang; Hongyan Zhang; Xiaojuan Sun; Lili Xu

    2011-01-01

    We induced human placenta-derived mesenchymal stem cells (hPMSCs) to differentiate into neural cells by adding chemical reagents,despite the fact that toxic chemicals induce cell shrinkage or cytoskeletal formation,which does not represent a proper cell differentiation process.The present study established a co-culture system with hPMSCs and neural cells and analyzed the influence of neural cells on hPMSC differentiation in a co-culture system.hPMSCs were isolated and purified from human full-term placenta using collagenase digestion.Fetal neural cells were co-cultured with hPMSCs for 48 hours using the Transwell co-culture system.hPMSCs co-cultured with neural cells exhibited a slender morphology with a filament.After 96 hours,hPMSCs expressed neuron-specific enolase,which suggested that co-culture of hPMSCs and neural cells induced neural differentiation of hPMSCs.

  18. The molecularly crowded cytoplasm of bacterialcCells : Dividing cells contrasted with viable but non-culturable (VBNC) bacterial cells

    NARCIS (Netherlands)

    Trevors, J. T.; van Elsas, J. D.; Bej, A. K.

    2013-01-01

    In this perspective, we discuss the cytoplasm in actively growing bacterial cells contrasted with viable but non-culturable (VBNC) cells. Actively growing bacterial cells contain a more molecularly crowded and organized cytoplasm, and are capable of completing their cell cycle resulting in cell divi

  19. Human disc cells in monolayer vs 3D culture: cell shape, division and matrix formation

    Directory of Open Access Journals (Sweden)

    Hanley Edward N

    2000-10-01

    Full Text Available Abstract Background The relationship between cell shape, proliferation, and extracellular matrix (ECM production, important aspects of cell behavior, is examined in a little-studied cell type, the human annulus cell from the intervertebral disc, during monolayer vs three-dimensional (3D culture. Results Three experimental studies showed that cells respond specifically to culture microenvironments by changes in cell shape, mitosis and ECM production: 1 Cell passages showed extensive immunohistochemical evidence of Type I and II collagens only in 3D culture. Chondroitin sulfate and keratan sulfate were abundant in both monolayer and 3D cultures. 2 Cells showed significantly greater proliferation in monolayer in the presence of platelet-derived growth factor compared to cells in 3D. 3 Cells on Matrigel™-coated monolayer substrates became rounded and formed nodular colonies, a finding absent during monolayer growth. Conclusions The cell's in vivo interactions with the ECM can regulate shape, gene expression and other cell functions. The shape of the annulus cell changes markedly during life: the young, healthy disc contains spindle shaped cells and abundant collagen. With aging and degeneration, many cells assume a strikingly different appearance, become rounded and are surrounded by unusual accumulations of ECM products. In vitro manipulation of disc cells provides an experimental window for testing how disc cells from given individuals respond when they are grown in environments which direct cells to have either spindle- or rounded-shapes. In vitro assessment of the response of such cells to platelet-derived growth factor and to Matrigel™ showed a continued influence of cell shape even in the presence of a growth factor stimulus. These findings contribute new information to the important issue of the influence of cell shape on cell behavior.

  20. Vegetal cell cultures under the effect of low intensity microwaves

    International Nuclear Information System (INIS)

    In order to study the microwave effect on the chlorophyll biosynthesis in Papaver rhoes in vitro cultures microwave radiation characterized by a power density of 0.9 mW/cm2 in a frequency range of 9. 75 - 10.75 GHz was used. P. rhoes in vitro cultures, were obtained from explants of leaves and flowers provided by adult individuals, grown in the Botanical garden of AL. I. Cuza University from Iasi. Murashige Skoog agarized medium with a suitable hormone balance was used to conduct cell culture development before as well as after exposure to microwaves. Assimilatory pigment levels (chlorophyll a, chlorophyll b and carotene pigments) have been evaluated by standard spectrophotometric technique. Student t-test (two tailed, pair) gave significant p-values for the modification of chlorophyll a and chlorophyll b levels after microwave treatment (p0.05). Microwave treatment seems to be able to stimulate assimilatory pigment biosynthesis in the vegetal cell cultures. An inhibitory effect may be associated to the phenotypic modifications noticed in the callus growth from 2 exposed vials - probably related to a non-thermal effect of microwaves in living tissues. Further study of sub-cultures derived from exposed vials is needed to clarify if microwaves of low power density are adequate for the stimulation of assimilatory pigment from chloroplast membranes. (authors)

  1. Unique cell culture systems for ground based research

    Science.gov (United States)

    Lewis, Marian L.

    1990-01-01

    The horizontally rotating fluid-filled, membrane oxygenated bioreactors developed at NASA Johnson for spacecraft applications provide a powerful tool for ground-based research. Three-dimensional aggregates formed by cells cultured on microcarrier beads are useful for study of cell-cell interactions and tissue development. By comparing electron micrographs of plant seedlings germinated during Shuttle flight 61-C and in an earth-based rotating bioreactor it is shown that some effects of microgravity are mimicked. Bioreactors used in the UAH Bioreactor Laboratory will make it possible to determine some of the effects of altered gravity at the cellular level. Bioreactors can be valuable for performing critical, preliminary-to-spaceflight experiments as well as medical investigations such as in vitro tumor cell growth and chemotherapeutic drug response; the enrichment of stem cells from bone marrow; and the effect of altered gravity on bone and muscle cell growth and function and immune response depression.

  2. Milk stimulates growth of prostate cancer cells in culture.

    Science.gov (United States)

    Tate, Patricia L; Bibb, Robert; Larcom, Lyndon L

    2011-11-01

    Concern has been expressed about the fact that cows' milk contains estrogens and could stimulate the growth of hormone-sensitive tumors. In this study, organic cows' milk and two commercial substitutes were digested in vitro and tested for their effects on the growth of cultures of prostate and breast cancer cells. Cows' milk stimulated the growth of LNCaP prostate cancer cells in each of 14 separate experiments, producing an average increase in growth rate of over 30%. In contrast, almond milk suppressed the growth of these cells by over 30%. Neither cows' milk nor almond milk affected the growth of MCF-7 breast cancer cells or AsPC-1 pancreatic cancer cells significantly. Soy milk increased the growth rate of the breast cancer cells. These data indicate that prostate and breast cancer patients should be cautioned about the possible promotional effects of commercial dairy products and their substitutes. PMID:22043817

  3. Lethal impacts of cigarette smoke in cultured tobacco cells

    Directory of Open Access Journals (Sweden)

    Kawano Tomonori

    2011-07-01

    Full Text Available Abstract Background In order to understand and generalize the toxic mechanism of cigarette smoke in living cells, comparison of the data between animal systems and other biological system such as microbial and plant systems is highly beneficial. Objective By employing the tobacco cells as model materials for cigarette smoke toxicity assay, the impacts of the combustion by-products such as nitrogen oxides could be highlighted as the toxic impacts of the plant-derived endogenous chemicals could be excluded in the plant cells. Methods Cigarette smoke-induced cell death was assessed in tobacco cell suspension cultures in the presence and absence of pharmacological inhibitors. Results Cigarette smoke was effective in induction of cell death. The smoke-induced cell death could be partially prevented by addition of nitric oxide (NO scavenger, suggesting the role for NO as the cell death mediator. Addition of NO donor to tobacco cells also resulted in development of partial cell death further confirming the role of NO as cell death mediator. Members of reactive oxygen species and calcium ion were shown to be protecting the cells from the toxic action of smoke-derived NO.

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

    Science.gov (United States)

    Marquette, Michele L.; Sognier, Marguerite A.

    2013-01-01

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

  5. Growing Three-Dimensional Cartilage-Cell Cultures

    Science.gov (United States)

    Spaulding, Glenn F.; Prewett, Tacey L.; Goodwin, Thomas J.

    1995-01-01

    Process for growing three-dimensional cultures of mammalian cartilage from normal mammalian cells devised. Effected using horizontal rotating bioreactor described in companion article, "Simplified Bioreactor for Growing Mammalian Cells" (MSC-22060). Bioreactor provides quiescent environment with generous supplies of nutrient and oxygen. Initiated with noncartilage cells. Artificially grown tissue resembles that in mammalian cartilage. Potential use in developing therapies for damage to cartilage by joint and back injuries and by such inflammatory diseases as arthritis and temporal-mandibular joint disease. Also used to test nonsteroid anti-inflammation medicines.

  6. The Effect of Spaceflight on Bone Cell Cultures

    Science.gov (United States)

    Landis, William J.

    1999-01-01

    Understanding the response of bone to mechanical loading (unloading) is extremely important in defining the means of adaptation of the body to a variety of environmental conditions such as during heightened physical activity or in extended explorations of space or the sea floor. The mechanisms of the adaptive response of bone are not well defined, but undoubtedly they involve changes occurring at the cellular level of bone structure. This proposal has intended to examine the hypothesis that the loading (unloading) response of bone is mediated by specific cells through modifications of their activity cytoskeletal elements, and/or elaboration of their extracellular matrices. For this purpose, this laboratory has utilized the results of a number of previous studies defining molecular biological, biochemical, morphological, and ultrastructural events of the reproducible mineralization of a primary bone cell (osteoblast) culture system under normal loading (1G gravity level). These data and the culture system then were examined following the use of the cultures in two NASA shuttle flights, STS-59 and STS-63. The cells collected from each of the flights were compared to respective synchronous ground (1G) control cells examined as the flight samples were simultaneously analyzed and to other control cells maintained at 1G until the time of shuttle launch, at which point they were terminated and studied (defined as basal cells). Each of the cell cultures was assayed in terms of metabolic markers- gene expression; synthesis and secretion of collagen and non-collagenous proteins, including certain cytoskeletal components; assembly of collagen into macrostructural arrays- formation of mineral; and interaction of collagen and mineral crystals during calcification of the cultures. The work has utilized a combination of biochemical techniques (radiolabeling, electrophoresis, fluorography, Western and Northern Blotting, and light microscopic immunofluorescence) and structural

  7. A simple technique for preparation of chicken-embryo-skin cell cultures.

    Science.gov (United States)

    Silim, A; El Azhary, M A; Roy, R S

    1982-01-01

    A simple, rapid technique was developed for preparing chicken-embryo-skin cell cultures utilizing trypsinization of the skin of intact 12-day-old chicken embryos. When cell cultures were inoculated with fowl pox virus, those that consisted of at least 80% epithelial cells yielded a higher virus titer than fibroblast cell cultures. PMID:6284112

  8. Human neuronal cells in culture: from concepts to basic methodology.

    Science.gov (United States)

    Silani, V; Pizzuti, A; Donato, M F; Falini, A; Bassani, R; Strada, O; Causarano, R I; Mariani, D; Villani, R M; Scarlato, G

    1990-01-01

    The paper reviews some conceptual and methodological aspects of the tissue culture models which, during the past three decades, demonstrated a remarkable mimicry of many important structures and functions of the mammalian Central Nervous System (CNS) and related peripheral sensory and motor elements. Emphasis is placed on an original human neuronal tissue culture model obtained from selective CNS areas. The different cell types were identified and the neurotrophic interactions preliminary characterized. Neuropathological findings suggest hypothesis that can be fully tested using in vitro human models of affected cerebral specific areas. PMID:2102114

  9. Long term maintenance of myeloid leukemic stem cells cultured with unrelated human mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Sawa Ito

    2015-01-01

    Full Text Available Mesenchymal stromal cells (MSCs support the growth and differentiation of normal hematopoietic stem cells (HSCs. Here we studied the ability of MSCs to support the growth and survival of leukemic stem cells (LSCs in vitro. Primary leukemic blasts isolated from the peripheral blood of 8 patients with acute myeloid leukemia (AML were co-cultured with equal numbers of irradiated MSCs derived from unrelated donor bone marrow, with or without cytokines for up to 6 weeks. Four samples showed CD34+CD38− predominance, and four were predominantly CD34+CD38+. CD34+ CD38− predominant leukemia cells maintained the CD34+ CD38− phenotype and were viable for 6 weeks when co-cultured with MSCs compared to co-cultures with cytokines or medium only, which showed rapid differentiation and loss of the LSC phenotype. In contrast, CD34+ CD38+ predominant leukemic cells maintained the CD34+CD38+ phenotype when co-cultured with MSCs alone, but no culture conditions supported survival beyond 4 weeks. Cell cycle analysis showed that MSCs maintained a higher proportion of CD34+ blasts in G0 than leukemic cells cultured with cytokines. AML blasts maintained in culture with MSCs for up to 6 weeks engrafted NSG mice with the same efficiency as their non-cultured counterparts, and the original karyotype persisted after co-culture. Chemosensitivity and transwell assays suggest that MSCs provide pro-survival benefits to leukemic blasts through cell–cell contact. We conclude that MSCs support long-term maintenance of LSCs in vitro. This simple and inexpensive approach will facilitate basic investigation of LSCs and enable screening of novel therapeutic agents targeting LSCs.

  10. Keratocytes Derived from Spheroid Culture of Corneal Stromal Cells Resemble Tissue Resident Keratocytes

    OpenAIRE

    Byun, Yong-Soo; Tibrewal, Sapna; Kim, Eunjae; Yco, Lisette; Sarkar, Joy; Ivanir, Yair; Liu, Chia-Yang; Sano, Cecile M.; Jain, Sandeep

    2014-01-01

    Purpose Corneal stromal cells transform to precursor cells in spheroid culture. We determined whether keratocytes derived from spheroid culture of murine corneal stromal cells resemble tissue resident keratocytes. Methods Spheroid culture was performed by seeding dissociated stromal cells onto ultra-low attachment plates containing serum-free mesenchymal stem cell culture medium. Spheroids were characterized with phenotype specific markers and stemness transcription factor genes. Spheroids an...

  11. Characterizing parameters of Jatropha curcas cell cultures for microgravity studies

    Science.gov (United States)

    Vendrame, Wagner A.; Pinares, Ania

    2013-06-01

    Jatropha (Jatropha curcas) is a tropical perennial species identified as a potential biofuel crop. The oil is of excellent quality and it has been successfully tested as biodiesel and in jet fuel mixes. However, studies on breeding and genetic improvement of jatropha are limited. Space offers a unique environment for experiments aiming at the assessment of mutations and differential gene expression of crops and in vitro cultures of plants are convenient for studies of genetic variation as affected by microgravity. However, before microgravity studies can be successfully performed, pre-flight experiments are necessary to characterize plant material and validate flight hardware environmental conditions. Such preliminary studies set the ground for subsequent spaceflight experiments. The objectives of this study were to compare the in vitro growth of cultures from three explant sources (cotyledon, leaf, and stem sections) of three jatropha accessions (Brazil, India, and Tanzania) outside and inside the petriGAP, a modified group activation pack (GAP) flight hardware to fit petri dishes. In vitro jatropha cell cultures were established in petri dishes containing a modified MS medium and maintained in a plant growth chamber at 25 ± 2 °C in the dark. Parameters evaluated were surface area of the explant tissue (A), fresh weight (FW), and dry weight (DW) for a period of 12 weeks. Growth was observed for cultures from all accessions at week 12, including subsequent plantlet regeneration. For all accessions differences in A, FW and DW were observed for inside vs. outside the PetriGAPs. Growth parameters were affected by accession (genotype), explant type, and environment. The type of explant influenced the type of cell growth and subsequent plantlet regeneration capacity. However, overall cell growth showed no abnormalities. The present study demonstrated that jatropha in vitro cell cultures are suitable for growth inside PetriGAPs for a period of 12 weeks. The parameters

  12. Isolation and culture of protoplasts of Ma-phut (Garcinia dulcis derived from cell suspension culture

    Directory of Open Access Journals (Sweden)

    Sompong Te-chato

    2008-09-01

    Full Text Available Friable callus induced from young leaves of Ma-phut on Murashige and Skoog (MS medium containing 3% sucrose,1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D, 0.5 mg/l benzyladenine (BA and 500 mg/l polyvinylpyrrolidone (PVP, was cultured in liquid medium with the same components. Various ages of cell suspension at weekly intervals were then incubated in various kinds and concentrations of cell wall digestion enzymes combined with 1% macerozyme R-10 on a rotary shaker at 100 rpm under 1500 lux illumination at 26±4oC. Purified protoplasts were cultured at various densities in MS medium (adjusted osmoticum to 0.4 M by mannitol supplemented with 3% sucrose and two types of auxin, 2,4-D and NAA at four concentrations (1, 2, 3 and 4 mg/l together with 1 mg/l BA. The results revealed that a four-day old cell suspension culture incubated in 2% cellulase Onozuka R-10 (CR10 in combination with 1% macerozyme R-10 gave an optimum result in both yield and viability of protoplasts at 5.7x106/1 ml PCV and 80%, respectively. Embedding protoplasts at a density of 2.5x105/ml in 0.2% phytagel containing MS medium supplemented with 3 mg/l NAA and 1 mg/l BA promoted the most effective division of the protoplasts (20%. The first division of the protoplasts was obtained after 2 days of culture and further divisions to form micro- and macro-colonies could be observed after 7-10 days of culture. However, callusformation and plantlet regeneration was not obtained.

  13. MEMS-based dynamic cell-to-cell culture platforms using electrochemical surface modifications

    International Nuclear Information System (INIS)

    MEMS-based biological platforms with the capability of both spatial placements and time releases of living cells for cell-to-cell culture experiments have been designed and demonstrated utilizing electrochemical surface modification effects. The spatial placement is accomplished by electrochemical surface modification of substrate surfaces to be either adhesive or non-adhesive for living cells. The time control is achieved by the electrical activation of the selective indium tin oxide co-culture electrode to allow the migration of living cells onto the electrode to start the cell-to-cell culture studies. Prototype devices have a three-electrode design with an electrode size of 50 × 50 µm2 and the separation gaps of 2 µm between them. An electrical voltage of −1.5 V has been used to activate the electrodes independently and sequentially to demonstrate the dynamic cell-to-cell culture experiments of NIH 3T3 fibroblast and Madin Darby canine kidney cells. As such, this MEMS platform could be a basic yet versatile tool to characterize transient cell-to-cell interactions

  14. The major bovine mastitis pathogens have different cell tropisms in cultures of bovine mammary gland cells

    NARCIS (Netherlands)

    Lammers, A.; Vorstenbosch, van C.J.; Erkens, J.H.F.; Smith, H.E.

    2001-01-01

    We previously showed that Staphylococcus aureus cells adhered mainly to an elongated cell type, present in cultures of bovine mammary gland cells. Moreover. we showed that this adhesion was mediated by binding to fibronectin. The same in vitro model was used here, to study adhesion of other importan

  15. Isolation and culture of porcine neural progenitor cells from embryos and pluripotent stem cells

    DEFF Research Database (Denmark)

    Rasmussen, Mikkel Aabech; Hall, Vanessa Jane; Hyttel, Poul

    2013-01-01

    The isolation and culture of neural progenitor cells (NPCs) from pluripotent stem cells has facilitated in vitro mechanistic studies of diseases related to the nervous system, as well as discovery of new medicine. In addition, NPCs are envisioned to play a crucial role in future cell replacement...

  16. Arsenite maintains germinative state in cultured human epidermal cells

    International Nuclear Information System (INIS)

    Arsenic is a well-known carcinogen for human skin, but its mechanism of action and proximal macromolecular targets remain to be elucidated. In the present study, low micromolar concentrations of sodium arsenite maintained the proliferative potential of epidermal keratinocytes, decreasing their exit from the germinative compartment under conditions that promote differentiation of untreated cells. This effect was observed in suspension and in post-confluent surface cultures as measured by colony-forming ability and by proportion of rapidly adhering colony-forming cells. Arsenite-treated cultures exhibited elevated levels of β1-integrin and β-catenin, two proteins enriched in cells with high proliferative potential. Levels of phosphorylated (inactive) glycogen synthase kinase 3β were higher in the treated cultures, likely accounting for the increased levels of transcriptionally available β-catenin. These findings suggest that arsenic could have co-carcinogenic and tumor co-promoting activities in the epidermis as a result of increasing the population and persistence of germinative cells targeted by tumor initiators and promoters. These findings also identify a critical signal transduction pathway meriting further exploration in pursuit of this phenomenon

  17. Culture and purification of human fetal olfactory bulb ensheathing cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective: To obtain high purity of human fetal olfactory bulb ensheathing cells (OB-hOECs) in vitro and to develop a simple and effective method for primary culture of OB-hOECs. Methods: OB-hOECs were cultured based on the differential rates of attachment of the various harvested cell types. Then the method was combined with arabinoside cytosine (Ara-C)inhibition, serum-free starvation or intermittent neurotrophin 3 (NT3) nutrition method to observe cell states in different cultural environments. The purity of OB-hOECs was assessed with immunocytochemical analysis. Results: OB-hOECs appeared bipolar and tripolar shape, with slender processes forming network. The purity of OECs reached 88% with the selective attachment method on day 6, and then fibroblast proliferated quickly and reduced the purity. When combined with the starvation method, the purity of OECs was 91% on day 6 and 86% on day 9, however, OECs were in a poor state. While combined with the NT3 method, the purity reached 95% on day 9 and 83% on day 12, respectively. The cells still remained in a good state. Conclusion: A combination of selective attachment and intermittent NT3 nutrition is an effective method to obtain OECs with higher purity and quality.

  18. Dexamethasone Modulation on Cultured Human Retinal Pigment Epithelial Cell

    Institute of Scientific and Technical Information of China (English)

    Bing Liu; Yannian; Hui Yusheng Wang; Hong Wei

    2001-01-01

    Purpose: Dexamethasone(DEX) was tested for its ability to modulate human retinal pigment epithelium (hRPE) cell proliferation in cell culture. Methods: DEX in different concentrations was added to cultured hRPE cells. The effects were measured with MTT method, 3H-thymidine(3H-TdR) incorporation and flow cytometw. Results: DEX increased survival rate and DNA synthesis from 32 mg/L to 320 mg/L under hRPE culture conditions, but paradoxically reduced them at 1 000 mg/L and 3 200 mg/L in dose and time dependent fashion by both MTT assay and 3 H-TdR incorporation. The cell numbers in S phase and G2/M phase increased 28.32 % at DEX concentration 320 mg/L, in contrast, reduced 41.84 % at 1 000 mg/L. Conclusion: DEX increased proliferation from 32 mg/L to 320 mg/L, and inhibited proliferation at concentrations greater than 320 mg/L. The inhibiting effect of DEX may happen in s phase and G2/M phase. Eye Sciernce 2001; 17:27 ~ 30.

  19. Enhancement effect of shikonin in cell suspension culture and transfermanant culture by radiation application

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jae Sung; Lee, Young Keun; Chung, Byung Yeoup; Lee, Young Bok; Hwang Hye Yeon

    2004-10-01

    The cell lines 679, 679-29 and 622-46 of L. erythrorhizon could be selected on LS agar medium for the production shikonin in cell suspension culture. The shikonin was increased moderately in suspension culture of cell line 622-46 in LS liquid medium containing BA 2 mg{center_dot}L{sup -1} and IAA 0.2 mg{center_dot}L{sup -1} in the dark, and was increased by adding 1 {mu}M Cu{sup 2+} and 100 {mu}M methyl jasmonate The accumulation of shikonin in the liquid medium was increased significantly by 2 Gy irradiation to callus of cell line 622-46 and culture in LS liquid medium containing BA 2 mg{center_dot}L{sup -1} and IAA 0.2 mg{center_dot}L{sup -1} in the dark and shikonin in cell debris was higher by 16 Gy irradiation. The activity of p-hydroxybenzoate geranyltransferase was increased by irradiation of 2 Gy and 16 Gy of {gamma} radiation. Seedling hypocotyles of L. erythrorhizon were infected with Agrogacterium rhizogenes strain 15834 harboring a binary vector with an intron bearing the GUS ({beta}-glucuronidase) gene driven by cauliflower mosaic virus (CaMV) 35S promotor as well as the HPT (hygromycin phosphotransferase) gene as the selection marker. Hairy roots isolated were hygromycin resistant and had integrated GUS gene in DNA. The root tip grown on M-9 medium showed normal pigment production pattern in border cells and root hairs.

  20. [Wound treatment with autogenous epidermal cell expansion culture].

    Science.gov (United States)

    Bonnekoh, B; Müller, R P; Mahrle, G; Steigleder, G K

    1988-11-11

    Sheets of autologous epidermal cells grown by expansion culture were used to cover small skin defects in seven patients with postoperative necroses, necroses due to temporal arteritis, varicose ulcers or after tangential excision of tattoos. Several transplantation techniques were used: backing of the cultured epithelia with vaseline gauze, Surfasoft, Adaptic, Silastic foil, culturing directly from Petriperm-foil. Meshed Silastic-foil proved to give the best support. Optimal take of the in-vitro epithelia (more than 80% of their surface area) was achieved only for fresh dermal wound-beds. The take was only moderate on chronic granulation tissue, but the transplants reduced the formation of fibrinous-necrotic material and favoured the formation of fresh granulation tissue. PMID:3181024

  1. In Vitro Biologic Activities of the Antimicrobials Triclocarban, Its Analogs, and Triclosan in Bioassay Screens: Receptor-Based Bioassay Screens

    OpenAIRE

    Ahn, Ki Chang; Zhao, Bin; Chen, Jiangang; Cherednichenko, Gennady; Sanmarti, Enio; Denison, Michael S.; Lasley, Bill; Pessah, Isaac N; Kültz, Dietmar; Chang, Daniel P.Y.; Gee, Shirley J.; Hammock, Bruce D.

    2008-01-01

    Background Concerns have been raised about the biological and toxicologic effects of the antimicrobials triclocarban (TCC) and triclosan (TCS) in personal care products. Few studies have evaluated their biological activities in mammalian cells to assess their potential for adverse effects. Objectives In this study, we assessed the activity of TCC, its analogs, and TCS in in vitro nuclear-receptor–responsive and calcium signaling bioassays. Materials and methods We determined the biological ac...

  2. Radiation Response of Cultured Human Cells Is Unaffected by Johrei

    Directory of Open Access Journals (Sweden)

    Zach Hall

    2007-01-01

    Full Text Available Johrei has been credited with healing thousands from radiation wounds after the Hiroshima and Nagasaki bombs in 1945. This alternative medical therapy is becoming increasingly popular in the United States, as are other Energy Medicine modalities that purport to influence a universal healing energy. Human brain cells were cultured and exposed to increasing doses of ionizing radiation. Experienced Johrei practitioners directed healing intentionality toward the cells for 30 min from a distance of 20 cm and the fate of the cells was observed by computerized time-lapse microscopy. Cell death and cell divisions were tallied every 30 min before, during and after Johrei treatment for a total of 22.5 h. An equal number of control experiments were conducted in which cells were irradiated but did not receive Johrei treatment. Samples were assigned to treatment conditions randomly and data analysis was conducted in a blinded fashion. Radiation exposure decreased the rate of cell division (cell cycle arrest in a dose-dependent manner. Division rates were estimated for each 30 min and averaged over 8 independent experiments (4 control and 4 with Johrei treatment for each of 4 doses of X-rays (0, 2, 4 and 8 Gy. Because few cell deaths were observed, pooled data from the entire observation period were used to estimate death rates. Analysis of variance did not reveal any significant differences on division rate or death rate between treatment groups. Only radiation dose was statistically significant. We found no indication that the radiation response of cultured cells is affected by Johrei treatment.

  3. Radiation response of cultured human cells is unaffected by Johrei.

    Science.gov (United States)

    Hall, Zach; Luu, Tri; Moore, Dan; Yount, Garret

    2007-06-01

    Johrei has been credited with healing thousands from radiation wounds after the Hiroshima and Nagasaki bombs in 1945. This alternative medical therapy is becoming increasingly popular in the United States, as are other Energy Medicine modalities that purport to influence a universal healing energy. Human brain cells were cultured and exposed to increasing doses of ionizing radiation. Experienced Johrei practitioners directed healing intentionality toward the cells for 30 min from a distance of 20 cm and the fate of the cells was observed by computerized time-lapse microscopy. Cell death and cell divisions were tallied every 30 min before, during and after Johrei treatment for a total of 22.5 h. An equal number of control experiments were conducted in which cells were irradiated but did not receive Johrei treatment. Samples were assigned to treatment conditions randomly and data analysis was conducted in a blinded fashion. Radiation exposure decreased the rate of cell division (cell cycle arrest) in a dose-dependent manner. Division rates were estimated for each 30 min and averaged over 8 independent experiments (4 control and 4 with Johrei treatment) for each of 4 doses of X-rays (0, 2, 4 and 8 Gy). Because few cell deaths were observed, pooled data from the entire observation period were used to estimate death rates. Analysis of variance did not reveal any significant differences on division rate or death rate between treatment groups. Only radiation dose was statistically significant. We found no indication that the radiation response of cultured cells is affected by Johrei treatment. PMID:17549235

  4. A Place to Call Home: Bioengineering Pluripotential Stem Cell Cultures

    Directory of Open Access Journals (Sweden)

    Mark Weingarten

    2015-03-01

    Full Text Available Pluripotent stem cells (PSCs have the power to revolutionize the future of cell-based therapies and regenerative medicine. However, stem/progenitor cell use in the clinical arsenal has been hampered by discrepancies resulting from stem cell engineering and expansion, as well as in their (mass differentiation in culture. Moreover, the manner in which external conditions affect PSC and induced-pluripotent stem cell lineage establishment as well as maturation remains controversial. In this review, we examine novel methods of cell engineering and the role of reprogramming transcription factors in PSC development. In addition, we explore the effect of external environmental signals on PSC cultivation and differentiation by elucidating key components of the primordial stem cell microenvironment, the blastocyst. Furthermore, we assess the effects of hypoxic conditions on DNA editing, gene expression, and protein function in PSC self-renewal and growth. Finally, we speculate on the principal use of gap junction subunit expression as relevant biomarkers of PSC fate. Improving bioreactor design and pertinent cell biomarker classification could vastly enhance manufactured stem cell yield and quality, thereby increasing the potency and safety of therapeutic cells to be used in regenerative medicine.

  5. Sunlight-induced killing of nondividing human cells in culture

    International Nuclear Information System (INIS)

    Nondividing populations of human diploid fibroblasts that are DNA excision repair proficient and repair deficient were exposed to mid-day summer sunlight and their survival determined based on their ability to remain attached to a culture vessel surface. Whereas mid- and far-UV wavelengths and sunlamp radiation cause a gradual degeneration and detachment of cells in a dose-dependent manner, sunlight does not promote cell killing in repair proficient cells. Detachment of repair deficient cells is promoted to a limited extent but only at sunlight exposure times that are low with respect to the amount of DNA damage induced. Repair proficient and deficient cells exposed to sunlight for longer times do not detach. Pyrimidine dimer levels in these sunlight irradiated cells were great enough to have promoted detachment had these levels been induced by UV (254 nm) alone. Other photodamage induced by these exposures evidently inhibits the dimer induced cell degeneration that leads to cell detachment. It was concluded that pyrimidine dimers are responsible for cell killing at short sunlight exposure times (80 min) cells are killed by a different mechanism. (author)

  6. Long-term Cultured Human Neural Stem Cells Undergo Spontaneous Transformation to Tumor-Initiating Cells

    Directory of Open Access Journals (Sweden)

    Weihua Wu, Qihua He, Xiaoxia Li, Xiaoyan Zhang, Aili Lu, Ruimin Ge, HongYing Zhen, Alfred E. Chang, Qiao Li, Li Shen

    2011-01-01

    Full Text Available In this report, we describe the spontaneous malignant transformation of long-term cultured human fetal striatum neural stem cells (hsNSCs, passage 17. After subcutaneous transplantation of long-term cultured hsNSCs into immunodeficient nude mice, 2 out of 15 mice formed xenografts which expressed neuroendocrine tumor markers CgA and NSE. T1 cells, a cell line that we derived from one of the two subcutaneous xenografts, have undergone continuous expansion in vitro. These T1 cells showed stem cell-like features and expressed neural stem cell markers nestin and CD133. The T1 cells were involved in abnormal karyotype, genomic instability and fast proliferation. Importantly, after long-term in vitro culture, the T1 cells did not result in subcutaneous xenografts, but induced intracranial tumor formation, indicating that they adjusted themselves to the intracranial microenvironment. We further found that the T1 cells exhibited an overexpressed level of EGFR, and the CD133 positive T1 cells showed a truncation mutation in the exons 2-7 of the EGFR (EGFRvIII gene. These results suggest that continuous expansion of neural stem cells in culture may lead to malignant spontaneous transformation. This phenomenon may be functionally related to EGFR by EGFRvIII gene mutation.

  7. Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

    Directory of Open Access Journals (Sweden)

    Magnusson Magnus K

    2010-07-01

    Full Text Available Abstract Background Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D co-culture assay. Methods Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. Results In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. Conclusion Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

  8. Co-culture with Sertoli cells promotes proliferation and migration of umbilical cord mesenchymal stem cells

    International Nuclear Information System (INIS)

    Highlights: ► Co-culture of Sertoli cells (SCs) with human umbilical cord mesenchymal stem cells (UCMSCs). ► Presence of SCs dramatically increased proliferation and migration of UCMSCs. ► Presence of SCs stimulated expression of Mdm2, Akt, CDC2, Cyclin D, CXCR4, MAPKs. -- Abstract: Human umbilical cord mesenchymal stem cells (hUCMSCs) have been recently used in transplant therapy. The proliferation and migration of MSCs are the determinants of the efficiency of MSC transplant therapy. Sertoli cells are a kind of “nurse” cells that support the development of sperm cells. Recent studies show that Sertoli cells promote proliferation of endothelial cells and neural stem cells in co-culture. We hypothesized that co-culture of UCMSCs with Sertoli cells may also promote proliferation and migration of UCMSCs. To examine this hypothesis, we isolated UCMSCs from human cords and Sertoli cells from mouse testes, and co-cultured them using a Transwell system. We found that UCMSCs exhibited strong proliferation ability and potential to differentiate to other cell lineages such as osteocytes and adipocytes. The presence of Sertoli cells in co-culture significantly enhanced the proliferation and migration potential of UCMSCs (P < 0.01). Moreover, these phenotypic changes were accompanied with upregulation of multiple genes involved in cell proliferation and migration including phospho-Akt, Mdm2, phospho-CDC2, Cyclin D1, Cyclin D3 as well as CXCR4, phospho-p44 MAPK and phospho-p38 MAPK. These findings indicate that Sertoli cells boost UCMSC proliferation and migration potential.

  9. Co-culture with Sertoli cells promotes proliferation and migration of umbilical cord mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Fenxi, E-mail: fxzhang0824@gmail.com [Department of Anatomy, Sanquan College, Xinxiang Medical University, Henan 453003, People' s Republic of China (China); Hong, Yan; Liang, Wenmei [Department of Histology and Embryology, Guiyang Medical University, Guizhou 550004, People' s Republic of China (China); Ren, Tongming [Department of Anatomy, Sanquan College, Xinxiang Medical University, Henan 453003, People' s Republic of China (China); Jing, Suhua [ICU Center, The Third Hospital of Xinxiang Medical University, Henan 453003, People' s Republic of China (China); Lin, Juntang [Stem Cell Center, Xinxiang Medical University, Henan 453003, People' s Republic of China (China)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer Co-culture of Sertoli cells (SCs) with human umbilical cord mesenchymal stem cells (UCMSCs). Black-Right-Pointing-Pointer Presence of SCs dramatically increased proliferation and migration of UCMSCs. Black-Right-Pointing-Pointer Presence of SCs stimulated expression of Mdm2, Akt, CDC2, Cyclin D, CXCR4, MAPKs. -- Abstract: Human umbilical cord mesenchymal stem cells (hUCMSCs) have been recently used in transplant therapy. The proliferation and migration of MSCs are the determinants of the efficiency of MSC transplant therapy. Sertoli cells are a kind of 'nurse' cells that support the development of sperm cells. Recent studies show that Sertoli cells promote proliferation of endothelial cells and neural stem cells in co-culture. We hypothesized that co-culture of UCMSCs with Sertoli cells may also promote proliferation and migration of UCMSCs. To examine this hypothesis, we isolated UCMSCs from human cords and Sertoli cells from mouse testes, and co-cultured them using a Transwell system. We found that UCMSCs exhibited strong proliferation ability and potential to differentiate to other cell lineages such as osteocytes and adipocytes. The presence of Sertoli cells in co-culture significantly enhanced the proliferation and migration potential of UCMSCs (P < 0.01). Moreover, these phenotypic changes were accompanied with upregulation of multiple genes involved in cell proliferation and migration including phospho-Akt, Mdm2, phospho-CDC2, Cyclin D1, Cyclin D3 as well as CXCR4, phospho-p44 MAPK and phospho-p38 MAPK. These findings indicate that Sertoli cells boost UCMSC proliferation and migration potential.

  10. Calcium exchange, structure, and function in cultured adult myocardial cells

    International Nuclear Information System (INIS)

    Cells digested from adult rat heart and cultured for 14 days demonstrate all the structural elements, in mature form, associated with the process of excitation-contraction (EC) coupling. The transverse tubular (TT) system is well developed with an extensive junctional sarcoplasmic reticulum (JSR). In nonphosphate-containing buffer contraction of the cells is lost as rapidly as zero extracellular Ca concentration ([Ca]0) solution is applied and a negative contraction staircase is produced on increase of stimulation frequency. Structurally and functionally the cells have the characteristics of adult cells in situ. 45Ca exchange and total 45Ca measurement in N-2-hydroxyethylpiperazine N'-2-ethanesulfonic acid (HEPES)-buffered perfusate define three components of cellular Ca: 1) a rapidly exchangeable component accounting for 36% of total Ca, 2) a slowly exchangeable component (t/sub 1/2/ 53 min) accounting for 7% total Ca, and 3) the remaining 57% cellular Ca is inexchangeable (demonstrates no significant exchange within 60 min). The slowly exchangeable component can be increased 10-fold within 60 min by addition of phosphate to the perfusate. The Ca distribution and exchange characteristics are little different from those of 3-day cultures of neonatal rat heart previously studied. The results suggest that the cells are representative of adult cells in situ and that both sarcolemmal-bound and sarcoplasmic reticular Ca contribute to the component of Ca that is rapidly exchangeable

  11. Aquaporin-1 Expressed in Cultured Human Trabecular Meshwork Cells

    Institute of Scientific and Technical Information of China (English)

    Mingkai Lin; Jian Ge; Yehong Zhuo; Yuqing Lan; Keming Yu; Jianliang Zheng

    2002-01-01

    Objective:To determine if aquaporin-1 could be detected in cultures of human trabecularshwork cells. Methods: Using primers specific for aquaporin-1, reverse transcription combined withpolymerase chain reaction (RT-PCR) yielded a product and its size with total RNAprepared from the human trabecular meshwork cells. SDS-PAGE and immunoblottingwere also used in this study to detect the specific water channel.Results: The presence of this product and its size (298 base pairs) are consistent withthat of an aquaporin-1 message in these cells. A band of 28 kD in agreement with themolecular size of aquaporin-1 was showed in a film by immunoblotting.Conclusion: The presence of aquaporin-1 in human trabecular meshwork cells, thepredominant cell-type of the primary outflow region of the human eye, suggests that waterchannels may be involved in the movement of aqueous fluid out of the eye. In addition,the existence of aquaporin-1 on cultures of human trabecular meshwork cells provides anin vitro model to study the endogenous expression of aquaporin-1 and its possible role inthe regulation of aqueous outflow.

  12. Methyl Jasmonate Represses Growth and Affects Cell Cycle Progression in Cultured Taxus Cells

    OpenAIRE

    Patil, Rohan A.; Lenka, Sangram K.; Normanly, Jennifer; Walker, Elsbeth L.; Roberts, Susan C.

    2014-01-01

    Methyl jasmonate (MeJA) elicitation is an effective strategy to induce and enhance synthesis of the anticancer agent paclitaxel (Taxol®) in Taxus cell suspension cultures; however, concurrent decreases in growth are often observed, which is problematic for large scale bioprocessing. Here, increased accumulation of paclitaxel in Taxus cuspidata suspension cultures with MeJA elicitation was accompanied by a concomitant decrease in cell growth, evident within the first three days post-elicitatio...

  13. Effects of simulated microgravity on mouse Sertoli cells in culture

    Science.gov (United States)

    Angela, Masini Maria; Prato, Paola; Linda, Scarabelli; Lanza, Cristina; Palmero, Silvio; Pointis, Georges; Ricci, Franco; Strollo, Felice

    With the advent of space flights questions concerning the effects of microgravity (0xG) on hu-man reproduction physiology have got priority Spermatogenesis is a complex, highly ordered process of cell division and differentiation by which spermatogonial cells give rise to mature spermatozoa. Sertoli cells play a crucial role in the development of germ cells and the regulation of spermatogenesis. In this study the influence of 0xG on Sertoli cells was evaluated. A Sertoli cell line from mouse testis (42GPA9) was analyzed for cytoskeletal (using the 3D reconstruction generated from a stack of confocal images) and SHBG changes by immunohistochemistry, for antioxidant agents by RT-PCR and for culture medium lactate concentrations by wet chemistry. Cells were cultured for 6, 24 and 48 hrs on a three-dimensional Random Positioning Machine (3D-RPM); static controls (1xG) were positioned on the supporting frame. At the end of each experiment, cultured cells were either fixed in paraformaldehyde or RNA-extracted or used for culture medium lactate measurements as needed. At 0xG Sertoli cytoskeleton got disorganized, microtubules fragmented and SHBG undetectable already after 24 hrs, with alterations wors-ening further until 48 hrs; various antioxidant systems (SOD, GST, PARP, MTs) appreciably increased during the first 24 hrs but significantly decreased at 48 hrs. No changes occurred in 1xG samples. At least initially, 0xG seems to perturb antioxidant protection strategies allowing the testes to support sperm production, thus generating an aging-like state of oxidative stress. Lactate production at 0xG slightly decreased only after 24 hrs. Further experiments need to be carried out in space to investigate upon steroidogenesis and germ cell differentiation within the testis, to rule out eventually pending male infertility consequences, which would be a problem nowadays, when life expectancy increases and male fertility might become a social issue often extending into 60 years

  14. An introduction to plant cell culture: the future ahead.

    Science.gov (United States)

    Loyola-Vargas, Víctor M; Ochoa-Alejo, Neftalí

    2012-01-01

    Plant cell, tissue, and organ culture (PTC) techniques were developed and established as an experimental necessity for solving important fundamental questions in plant biology, but they currently represent very useful biotechnological tools for a series of important applications such as commercial micropropagation of different plant species, generation of disease-free plant materials, production of haploid and doublehaploid plants, induction of epigenetic or genetic variation for the isolation of variant plants, obtention of novel hybrid plants through the rescue of hybrid embryos or somatic cell fusion from intra- or intergeneric sources, conservation of valuable plant germplasm, and is the keystone for genetic engineering of plants to produce disease and pest resistant varieties, to engineer metabolic pathways with the aim of producing specific secondary metabolites or as an alternative for biopharming. Some other miscellaneous applications involve the utilization of in vitro cultures to test toxic compounds and the possibilities of removing them (bioremediation), interaction of root cultures with nematodes or mycorrhiza, or the use of shoot cultures to maintain plant viruses. With the increased worldwide demand for biofuels, it seems that PTC will certainly be fundamental for engineering different plants species in order to increase the diversity of biofuel options, lower the price marketing, and enhance the production efficiency. Several aspects and applications of PTC such as those mentioned above are the focus of this edition. PMID:22610615

  15. A modular suite of hardware enabling spaceflight cell culture research

    Science.gov (United States)

    Hoehn, Alexander; Klaus, David M.; Stodieck, Louis S.

    2004-01-01

    BioServe Space Technologies, a NASA Research Partnership Center (RPC), has developed and operated various middeck payloads launched on 23 shuttle missions since 1991 in support of commercial space biotechnology projects. Modular cell culture systems are contained within the Commercial Generic Bioprocessing Apparatus (CGBA) suite of flight-qualified hardware, compatible with Space Shuttle, SPACEHAB, Spacelab and International Space Station (ISS) EXPRESS Rack interfaces. As part of the CGBA family, the Isothermal Containment Module (ICM) incubator provides thermal control, data acquisition and experiment manipulation capabilities, including accelerometer launch detection for automated activation and thermal profiling for culture incubation and sample preservation. The ICM can accommodate up to 8 individually controlled temperature zones. Command and telemetry capabilities allow real-time downlink of data and video permitting remote payload operation and ground control synchronization. Individual cell culture experiments can be accommodated in a variety of devices ranging from 'microgravity test tubes' or standard 100 mm Petri dishes, to complex, fed-batch bioreactors with automated culture feeding, waste removal and multiple sample draws. Up to 3 levels of containment can be achieved for chemical fixative addition, and passive gas exchange can be provided through hydrophobic membranes. Many additional options exist for designing customized hardware depending on specific science requirements.

  16. Gene probes to detect cross-culture contamination in hormone producing cell lines

    DEFF Research Database (Denmark)

    Matsuba, I; Lernmark, A; Madsen, Ole Dragsbæk;

    1988-01-01

    Cross-culture contamination of cell lines propagated in continuous culture is a frequent event and particularly difficult to resolve in cells expressing similar phenotypes. We demonstrate that DNA-DNA hybridization to blotted endonuclease-digested cell DNA effectively detects cross-culture contam...... effective use of gene probes to control the origin of cell cultures.......Cross-culture contamination of cell lines propagated in continuous culture is a frequent event and particularly difficult to resolve in cells expressing similar phenotypes. We demonstrate that DNA-DNA hybridization to blotted endonuclease-digested cell DNA effectively detects cross......-culture contamination to monitor inter-species as well as intra-species cross contamination. An insulin-producing cell-line, Clone-16, originally cloned from a human fetal endocrine pancreatic cell line did not produce human c-peptide as anticipated. DNA from these cells showed no hybridization to the human ALU...

  17. Handbook of plant cell culture. Volume 2. Crop species

    Energy Technology Data Exchange (ETDEWEB)

    Sharp, W.R.; Evans, D.A.; Ammirato, P.V.; Yamada, Y. (eds.)

    1984-01-01

    In this volume the state-of-the-art plant cell culture techniques described in the first volume are applied to several agricultural and horticultural crops. In 21 chapters, they include maize, oats, wheat, beans, red clover and other forage legumes, asparagus, celery, cassava, sweet potato, banana, pawpaw, apple, grapes, conifers, date palm, rubber, sugarcane and tobacco. Each chapter contains (1) detailed protocols to serve as the foundation for current research, (2) a critical review of the literature, and (3) in-depth evaluations of the potential shown by plant cell culture for crop improvement. The history and economic importance of each crop are discussed. This volume also includes an essay, ''Oil from plants'', by M. Calvin.

  18. Gill cell culture systems as models for aquatic environmental monitoring.

    Science.gov (United States)

    Bury, Nic R; Schnell, Sabine; Hogstrand, Christer

    2014-03-01

    A vast number of chemicals require environmental safety assessments for market authorisation. To ensure acceptable water quality, effluents and natural waters are monitored for their potential harmful effects. Tests for market authorisation and environmental monitoring usually involve the use of large numbers of organisms and, for ethical, cost and logistic reasons, there is a drive to develop alternative methods that can predict toxicity to fish without the need to expose any animals. There is therefore a great interest in the potential to use cultured fish cells in chemical toxicity testing. This review summarises the advances made in the area and focuses in particular on a system of cultured fish gill cells grown into an epithelium that permits direct treatment with water samples. PMID:24574380

  19. Long-term Cultured Human Neural Stem Cells Undergo Spontaneous Transformation to Tumor-Initiating Cells

    OpenAIRE

    Weihua Wu, Qihua He, Xiaoxia Li, Xiaoyan Zhang, Aili Lu, Ruimin Ge, HongYing Zhen, Alfred E. Chang, Qiao Li, Li Shen

    2011-01-01

    In this report, we describe the spontaneous malignant transformation of long-term cultured human fetal striatum neural stem cells (hsNSCs, passage 17). After subcutaneous transplantation of long-term cultured hsNSCs into immunodeficient nude mice, 2 out of 15 mice formed xenografts which expressed neuroendocrine tumor markers CgA and NSE. T1 cells, a cell line that we derived from one of the two subcutaneous xenografts, have undergone continuous expansion in vitro. These T1 cells showed stem ...

  20. Efficient algal bioassay based on short-term photosynthetic response

    International Nuclear Information System (INIS)

    A procedure is described for measuring the effects of toxicants on algal photosynthesis (carbon-14 bicarbonate (H14CO3)uptake) in 4-h experiments. The results for individual aromatic compounds and the water-soluble fraction (WSF) of a synthetic oil are presented as examples of applications of the bioassay. The toxicity of the WSF varied among the seven algal species tested, and the responses of some species were pH-dependent. With Selenastrum capricornutum as the test organism, the bioassay results were unaffected by variations in pH from 7.0 to 9.0, light intensity from 40 to 200 μeinsteins m-2 s-1, culture density up to 0.5 mg chlorophyll a per litre, and agitation up to 100 rpm. The photosynthesis bioassay is simpler and faster (4 h versus 4 to 14 days), uses smaller culture volumes, and requires less space than static culture-growth tests. One person can conveniently test four materials per day, and the entire procedure, including preparation, exposure, and analysis, takes less than two days. The short incubation time reduces bottle effects such as pH changes, accumulation of metabolic products, nutrient depletion, and bacterial growth. Processes that remove or alter the test materials are also minimized. The data presented here indicate that algal photosynthesis is inhibited at toxicant concentrations similar to those that cause acute effects in aquatic animals. A model of a pelagic ecosystem is used to demonstrate that even temporary (seven-day) inhibition of algal photosynthesis can have a measurable impact on other trophic levels, particularly if the other trophic levels are also experiencing toxic effects. 25 references, 6 figures, 1 table

  1. Ion-selective microelectrode arrays for cell culture monitoring

    OpenAIRE

    Generelli, Silvia; De Rooij, Nicolas-F.

    2008-01-01

    The design, microfabrication and characterization of a platform comprising an array of ion-selective microelectrodes (µISE) aimed at in vitro cellular physiology and toxicology is described. This study focusses on K+ and Ca2+ monitoring in cell culture environments. A potential promising application of such a platform is based on recent findings in molecular biology, revealing connections between certain diseases, as for example some types of cancer or parkinsonism, and a malfunction in cellu...

  2. Transparent, biocompatible nanostructured surfaces for cancer cell capture and culture

    Directory of Open Access Journals (Sweden)

    Cheng BR

    2014-05-01

    Full Text Available Boran Cheng,1,* Zhaobo He,2,* Libo Zhao,2,* Yuan Fang,1 Yuanyuan Chen,1 Rongxiang He,2 Fangfang Chen,1 Haibin Song,1 Yuliang Deng,2 Xingzhong Zhao,2 Bin Xiong1 1Department of Oncology, Zhongnan Hospital of Wuhan University, Hubei Key Laboratory of Tumor Biological Behaviors, Hubei Cancer Clinical Study Center, Wuhan, Hubei, People’s Republic of China; 2Key Laboratory of Artificial Micro- and Nano-Structures of Ministry of Education, School of Physics and Technology, Wuhan University, Wuhan, Hubei, People’s Republic of China *These authors contributed equally to this work Abstract: Circulating tumor cells (CTCs in the blood which have detached from both the primary tumor and any metastases may be considered as a “liquid biopsy” and are expected to replace tumor biopsies in the monitoring of treatment response and determining patient prognosis. Here, we introduce a facile and efficient CTC detection material made of hydroxyapatite/chitosan (HA/CTS, which is beneficial because of its transparency and excellent biological compatibility. Atomic force microscopy images show that the roughness of the HA/CTS nanofilm (HA/CTSNF substrates can be controlled by changing the HA:CTS ratio. Enhanced local topographic interactions between nano-components on cancer cell membranes, and the antibody coated nanostructured substrate lead to improved CTC capture and separation. This remarkable nanostructured substrate has the potential for CTC culture in situ and merits further analysis. CTCs captured from artificial blood samples were observed in culture on HA/CTSNF substrates over a period of 14 days by using conventional staining methods (hematoxylin eosin and Wright’s stain. We conclude that these substrates are multifunctional materials capable of isolating and culturing CTCs for subsequent studies. Keywords: cell capture, cell culture, nanofilms, hydroxyapatite/chitosan

  3. Autoradiographic investigations on cell shape-mediated growth regulation of lens epithelial cells in culture

    International Nuclear Information System (INIS)

    An autoradiographic method is described which is well suited for the determination of the labelling index in flattened as well as rounded cells. Using this method DNA synthesis of lens epithelial cells in culture was found to be dependent on cell attachment, cell flattening and intact microfilaments. Thus previous results on cell shape-mediated growth regulation could be confirmed. Moreover, considering the labelling index it was possible to conclude that cell rounding or a disintegration of microfilaments did not impair ongoing DNA synthesis but did prevent cells from entering the S-phase of the cycle. (author)

  4. Propagation and isolation of ranaviruses in cell culture

    DEFF Research Database (Denmark)

    Ariel, Ellen; Nicolajsen, Nicole; Christophersen, Maj-Britt;

    2009-01-01

    The optimal in vitro propagation procedure for a panel of ranavirus isolates and the best method for isolation of Epizootic haematopoietic necrosis virus (EHNV) from organ material in cell-culture were investigated. The panel of ranavirus isolates included: Frog virus 3 (FV3), Bohle iridovirus (BIV......), Pike-perch iridovirus (PPIV), European catfish virus (ECV), European sheatfish virus (ESV), EHNV, Doctor fish virus (DFV), Guppy virus 6 (GF6), short-finned eel virus (SERV) and Rana esculenta virus Italy 282/102 (REV 282/102). Each isolate was titrated in five cell lines: bluegill fry (BF-2...

  5. [Inhibition of adenovirus reproduction in cell culture by specific antibodies].

    Science.gov (United States)

    Povnytsia, O Iu; Nosach, L M; Zhovnovata, V L; Zahorodnia, S D; Vantsak, N P; Tokarchuk, L V; Polishchuk, O M; Diachenko, N S

    2009-01-01

    The capacity of specific antibodies to inhibit the reproduction of homo- and heterologous adenoviruses in Hela cell added to culture medium after virus adsorption was studied. The inhibiting effect of polyclonal antivirus and monospecific antihexone antibodies to homo- and heterologous adenoviruses was shown. The effect was more expressed when using antibodies to homologous antibodies. The intensity of inhibition depended on antibodies concentration in the medium and infecting dose of the virus. Essential reduction of the quantity of infected cells and a decrease of the titer of adenovirus synthesized in the presence of homo- and heterologous antibodies was shown but adenovirus reproduction was not inhibited completely. PMID:19663330

  6. In Vitro Cell Culture Infectivity Assay for Human Noroviruses

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M.; Honer Zu Bentrup, Kerstin A.; Orosz Coghlan, Patricia A.; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza; Nickerson, Cheryl A.

    2007-01-30

    Human noroviruses (NoV) cause severe, self-limiting gastroenteritis that typically lasts 24 - 48 hours. The true nature of NoV pathogenesis remains unknown due to the lack of suitable tissue culture or animal models. Here we show, for the first time, that NoV can infect and replicate in an organoid, three-dimensional (3-D) model of human small intestinal epithelium (INT-407). Cellular differentiation for this model was achieved by growing the cells in 3-D on porous collagen I-coated microcarrier beads under conditions of physiological fluid shear in rotating wall vessel bioreactors. Microscopy, PCR, and fluorescent in-situ hybridization were employed to provide evidence of NoV infection. CPE and norovirus RNA was detected at each of the five cell passages for both genogroup I and II viruses. Our results demonstrate that the highly differentiated 3-D cell culture model can support the natural growth of human noroviruses, whereas previous attempts using differentiated monolayer cultures failed.

  7. A Three-Dimensional Cell Culture Model To Study Enterovirus Infection of Polarized Intestinal Epithelial Cells.

    Science.gov (United States)

    Drummond, Coyne G; Nickerson, Cheryl A; Coyne, Carolyn B

    2016-01-01

    Despite serving as the primary entry portal for coxsackievirus B (CVB), little is known about CVB infection of the intestinal epithelium, owing at least in part to the lack of suitable in vivo models and the inability of cultured cells to recapitulate the complexity and structure associated with the gastrointestinal (GI) tract. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of Caco-2 cells to model CVB infection of the gastrointestinal epithelium. We show that Caco-2 cells grown in 3-D using the rotating wall vessel (RWV) bioreactor recapitulate many of the properties of the intestinal epithelium, including the formation of well-developed tight junctions, apical-basolateral polarity, brush borders, and multicellular complexity. In addition, transcriptome analyses using transcriptome sequencing (RNA-Seq) revealed the induction of a number of genes associated with intestinal epithelial differentiation and/or intestinal processes in vivo when Caco-2 cells were cultured in 3-D. Applying this model to CVB infection, we found that although the levels of intracellular virus production were similar in two-dimensional (2-D) and 3-D Caco-2 cell cultures, the release of infectious CVB was enhanced in 3-D cultures at early stages of infection. Unlike CVB, the replication of poliovirus (PV) was significantly reduced in 3-D Caco-2 cell cultures. Collectively, our studies show that Caco-2 cells grown in 3-D using the RWV bioreactor provide a cell culture model that structurally and transcriptionally represents key aspects of cells in the human GI tract and can thus be used to expand our understanding of enterovirus-host interactions in intestinal epithelial cells. IMPORTANCE Coxsackievirus B (CVB), a member of the enterovirus family of RNA viruses, is associated with meningitis, pericarditis, diabetes, dilated cardiomyopathy, and myocarditis, among other pathologies. CVB is transmitted via the fecal-oral route and encounters the

  8. A Three-Dimensional Cell Culture Model To Study Enterovirus Infection of Polarized Intestinal Epithelial Cells

    Science.gov (United States)

    Drummond, Coyne G.

    2015-01-01

    ABSTRACT Despite serving as the primary entry portal for coxsackievirus B (CVB), little is known about CVB infection of the intestinal epithelium, owing at least in part to the lack of suitable in vivo models and the inability of cultured cells to recapitulate the complexity and structure associated with the gastrointestinal (GI) tract. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of Caco-2 cells to model CVB infection of the gastrointestinal epithelium. We show that Caco-2 cells grown in 3-D using the rotating wall vessel (RWV) bioreactor recapitulate many of the properties of the intestinal epithelium, including the formation of well-developed tight junctions, apical-basolateral polarity, brush borders, and multicellular complexity. In addition, transcriptome analyses using transcriptome sequencing (RNA-Seq) revealed the induction of a number of genes associated with intestinal epithelial differentiation and/or intestinal processes in vivo when Caco-2 cells were cultured in 3-D. Applying this model to CVB infection, we found that although the levels of intracellular virus production were similar in two-dimensional (2-D) and 3-D Caco-2 cell cultures, the release of infectious CVB was enhanced in 3-D cultures at early stages of infection. Unlike CVB, the replication of poliovirus (PV) was significantly reduced in 3-D Caco-2 cell cultures. Collectively, our studies show that Caco-2 cells grown in 3-D using the RWV bioreactor provide a cell culture model that structurally and transcriptionally represents key aspects of cells in the human GI tract and can thus be used to expand our understanding of enterovirus-host interactions in intestinal epithelial cells. IMPORTANCE Coxsackievirus B (CVB), a member of the enterovirus family of RNA viruses, is associated with meningitis, pericarditis, diabetes, dilated cardiomyopathy, and myocarditis, among other pathologies. CVB is transmitted via the fecal-oral route and

  9. Ethanolamine metabolism in cultured bovine aortic endothelial cells

    International Nuclear Information System (INIS)

    The role of extracellular ethanolamine in phospholipid synthesis was examined in cultured bovine aortic endothelial cells. Serine and ethanolamine were both readily accumulated by these cells and incorporated into phospholipid. Exposing cells to extracellular ethanolamine for 4-6 weeks had no effect on cell growth, yet increased the phosphatidylethanolamine content of these cells by 31% as compared to control cells. The intracellular content of ethanolamine was measured by high performance liquid chromatography, and results showed that the ethanolamine-treated cells contained a significantly greater amount of free ethanolamine compared to control cells. Ethanolamine-treated cells also had decreased accumulation and incorporation into lipid of [3H]ethanolamine throughout a 48-h incubation and increased K'm and V'max parameters of ethanolamine transport as compared to control cells. Studies were also done to examine the effect of ethanolamine on the generation of free ethanolamine from phosphatidylserine. In pulse-chase experiments with [3H]serine, a physiological concentration of ethanolamine decreased the amount of 3H-labeled phosphatidylethanolamine produced from 3H-labeled phosphatidylserine by 12 h as compared to the amount of 3H-labeled phosphatidyl-ethanolamine produced in the absence of ethanolamine in the chase incubation. Furthermore, ethanolamine-treated cells accumulated 20% less labeled ethanolamine in the aqueous pool from [3H]serine after 24 h of incubation than did control cells. These results can be explained by isotope dilution with the ethanolamine pool that accumulates in these cells with time when exposed to media supplemented with a physiological concentration of ethanolamine and by an effect of ethanolamine on ethanolamine generation from phosphatidylserine

  10. Renal mesangial cell cultures as a model for study of erythropoietin production.

    OpenAIRE

    Kurtz, Armin; Jelkmann, W; Sinowatz, F.; Bauer, Christian

    1983-01-01

    Mesangial cells derived from isolated glomeruli of rat kidney were grown as homogeneous cell lines in culture. They released, into the culture medium, erythropoietin that had free terminal galactosyl residues and was therefore not active in vivo. The production of erythropoietin by these cells was significantly enhanced by either lowering the PO2 in the incubation atmosphere or by adding cobalt chloride to the culture medium. Therefore, mesangial cells in culture may be considered as an in vi...

  11. An Assessment of Cell Culture Plate Surface Chemistry for in Vitro Studies of Tissue Engineering Scaffolds

    OpenAIRE

    Alexander Röder; Elena García-Gareta; Christina Theodoropoulos; Nikola Ristovski; Keith A. Blackwood; Woodruff, Maria A.

    2015-01-01

    The use of biopolymers as a three dimensional (3D) support structure for cell growth is a leading tissue engineering approach in regenerative medicine. Achieving consistent cell seeding and uniform cell distribution throughout 3D scaffold culture in vitro is an ongoing challenge. Traditionally, 3D scaffolds are cultured within tissue culture plates to enable reproducible cell seeding and ease of culture media change. In this study, we compared two different well-plates with different surface ...

  12. THE STUDY OF VIRUSES REPRODUCTION IN CELL CULTURES BY THE METHOD OF MATHEMATICAL MODELING

    OpenAIRE

    N. A. Kontarov; S. A. Grishunina; N. V. Balaev; N. V. Yuminova; V. V. Zverev

    2014-01-01

    Abstract. In the study the mathematical analysis of viruses reproduction in cell culture using the Marchuk’ mathematical model to predict  reproduction of virus in one or another cell cultures has been conducted. The obtained theoretical results are corresponded to the experimental data on reproduction of rubella virus in cell cultures RK-13 and BHK-21. The sum of theoretical and experimental results can be used to select the optimal cell cultures for virus cumulation.

  13. Benchmarking of commercially available CHO cell culture media for antibody production

    OpenAIRE

    Reinhart, David; Damjanovic, Lukas; Kaisermayer, Christian; Kunert, Renate

    2015-01-01

    In this study, eight commercially available, chemically defined Chinese hamster ovary (CHO) cell culture media from different vendors were evaluated in batch culture using an IgG-producing CHO DG44 cell line as a model. Medium adaptation revealed that the occurrence of even small aggregates might be a good indicator of cell growth performance in subsequent high cell density cultures. Batch experiments confirmed that the culture medium has a significant impact on bioprocess performance, but hi...

  14. Pigment Cell Differentiation in Sea Urchin Blastula-Derived Primary Cell Cultures

    OpenAIRE

    Natalya V. Ageenko; Konstantin V. Kiselev; Dmitrenok, Pavel S.; Odintsova, Nelly A.

    2014-01-01

    The quinone pigments of sea urchins, specifically echinochrome and spinochromes, are known for their effective antioxidant, antibacterial, antifungal, and antitumor activities. We developed in vitro technology for inducing pigment differentiation in cell culture. The intensification of the pigment differentiation was accompanied by a simultaneous decrease in cell proliferation. The number of pigment cells was two-fold higher in the cells cultivated in the coelomic fluids of injured sea urchin...

  15. Cryopreservation of primary cell cultures of marine invertebrates.

    Science.gov (United States)

    Odintsova, N; Kiselev, K; Sanina, N; Kostetsky, E

    2001-01-01

    Primary cell cultures obtained from somatic and larval tissues of bivalve molluscs and from embryos of sea urchins were frozen to -196 degrees C by two-step freezing using 10% dimethyl sulfoxide (DMSO) or/and trehalose (3-30 mg/ml) as cryoprotectants. We estimated both cell viability and the RNA synthetic activity after freeze-thaw. Total lipid extracts from the tissues of echinoderms examined as possible cryoprotective agents demonstrated a weak cryoprotective capacity. Mussel lipid extract was found to possess a considerable cryoprotective activity. Cryoprotective capacity of tested lipids correlated with their thermotropic behaviour. DMSO + trehalose combination was shown to be a favourable cryoprotectant and sea urchin blastula cells the most freezing-tolerant cells. PMID:11788872

  16. Flow field measurements in the cell culture unit

    Science.gov (United States)

    Walker, Stephen; Wilder, Mike; Dimanlig, Arsenio; Jagger, Justin; Searby, Nancy

    2002-01-01

    The cell culture unit (CCU) is being designed to support cell growth for long-duration life science experiments on the International Space Station (ISS). The CCU is a perfused loop system that provides a fluid environment for controlled cell growth experiments within cell specimen chambers (CSCs), and is intended to accommodate diverse cell specimen types. Many of the functional requirements depend on the fluid flow field within the CSC (e.g., feeding and gas management). A design goal of the CCU is to match, within experimental limits, all environmental conditions, other than the effects of gravity on the cells, whether the hardware is in microgravity ( micro g), normal Earth gravity, or up to 2g on the ISS centrifuge. In order to achieve this goal, two steps are being taken. The first step is to characterize the environmental conditions of current 1g cell biology experiments being performed in laboratories using ground-based hardware. The second step is to ensure that the design of the CCU allows the fluid flow conditions found in 1g to be replicated from microgravity up to 2g. The techniques that are being used to take these steps include flow visualization, particle image velocimetry (PIV), and computational fluid dynamics (CFD). Flow visualization using the injection of dye has been used to gain a global perspective of the characteristics of the CSC flow field. To characterize laboratory cell culture conditions, PIV is being used to determine the flow field parameters of cell suspension cultures grown in Erlenmeyer flasks on orbital shakers. These measured parameters will be compared to PIV measurements in the CSCs to ensure that the flow field that cells encounter in CSCs is within the bounds determined for typical laboratory experiments. Using CFD, a detailed simulation is being developed to predict the flow field within the CSC for a wide variety of flow conditions, including microgravity environments. Results from all these measurements and analyses of the

  17. Medium for development of bee cell cultures (Apis mellifera: Hymenoptera: Apidae)

    Science.gov (United States)

    A bee cell culture system was developed. A medium, WH2, for the production of cell cultures from hymenopteran species such as honey bee, Apis mellifera L. (Hymenoptera: Apidae) was developed. Multiple bee cell cultures were produced when using bee larvae and pupae as starting material and the modif...

  18. Culture of porcine hepatocytes or bile duct epithelial cells by inductive serum-free media

    Science.gov (United States)

    A serum-free, feeder-cell-dependent, selective culture system for the long-term culture of porcine hepatocytes or cholangiocytes was developed. Liver cells were isolated from 1 wk old pigs or young adult pigs (25 and 63 kg live weight) and were placed in primary culture on feeder-cell layers of mit...

  19. Human alveolar epithelial type II cells in primary culture.

    Science.gov (United States)

    Mao, Pu; Wu, Songling; Li, Jianchun; Fu, Wei; He, Weiqun; Liu, Xiaoqing; Slutsky, Arthur S; Zhang, Haibo; Li, Yimin

    2015-02-01

    Alveolar epithelial type II (AEII) cells are a key structure and defender in the lung but also are the targets in many lung diseases, including acute respiratory distress syndrome, ventilator-induced lung injury, and pulmonary fibrosis. We sought to establish an optimized method for high yielding and long maintenance of characteristics of primary human AEII cells to facilitate the investigation of the mechanisms of lung diseases at the cellular and molecular levels. Adult human peripheral normal lung tissues of oncologic patients undergoing lung resection were collected. The AEII cells were isolated and identified by the expression of pro-surfactant protein (SP)C, epithelial sodium channel (αENaC) and cytokeratin (CK)-8, the lamellar bodies specific for AEII cells, and confirmed by the histology using electron microscopy. The phenotype of AEII cells was characterized by the expression of surfactant proteins (SP-A, SP-B, SP-C, SP-D), CK-8, KL-6, αENaC, and aquaporin (AQP)-3, which was maintained over 20 days. The biological activity of the primary human AEII cells producing SP-C, cytokines, and intercellular adhesion molecule-1 was vigorous in response to stimulation with tumor necrosis factor-α. We have modified previous methods and optimized a method for isolation of high purity and long maintenance of the human AEII cell phenotype in primary culture. This method provides an important tool for studies aiming at elucidating the molecular mechanisms of lung diseases exclusively in AEII cells. PMID:25677546

  20. A Shrinkage Estimator for Combination of Bioassays

    Institute of Scientific and Technical Information of China (English)

    Jian Xiong; D.G. Chen; Zhen-hai Yang

    2007-01-01

    A shrinkage estimator and a maximum likelihood estimator are proposed in this paper for combination of bioassays. The shrinkage estimator is obtained in closed form which incorporates prior information just on the common log relative potency after the homogeneity test for combination of bioassays is accepted. It is a practical improvement over other estimators which require iterative procedure to obtain the estimator for the relative potency. A real data is also used to show the superiorities for the newly-proposed procedures.

  1. Curious results with palladium- and platinum-carrying polymers in mass cytometry bioassays and an unexpected application as a dead cell stain.

    Science.gov (United States)

    Majonis, Daniel; Ornatsky, Olga; Kinach, Robert; Winnik, Mitchell A

    2011-11-14

    We describe the synthesis of metal-chelating polymers (MCPs) with four different pendant polyaminocarboxylate ligands (EDTA, DTPA, TTHA, DOTA) and an orthogonal end-group, either a fluorescein molecule or a bismaleimide linker for antibody attachment. Polymer characterization by a combination of (1)H NMR, UV/vis absorption measurements, and thermal gravimetric analysis (TGA) indicated that each chain of the fluorescein-terminated polymers contained one dye molecule. These polymer samples were loaded with three different types of lanthanide ions as well as palladium and platinum ions. The numbers of metal atoms per chain were determined by a combination of UV/vis and conventional ICP-MS measurements. The experiments with lanthanide ions demonstrated that a net anionic charge on the polymer is important for water solubility. These experiments also showed that at least one type of lanthanide ion (La(3+)) is capable of forming a bimetallic complex with pendant DTPA groups. Conditions were developed for loading these polymers with palladium and platinum ions. While these polymers could be conjugated to antibodies, the presence of Pd or Pt ions in the polymer interfered with the ability of the antibody to recognize its antigen. For example, a goat anti-mouse (secondary) antibody labeled with polymers that contain Pd or Pt no longer recognized a primary antibody in a sandwich assay. In mass cytometry assays, these Pd- or Pt-containing MCPs were very effective in recognizing dead cells and provide a new and robust assay for distinguishing live cells from dead cells. PMID:21955116

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

    International Nuclear Information System (INIS)

    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

  3. Bioassay criteria for environmental restoration workers

    International Nuclear Information System (INIS)

    Environmental restoration (ER) work at the U. S. Department of Energy Hanford Site posed questions concerning when to perform bioassay monitoring of workers for potential intakes of radioactivity. Application of criteria originally developed for use inside radionuclide processing facilities to ER work resulted in overly restrictive bioassay requirements. ER work typically involves site characterization or, excavating large quantities of potentially contaminated soil, rather than working with concentrated quantities of radioactivity as in a processing facility. An improved approach, tailored to ER work, provided soil contamination concentrations above which worker bioassay would be required. Soil concentrations were derived assuming acute or chronic intakes of 2% of an Annual Limit on Intake (ALI), or a potential committed effective dose equivalent of 100 mrem, and conservative dust loading of air from the work. When planning ER work, the anticipated soil concentration and corresponding need for bioassay could be estimated from work-site historical records. Once site work commenced, soil sampling and work-place surveys could be used to determine bioassay needs. This approach substantially reduced the required number of bioassay samples with corresponding reductions in analytical costs, schedules, and more flexible work-force management. (Work supported by the US Department of Energy under contract DOE-AC06-76RLO 1830.)

  4. Establishment of automated culture system for murine induced pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Koike Hiroyuki

    2012-11-01

    Full Text Available Abstract Background Induced pluripotent stem (iPS cells can differentiate into any cell type, which makes them an attractive resource in fields such as regenerative medicine, drug screening, or in vitro toxicology. The most important prerequisite for these industrial applications is stable supply and uniform quality of iPS cells. Variation in quality largely results from differences in handling skills between operators in laboratories. To minimize these differences, establishment of an automated iPS cell culture system is necessary. Results We developed a standardized mouse iPS cell maintenance culture, using an automated cell culture system housed in a CO2 incubator commonly used in many laboratories. The iPS cells propagated in a chamber uniquely designed for automated culture and showed specific colony morphology, as for manual culture. A cell detachment device in the system passaged iPS cells automatically by dispersing colonies to single cells. In addition, iPS cells were passaged without any change in colony morphology or expression of undifferentiated stem cell markers during the 4 weeks of automated culture. Conclusions Our results show that use of this compact, automated cell culture system facilitates stable iPS cell culture without obvious effects on iPS cell pluripotency or colony-forming ability. The feasibility of iPS cell culture automation may greatly facilitate the use of this versatile cell source for a variety of biomedical applications.

  5. Cell Culture in Microgravity: Opening the Door to Space Cell Biology

    Science.gov (United States)

    Pellis, Neal R.; Dawson, David L. (Technical Monitor)

    1999-01-01

    Adaptational response of human cell populations to microgravity is investigated using simulation, short-term Shuttle experiments, and long-term microgravity. Simulation consists of a clinostatically-rotated cell culture system. The system is a horizontally-rotated cylinder completely filled with culture medium. Low speed rotation results in continuous-fall of the cells through the fluid medium. In this setting, cells: 1) aggregate, 2) propagate in three dimensions, 3) synthesize matrix, 4) differentiate, and 5) form sinusoids that facilitate mass transfer. Space cell culture is conducted in flight bioreactors and in static incubators. Cells grown in microgravity are: bovine cartilage, promyelocytic leukemia, kidney proximal tubule cells, adrenal medulla, breast and colon cancer, and endothelium. Cells were cultured in space to test specific hypotheses. Cartilage cells were used to determine structural differences in cartilage grown in space compared to ground-based bioreactors. Results from a 130-day experiment on Mir revealed that cartilage grown in space was substantially more compressible due to insufficient glycosaminoglycan in the matrix. Interestingly, earth-grown cartilage conformed better to the dimensions of the scaffolding material, while the Mir specimens were spherical. The other cell populations are currently being analyzed for cell surface properties, gene expression, and differentiation. Results suggest that some cells spontaneously differentiate in microgravity. Additionally, vast changes in gene expression may occur in response to microgravity. In conclusion, the transition to microgravity may constitute a physical perturbation in cells resulting in unique gene expressions, the consequences of which may be useful in tissue engineering, disease modeling, and space cell biology.

  6. Digital microfluidics for automated hanging drop cell spheroid culture.

    Science.gov (United States)

    Aijian, Andrew P; Garrell, Robin L

    2015-06-01

    Cell spheroids are multicellular aggregates, grown in vitro, that mimic the three-dimensional morphology of physiological tissues. Although there are numerous benefits to using spheroids in cell-based assays, the adoption of spheroids in routine biomedical research has been limited, in part, by the tedious workflow associated with spheroid formation and analysis. Here we describe a digital microfluidic platform that has been developed to automate liquid-handling protocols for the formation, maintenance, and analysis of multicellular spheroids in hanging drop culture. We show that droplets of liquid can be added to and extracted from through-holes, or "wells," and fabricated in the bottom plate of a digital microfluidic device, enabling the formation and assaying of hanging drops. Using this digital microfluidic platform, spheroids of mouse mesenchymal stem cells were formed and maintained in situ for 72 h, exhibiting good viability (>90%) and size uniformity (% coefficient of variation digital microfluidic platform provides a viable tool for automating cell spheroid culture and analysis. PMID:25510471

  7. Effects of methyl isocyanate on rat muscle cells in culture.

    Science.gov (United States)

    Anderson, D; Goyle, S; Phillips, B J; Tee, A; Beech, L; Butler, W H

    1988-04-01

    Since the Bhopal disaster, in which the causal agent was methyl isocyanate (MIC), exposed people have complained of various disorders including neuromuscular dysfunction. In an attempt to gain some information about the response of muscle tissue to MIC its effects were investigated in cells in culture isolated from muscle of 2 day old rats. After treatment with a range of MIC concentrations (0.025-0.5 microliter/5 ml culture) the total number of nuclei of the two main cell types (fibroblasts and myoblasts) and the number of nuclei in muscle fibres (myotubes) were recorded. At lower doses which had little effect on the total number of nuclei, the formation of muscle fibres--that is, fusion of muscle cells--was prevented as the proportion of nuclei in myotubes was decreased. At higher doses both cell types were killed. This would suggest either an effect on muscle differentiation or a selective toxicity towards myoblasts. The observations were supported by light and electron microscopy. PMID:3378004

  8. Thymic epithelial cells. I. Expression of strong suppressive (veto) activity in mouse thymic epithelial cell cultures

    DEFF Research Database (Denmark)

    Claesson, Mogens Helweg; Ropke, C

    1990-01-01

    We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level. It is...... concluded that the epithelial stromal cells of the thymus, by acting as veto cells, may be responsible for the negative intrathymic selection of self-reactive thymocytes leading to elimination of the vast majority of immature thymic lymphocytes....

  9. Cultured stem cells are sensitive to gravity changes

    Science.gov (United States)

    Buravkova, L. B.; Romanov, Yu. A.; Konstantinova, N. A.; Buravkov, S. V.; Gershovich, Yu. G.; Grivennikov, I. A.

    2008-09-01

    Stem and precursor cells play an important role in development and regeneration. The state of these cells is regulated by biochemical substances, mechanical stimuli and cellular interactions. To estimate gravity effects we used two types of cultured stem cells: human mesenchymal stromal cells (hMSCs) from bone marrow and mice embryonic stem (mESC) line R1. Gravity changes were simulated by long-term (4-7 days) slow clinorotation and leaded to decreased hMSC proliferation, changes of cell morphology and modified F-actin cytoskeleton. We did not find the shifts in cell phenotype except for decreased expression of HLA 1 and CD105 but excretion of IL-6 into medium increased significantly. Remodeling of cytoskeleton started after first 4 h and was similar to preapoptotic changes. This data suggested the modification in cell adhesion and possible commitment of hMSC. It was observed that expression of alkaline phosphatase by MSC in osteogenic medium was more intensive in control. On the contrary, clinorotation did not change formation of mESC colonies and increased proliferation activity in LIF+-medium. However, the number of embryonic bodies after clinorotation was less than in static control. It is suggested that ESCs kept the viability and proliferative potential but decreased the differentiation ability after changes in gravity stimulation.

  10. The chemopotential effect of Annona muricata leaves against azoxymethane-induced colonic aberrant crypt foci in rats and the apoptotic effect of Acetogenin Annomuricin E in HT-29 cells: a bioassay-guided approach.

    Science.gov (United States)

    Zorofchian Moghadamtousi, Soheil; Rouhollahi, Elham; Karimian, Hamed; Fadaeinasab, Mehran; Firoozinia, Mohammad; Ameen Abdulla, Mahmood; Abdul Kadir, Habsah

    2015-01-01

    Annona muricata has been used in folk medicine for the treatment of cancer and tumors. This study evaluated the chemopreventive properties of an ethyl acetate extract of A. muricata leaves (EEAML) on azoxymethane-induced colonic aberrant crypt foci (ACF) in rats. Moreover, the cytotoxic compound of EEAML (Annomuricin E) was isolated, and its apoptosis-inducing effect was investigated against HT-29 colon cancer cell line using a bioassay-guided approach. This experiment was performed on five groups of rats: negative control, cancer control, EEAML (250 mg/kg), EEAML (500 mg/kg) and positive control (5-fluorouracil). Methylene blue staining of colorectal specimens showed that application of EEAML at both doses significantly reduced the colonic ACF formation compared with the cancer control group. Immunohistochemistry analysis showed the down-regulation of PCNA and Bcl-2 proteins and the up-regulation of Bax protein after administration of EEAML compared with the cancer control group. In addition, an increase in the levels of enzymatic antioxidants and a decrease in the malondialdehyde level of the colon tissue homogenates were observed, suggesting the suppression of lipid peroxidation. Annomuricin E inhibited the growth of HT-29 cells with an IC50 value of 1.62 ± 0.24 μg/ml after 48 h. The cytotoxic effect of annomuricin E was further substantiated by G1 cell cycle arrest and early apoptosis induction in HT-29 cells. Annomuricin E triggered mitochondria-initiated events, including the dissipation of the mitochondrial membrane potential and the leakage of cytochrome c from the mitochondria. Prior to these events, annomuricin E activated caspase 3/7 and caspase 9. Upstream, annomuricin E induced a time-dependent upregulation of Bax and downregulation of Bcl-2 at the mRNA and protein levels. In conclusion, these findings substantiate the usage of A. muricata leaves in ethnomedicine against cancer and highlight annomuricin E as one of the contributing compounds in the

  11. A Colorimetric Bioassay for Perchlorate

    Science.gov (United States)

    Heinnickel, M. L.; Smith, S.; Coates, J. D.

    2007-12-01

    Recognition of perchlorate (ClO4-) as a widespread contaminant across the United States and its potential adverse affects towards human health has motivated the EPA to place ClO4- on its contaminant candidate list for drinking water supplies. While a federal MCL has not yet been set, a recommended public health goal of 1 ppb (μg.L-1) was established by the US EPA in 2002. To date, methods of detection require use of sensitive ion chromatographic equipment that are expensive, time consuming, and require highly trained personnel for use. Our studies are focused on the development of a highly sensitive, simple, and robust colorimetric bioassay based on the primary enzyme involved in microbial ClO4- reduction, the perchlorate reductase (Pcr). A previously published assay used reduced methyl viologen (MV, the dye is reduced with sodium hydrosulfite) as an electron donor to demonstrate Pcr activity. The assay directly correlates the amount of MV oxidized with the amount of ClO4- reduced by assuming a transfer of four electrons. To test this assumption, we compared actual concentrations of MV oxidized to ClO4- reduced in this assay. ClO4- concentrations were determined using a Dionex ICS-500 ion chromatography system, while MV concentrations were determined using a standard curve generated at 578 nm. Comparisons between the two revealed that twelve molecules of MV were oxidized for each molecule of ClO4- reduced. The oxidation of these additional eight MV molecules is explained by the interaction of the dye with chlorite (the product of the Pcr reaction) and other contaminants that could be present in the enzyme prep. This unsettling result indicated this assay would be problematic for the detection of ClO4- in soil, which has many chemicals that could react with MV. To improve upon this assay, we have tried to reduce ClO4- using less reactive dyes and reductants. The reductants ascorbic acid, NADH, and dithiothreitol drive Pcr catalyzed ClO4- reduction, however, they

  12. Glycosylation-mediated phenylpropanoid partitioning in Populus tremuloides cell cultures

    Directory of Open Access Journals (Sweden)

    Babst Benjamin A

    2009-12-01

    Full Text Available Abstract Background Phenylpropanoid-derived phenolic glycosides (PGs and condensed tannins (CTs comprise large, multi-purpose non-structural carbon sinks in Populus. A negative correlation between PG and CT concentrations has been observed in several studies. However, the molecular mechanism underlying the relationship is not known. Results Populus cell cultures produce CTs but not PGs under normal conditions. Feeding salicyl alcohol resulted in accumulation of salicins, the simplest PG, in the cells, but not higher-order PGs. Salicin accrual reflected the stimulation of a glycosylation response which altered a number of metabolic activities. We utilized this suspension cell feeding system as a model for analyzing the possible role of glycosylation in regulating the metabolic competition between PG formation, CT synthesis and growth. Cells accumulated salicins in a dose-dependent manner following salicyl alcohol feeding. Higher feeding levels led to a decrease in cellular CT concentrations (at 5 or 10 mM, and a negative effect on cell growth (at 10 mM. The competition between salicin and CT formation was reciprocal, and depended on the metabolic status of the cells. We analyzed gene expression changes between controls and cells fed with 5 mM salicyl alcohol for 48 hr, a time point when salicin accumulation was near maximum and CT synthesis was reduced, with no effect on growth. Several stress-responsive genes were up-regulated, suggestive of a general stress response in the fed cells. Salicyl alcohol feeding also induced expression of genes associated with sucrose catabolism, glycolysis and the Krebs cycle. Transcript levels of phenylalanine ammonia lyase and most of the flavonoid pathway genes were reduced, consistent with down-regulated CT synthesis. Conclusions Exogenous salicyl alcohol was readily glycosylated in Populus cell cultures, a process that altered sugar utilization and phenolic partitioning in the cells. Using this system, we

  13. In vitro cell culture lethal dose submitted to gamma radiation

    International Nuclear Information System (INIS)

    The present study was designed to evaluate the in vitro effect of gamma radiation in cell culture of mouse connective tissue exposed to different doses of gamma radiation and under several conditions. The cell viability was analyzed by neutral red uptake methodology. This assay was developed for establish a methodology to be used in the future in the study of resveratrol radioprotection. Resveratrol (3,4',5- trihydroxystilbene), a phenolic phytoalexin that occurs naturally in some spermatophytes, such as grapevines, in response to injury as fungal infections and exposure to ultraviolet light. In the wines this compound is found at high levels and is considered one of the highest antioxidant constituents. The intense antioxidant potential of resveratrol provides many pharmacological activities including cardioprotection, chemoprevention and anti-tumor effects. Our results demonstrated that 60Co gamma radiation lethal dose (LD50) on NCTC clone 929 cells was about 340Gy. (author)

  14. Ribavirin Inhibits Parrot Bornavirus 4 Replication in Cell Culture.

    Science.gov (United States)

    Musser, Jeffrey M B; Heatley, J Jill; Koinis, Anastasia V; Suchodolski, Paulette F; Guo, Jianhua; Escandon, Paulina; Tizard, Ian R

    2015-01-01

    Parrot bornavirus 4 is an etiological agent of proventricular dilatation disease, a fatal neurologic and gastrointestinal disease of psittacines and other birds. We tested the ability of ribavirin, an antiviral nucleoside analog with antiviral activity against a range of RNA and DNA viruses, to inhibit parrot bornavirus 4 replication in duck embryonic fibroblast cells. Two analytical methods that evaluate different products of viral replication, indirect immunocytochemistry for viral specific nucleoprotein and qRT-PCR for viral specific phosphoprotein gene mRNA, were used. Ribavirin at concentrations between 2.5 and 25 μg/mL inhibited parrot bornavirus 4 replication, decreasing viral mRNA and viral protein load, in infected duck embryonic fibroblast cells. The addition of guanosine diminished the antiviral activity of ribavirin suggesting that one possible mechanism of action against parrot bornavirus 4 may likely be through inosine monophosphate dehydrogenase inhibition. This study demonstrates parrot bornavirus 4 susceptibility to ribavirin in cell culture. PMID:26222794

  15. In vitro cell culture lethal dose submitted to gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Moreno, Carolina S.; Rogero, Sizue O.; Rogero, Jose Roberto [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)], e-mail: carolina_sm@hotmail.com; Ikeda, Tamiko I.; Cruz, Aurea S. [Instituto Adolfo Lutz, Sao Paulo, SP (Brazil)

    2009-07-01

    The present study was designed to evaluate the in vitro effect of gamma radiation in cell culture of mouse connective tissue exposed to different doses of gamma radiation and under several conditions. The cell viability was analyzed by neutral red uptake methodology. This assay was developed for establish a methodology to be used in the future in the study of resveratrol radioprotection. Resveratrol (3,4',5- trihydroxystilbene), a phenolic phytoalexin that occurs naturally in some spermatophytes, such as grapevines, in response to injury as fungal infections and exposure to ultraviolet light. In the wines this compound is found at high levels and is considered one of the highest antioxidant constituents. The intense antioxidant potential of resveratrol provides many pharmacological activities including cardioprotection, chemoprevention and anti-tumor effects. Our results demonstrated that {sup 60}Co gamma radiation lethal dose (LD50) on NCTC clone 929 cells was about 340Gy. (author)

  16. Sulphur XANES Analysis of Cultured Human Prostate Cancer Cells

    International Nuclear Information System (INIS)

    Prostate cancer is one of the most commonly diagnosed cancers in men throughout the world. It is believed that changes to the structure of protein binding sites, altering its metabolism, may play an important role in carcinogenesis. Sulphur, often present in binding sites, can influence such changes through its chemical speciation. Hence there is a need for precise investigation of coordination environment of sulphur. X-ray absorption near edge structure spectroscopy offers such possibility. Cell culture samples offer histologically well defined areas of good homogeneity, suitable for successful and reliable X-ray absorption near edge structure analysis. This paper presents sulphur speciation data collected from three different human prostate cancer cell lines (PC-3, LNCaP and DU-145). Sulphur X-ray absorption near edge structure analysis was performed on K-edge structure. The spectra of cells were compared with those of cancerous tissue and with organic substances as well as inorganic compounds. (authors)

  17. Unbiased analysis by high throughput sequencing of the viral diversity in fetal bovine serum and trypsin used in cell culture.

    Science.gov (United States)

    Gagnieur, Léa; Cheval, Justine; Gratigny, Marlène; Hébert, Charles; Muth, Erika; Dumarest, Marine; Eloit, Marc

    2014-05-01

    Fetal bovine serum (FBS) and trypsin are reagents used in cell culture and have been the source of viral contamination of pharmaceutical products. We performed high throughput sequencing (HTS) of two pools of commercial batches of FBS and three commercial batches of trypsin. Taxonomies were assigned by comparing sequences of contigs and singletons to the entire NCBI nucleic acid and protein databases. The same major viral species were evidenced between batches of a given reagent but the proportion of viral reads among total reads varied markedly between samples (from 0.002% to 22.7%). In FBS, the sequences found were mainly from bovine viral diarrhea virus (BVDV) 1 to 3 and bovine parvovirus 3 (BPV3). The BVDV sequences derived from FBS showed only minor discrepancies with primers generally used for the screening of BVDV. Viral sequences in trypsin were mainly from porcine circovirus type 2. Other known viral sequences at lower read counts and potential new viral species (bovine parvovirus and bovine pegivirus) were evidenced. The load of some known and new viruses detected by HTS could be quantified by qPCR. Results of HTS provide a framework for evaluating the pertinence of control measures including the design of PCRs, bioassays and inactivation procedures. PMID:24661556

  18. The Biological Study of the Cultured Human Lens Epithelial Cells in Vitro

    Institute of Scientific and Technical Information of China (English)

    1994-01-01

    The human lens epithelial cells (HLE) cultured in vitro was established in normal and cataractous lenses. The biological feature, histological characteristics and the ultrastructure of the cultured HLE cells were investigated. The results reveal that the proliferative capacity of the culutured HLE cells is reversely proportional to the donour age; the cultured HLE cells has the limited proliferative capacity in vitro. The relieve of the contact inhibition is the effective trigger of the HLE cell prolife...

  19. Bladder cancer cell in co-culture induces human stem cell differentiation to urothelial cells through paracrine FGF10 signaling

    OpenAIRE

    Chung, Seyung S.; Koh, Chester J.

    2013-01-01

    FGF10 is required for embryonic epidermal morphogenesis including brain development, lung morphogenesis, and initiation of limb bud formation. In this study, we investigated the role of FGF10 as a lead induction factor for stem cell differentiation toward urothelial cell. To this end, human multi-potent stem cell in vitro system was employed. Human amniotic fluid stem cells were co-cultured with immortalized bladder cancer lines to induce directed differentiation into urothelial cells. Urothe...

  20. Optimization of Storage Temperature for Cultured ARPE-19 Cells

    Directory of Open Access Journals (Sweden)

    Lara Pasovic

    2013-01-01

    Full Text Available Purpose. The establishment of future retinal pigment epithelium (RPE replacement therapy is partly dependent on the availability of tissue-engineered RPE cells, which may be enhanced by the development of suitable storage methods for RPE. This study investigates the effect of different storage temperatures on the viability, morphology, and phenotype of cultured RPE. Methods. ARPE-19 cells were cultured under standard conditions and stored in HEPES-buffered MEM at nine temperatures (4°C, 8°C, 12°C, 16°C, 20°C, 24°C, 28°C, 32°C, and 37°C for seven days. Viability and phenotype were assessed by a microplate fluorometer and epifluorescence microscopy, while morphology was analyzed by scanning electron microscopy. Results. The percentage of viable cells preserved after storage was highest in the 16°C group (48.7%±9.8%; P<0.01 compared to 4°C, 8°C, and 24°C–37°C; P<0.05 compared to 12°C. Ultrastructure was best preserved at 12°C, 16°C, and 20°C. Expression of actin, ZO-1, PCNA, caspase-3, and RPE65 was maintained after storage at 16°C compared to control cells that were not stored. Conclusion. Out of nine temperatures tested between 4°C and 37°C, storage at 12°C, 16°C, and 20°C was optimal for maintenance of RPE cell viability, morphology, and phenotype. The preservation of RPE cells is critically dependent on storage temperature.

  1. Responsiveness of fetal rat brain cells to glia maturation factor during neoplastic transformation in cell culture

    DEFF Research Database (Denmark)

    Haugen, A; Laerum, O D; Bock, E

    1981-01-01

    The effect of partially purified extracts from adult pig brains containing a glia maturation protein factor (BE) has been investigated on neural cells during carcinogenesis. Pregnant BD IX-rats were given a single transplacental dose of the carcinogen ethylnitrosourea (EtNU) on the 18th day of...... gestation. The brains of the treated fetuses were transferred to cell culture and underwent neoplastic transformation with a characteristic sequence of phenotypic alterations which could be divided into five different stages. During the first 40 days after explantation (stage I & II) BE induced...... appreciable effect on GFA-content was seen any longer, although some few weakly GFA positive cells could be observed in all permanent cell lines. Fetal rat brain cells therefore seem to become less responsive to this differentiation inducer during neoplastic transformation in cell culture....

  2. Surface modification of uniaxial cyclic strain cell culture platform with temperature-responsive polymer for cell sheet detachment†

    OpenAIRE

    Lee, E L; Bendre, H. H.; Kalmykov, A.; Wong, J Y

    2015-01-01

    Current cell sheet-based blood vessels lack biomimetic structure and require excessively long culture times that may compromise smooth muscle cell phenotype. We modified a commercially available product for uniaxial cell sheet conditioning with thermoresponsive copolymers. Thus, culture of detachable conditioned cell sheets is shortened while retaining structural integrity and contractility.

  3. Epithelial Cell Culture from Human Adenoids: A Functional Study Model for Ciliated and Secretory Cells

    Directory of Open Access Journals (Sweden)

    Claudia González

    2013-01-01

    Full Text Available Background. Mucociliary transport (MCT is a defense mechanism of the airway. To study the underlying mechanisms of MCT, we have both developed an experimental model of cultures, from human adenoid tissue of ciliated and secretory cells, and characterized the response to local chemical signals that control ciliary activity and the secretion of respiratory mucins in vitro. Materials and Methods. In ciliated cell cultures, ciliary beat frequency (CBF and intracellular Ca2+ levels were measured in response to ATP, UTP, and adenosine. In secretory cultures, mucin synthesis and secretion were identified by using immunodetection. Mucin content was taken from conditioned medium and analyzed in the presence or absence of UTP. Results. Enriched ciliated cell monolayers and secretory cells were obtained. Ciliated cells showed a basal CBF of 10.7 Hz that increased significantly after exposure to ATP, UTP, or adenosine. Mature secretory cells showed active secretion of granules containing different glycoproteins, including MUC5AC. Conclusion. Culture of ciliated and secretory cells grown from adenoid epithelium is a reproducible and feasible experimental model, in which it is possible to observe ciliary and secretory activities, with a potential use as a model to understand mucociliary transport control mechanisms.

  4. A specific Fc gamma receptor on cultured rat mesangial cells

    International Nuclear Information System (INIS)

    Mesangial cells represent specialized pericytes in the renal glomerulus that contribute to the regulation of a variety of glomerular functions. Recently we and others have shown that cultured mesangial cells bind and take up immune complexes in an Fc-dependent manner leading in turn to generation of PGE2, reactive oxygen, and platelet-activating factor. The present studies were designed to further characterize potential Fc-gamma R on mesangial cells. Binding assays with either monomeric or heat aggregated (HA) [125I] labeled rat subclass-specific IgG were performed at 4 degrees C for 2 h on subcultured rat mesangial cells. Monomeric rat IgG2a, IgG2b, IgG1 and HA IgG2a bound only nonspecifically. Saturable Fc-dependent binding occurred for HA IgG2b and HA IgG1 though maximal binding and affinity were much higher for IgG2b. The presence of an Fc-gamma R was confirmed by surface protein iodination of mesangial cells (MC) and immunoprecipitation with either a polyclonal or mAb 2.4G2 prepared against murine Fc-gamma R. Both antibodies precipitated a 45-kDa iodinated protein band from cultured rat MC that comigrated with that from murine macrophage J774 cells on SDS-PAGE. This protein band also reacted with the polyclonal anti Fc-gamma R antibody on immunoblots. In contrast rat renal papillary epithelial cells were negative. The 45-kDa protein recognized by the rat anti-Fc-gamma R antibody 2.4G2 probably represents the binding site for HA IgG2b, as the 2.4G2 antibody also blocked binding of HA IgG2b. By immunofluorescence microscopy all MC stained positively with the polyclonal anti-Fc-gamma R antibody. A cDNA probe for the Fc-gamma RII-alpha on murine macrophages hybridized to mRNA from cultured rat MC which was of the same size (though less abundant) as that from J774 macrophages

  5. A specific Fc gamma receptor on cultured rat mesangial cells

    Energy Technology Data Exchange (ETDEWEB)

    Santiago, A.; Satriano, J.; DeCandido, S.; Holthofer, H.; Schreiber, R.; Unkeless, J.; Schlondorff, D. (Albert Einstein College of Medicine, NY (USA))

    1989-10-15

    Mesangial cells represent specialized pericytes in the renal glomerulus that contribute to the regulation of a variety of glomerular functions. Recently we and others have shown that cultured mesangial cells bind and take up immune complexes in an Fc-dependent manner leading in turn to generation of PGE2, reactive oxygen, and platelet-activating factor. The present studies were designed to further characterize potential Fc-gamma R on mesangial cells. Binding assays with either monomeric or heat aggregated (HA) (125I) labeled rat subclass-specific IgG were performed at 4 degrees C for 2 h on subcultured rat mesangial cells. Monomeric rat IgG2a, IgG2b, IgG1 and HA IgG2a bound only nonspecifically. Saturable Fc-dependent binding occurred for HA IgG2b and HA IgG1 though maximal binding and affinity were much higher for IgG2b. The presence of an Fc-gamma R was confirmed by surface protein iodination of mesangial cells (MC) and immunoprecipitation with either a polyclonal or mAb 2.4G2 prepared against murine Fc-gamma R. Both antibodies precipitated a 45-kDa iodinated protein band from cultured rat MC that comigrated with that from murine macrophage J774 cells on SDS-PAGE. This protein band also reacted with the polyclonal anti Fc-gamma R antibody on immunoblots. In contrast rat renal papillary epithelial cells were negative. The 45-kDa protein recognized by the rat anti-Fc-gamma R antibody 2.4G2 probably represents the binding site for HA IgG2b, as the 2.4G2 antibody also blocked binding of HA IgG2b. By immunofluorescence microscopy all MC stained positively with the polyclonal anti-Fc-gamma R antibody. A cDNA probe for the Fc-gamma RII-alpha on murine macrophages hybridized to mRNA from cultured rat MC which was of the same size (though less abundant) as that from J774 macrophages.

  6. From Three-Dimensional Cell Culture to Organs-on-Chips

    OpenAIRE

    Huh, Dongeun; Hamilton, Geraldine A.; Ingber, Donald E.

    2011-01-01

    Three-dimensional (3D) cell culture models have recently garnered great attention because they often promote levels of cell differentiation and tissue organization not possible in conventional two-dimensional (2D) culture systems. Here, we review new advances in 3D culture that leverage microfabrication technologies from the microchip industry and microfluidics approaches to create cell culture microenvironments that both support tissue differentiation and recapitulate the tissue-tissue inter...

  7. Human dental pulp stem cells cultured in serum-free supplemented medium

    OpenAIRE

    Bonnamain, Virginie; Thinard, Reynald; Sergent-Tanguy, Solène; Huet, Pascal; Bienvenu, Géraldine; Naveilhan, Philippe; Farges, Jean-Christophe; Alliot-Licht, Brigitte

    2013-01-01

    Growing evidence show that human dental pulp stem cells (DPSCs) could provide a source of adult stem cells for the treatment of neurodegenerative pathologies. In this study, DPSCs were expanded and cultured with a protocol generally used for the culture of neural stem/progenitor cells. Methodology: DPSC cultures were established from third molars. The pulp tissue was enzymatically digested and cultured in serum-supplemented basal medium for 12 h. Adherent (ADH) and non-adherent (non-ADH) ...

  8. Characterisation of resident multipotent vascular stem cell derived smooth muscle cells in culture

    OpenAIRE

    Kennedy, Eimear

    2015-01-01

    The origin of the vascular Smooth Muscle Cell (SMC) involved with vascular remodelling is very controversial. The theory that SMCs can dedifferentiate is long standing. However, in more recent years this idea has been challenged with the emergence of resident progenitor stem cells in the vascular wall. Here, a population of primary Multipotent Vascular Stem Cells (MVSCs) were isolated using explant culture from the medial layer of rat aortic tissue. MVSCs were characterised for multipotency b...

  9. Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency

    OpenAIRE

    Nii, Takenobu; Kohara, Hiroshi; Marumoto, Tomotoshi; Sakuma, Tetsushi; Yamamoto, Takashi; Tani, Kenzaburo

    2016-01-01

    Abstract Efficient gene transfer into human pluripotent stem cells (hPSCs) holds great promise for regenerative medicine and pharmaceutical development. In the past decade, various methods were developed for gene transfer into hPSCs; however, hPSCs form tightly packed colonies, making gene transfer difficult. In this study, we established a stable culture method of hPSCs at a single-cell state to reduce cell density and investigated gene transfection efficiency followed by gene editing effici...

  10. A biocompatible micro cell culture chamber for culturing and on-line monitoring of Eukaryotic cells

    DEFF Research Database (Denmark)

    Stangegaard, Michael

    2006-01-01

    Visualisering af cellulære processer over længere tidsperioder har været besværliggjort af cellernes krav til varme, fugtighed og et fysiologisk pH balanceret medie. Fremskridt indenfor mikro teknologi har muliggjort fabrikation af miniaturiserede celle kultur anordninger der er i stand til at ho...

  11. Further characterization of the adhesive-tumor-cell culture system for measuring the radiosensitivity of human tumor primary cultures

    International Nuclear Information System (INIS)

    This study extends the use of the adhesive-tumor-cell culture system to include: over 100 sensitivity measurements at 2.0 Gy; tumorgenicity determinations in nude mice; and flow cytometry of the cells grown in the system. The malignant nature of the growing cells was proved by injecting cells into nude mice. Tumors resulted in 60% of the cases and the histology of each xenograft was similar to that of the human tumor. Flow cytometry was used to obtain DNA histograms of the original cell suspension and of cultures during the two week culture period in order to obtain quantitative information about the growth of aneuploid versus diploid populations. The results thus far demonstrate that 95% of aneuploid populations yield aneuploid growth; of the first 20 cases studied, only one suspension with an aneuploid peak resulted in diploid growth. Of further interest was the observation that it is not unusual for a minor aneuploid population to become the predominate growth fraction after two weeks in culture. These results demonstrate that the adhesive-tumor-cell culture system supports the growth of malignant cells, that multiple cell populations exist in cell suspensions derived from solid tumors, and that differences exist between the radiosensitivity of cells at 2.0 Gy in different histology types

  12. Identification of a population of cells with hematopoietic stem cell properties in mouse aorta-gonad-mesonephros cultures

    International Nuclear Information System (INIS)

    The aorta-gonad-mesonephros (AGM) region is a primary source of definitive hematopoietic cells in the midgestation mouse embryo. In cultures of dispersed AGM regions, adherent cells containing endothelial cells are observed first, and then non-adherent hematopoietic cells are produced. Here we report on the characterization of hematopoietic cells that emerge in the AGM culture. Based on the expression profiles of CD45 and c-Kit, we defined three cell populations: CD45low c-Kit+ cells that had the ability to form hematopoietic cell colonies in methylcellulose media and in co-cultures with stromal cells; CD45low c-Kit- cells that showed a granulocyte morphology; CD45high c-Kitlow/- that exhibited a macrophage morphology. In co-cultures of OP9 stromal cells and freshly prepared AGM cultures, CD45low c-Kit+ cells from the AGM culture had the abilities to reproduce CD45low c-Kit+ cells and differentiate into CD45low c-Kit- and CD45high c-Kitlow/- cells, whereas CD45low c-Kit- and CD45high c-Kitlow/- did not produce CD45low c-Kit+ cells. Furthermore, CD45low c-Kit+ cells displayed a long-term repopulating activity in adult hematopoietic tissue when transplanted into the liver of irradiated newborn mice. These results indicate that CD45low c-Kit+ cells from the AGM culture have the potential to reconstitute multi-lineage hematopoietic cells

  13. Dibutyryl cyclic AMP reduces the radiosensitivity of cultured endothelial cells

    International Nuclear Information System (INIS)

    The purpose of this study was to determine whether dibutyryl cyclic AMP modifies the radiosensitivity of confluent monolayers of bovine aortic endothelial cells (BAEC). Three indices of BAEC function were monitored from 4-24 hrs after exposure to 1-10 Gy of 60Co gamma rays: the release of 51Cr from prelabeled cells, and release of lactate dehydrogenase (LDH) and plasminogen activator (PLA) into the culture medium. There was a time- and radiation dose-dependent increase in 51Cr, LDH and PLA release from the BAEC, detectable within 12 hrs after 5 Gy or higher, and by 24 hrs after 1 Gy or higher. This increased release was accompanied by a radiation dose-dependent decrease in 51Cr and LDH, and an increase in PLA activity in the lysate of cells adherent to the monolayer at 24 hrs. The continuous presence of cAMP from 1 hr before to 24 hrs after irradiation reduced all of these radiation reactions, although mM concentrations of cAMP were required for significant sparing. The presence of cAMP from 1 hr before to 10 min after irradiation had no effect on BAEC sensitivity, whereas cAMP added 10 min after irradiation was fully as effective as continuously administered drug. Thus, cultured BAEC exhibit membrane dysfunction within 24 hrs after clinically relevant radiation doses, and this dysfunction is ameliorated by cAMP present after irradiation

  14. Efficient flotation of yeast cells grown in batch culture.

    Science.gov (United States)

    Palmieri, M C; Greenhalf, W; Laluce, C

    1996-05-01

    A fast flotation assay was used to select new floating yeast strains. The flotation ability did not seem to be directly correlated to total extracellular protein concentration of the culture. However, the hydrophobicity of the cell was definitely correlated to the flotation capacity. The Saccharomyces strains (FLT strains) were highly hydrophobic and showed an excellent flotation performance in batch cultures without additives (flotation agents) and with no need for a special flotation chamber or flotation column. A stable and well-organized structure was evident in the dried foam as shown by scanning electron microscopy which revealed its unique structure showing mummified cells (dehydrated) attached to each other. The attachment among the cells and the high protein concentration of the foams indicated that proteins might be involved in the foam formation. The floating strains (strains FLT) which were not flocculent and showed no tendency to aggregate, were capable of growing and producing ethanol in a synthetic medium containing high glucose concentration as a carbon source. The phenomenon responsible for flotation seems to be quite different from the flocculation phenomenon. PMID:18626952

  15. Cell thickness of UV absorption by the cell: relation to UV action spectrum shift in mammalian cells in culture

    International Nuclear Information System (INIS)

    By means of reconstruction of series half - thin transverse sections the three - dimensional morphometry of SPEV cells for a series of their specific states in culture is performed: for exponential growth in a monolayer, in a merged monolayer, in the mitosis phase, for giant cells and suspension cells. In the monolayer the cell thickness in its central part depended mainly on the nucleus thickness and in average changed but slightly despite a wide range of changes in volumes of nuclei and cells and their density in culture. The cell thickness has noticeably increased in mitosis. For the above states of cells UV radiation absorption spectra are determined. It is shown that a certain shift of action spectrus of death of mammalian cells as compared with that for bacterial cell can be a seguence of selfshielding and not differences in the nature of active chromophores

  16. EFFECT OF MACROLIDE ANTIBIOTICS ON VARIOUS CELL CULTURES IN VITRO: 1. CELL MORPHOLOGY

    Directory of Open Access Journals (Sweden)

    Renáta Kováčová

    2012-08-01

    Full Text Available The aim of our study was to evaluate the cytotoxicity of macrolide antibiotics (tilmicosin, tylosin and spiramycin of various concentrations on different cell cultures in vitro. Cellular lines from animal tissues (VERO cells - kidney cells of Macacus rhesus, FE cells - feline embryonal cells, BHK 21 cellular line from young hamster kidneys were used. Tilmicosin effect: BHK cells are most sensitive, significant decrease in vital cells occurs already at the concentration of 50 μg.ml-1. VERO cells were most resistant, significant decrease of vital cells was observed only at the concentration of 300 μg.ml-1. Tylosin effect: BHK cells can be considered most sensitive, since at concentrations higher than 500 μg.ml-1, no vital cells were observed. At the concentration of 1000 μg.ml-1 were 3.13% of vital and 70.52% of subvital FE cells. In Vero cells, we observed a significant decrease at the concentration of 750 μg.ml-1. Spiramycin effect: Significant decrease of vital BHK cells was observed at the concentration of 150 μg.ml-1, at the concentration of 300 μg.ml-1, no vital cells and only 7.53% of subvital cells were observed. At the concentration of 500 μg.ml-1 reported 10.34% of vital FE cells. At the concentration of 500 μg.ml-1 22.48% of vital and 71.16% of subvital VERO cells were recorded.

  17. Comparative cytogenetic analysis of cultures of irradiated lymphocytes and mixed cultures of irradiated and nonirradiated cells

    International Nuclear Information System (INIS)

    Role is discussed of interphase death and mitotic delay of irradiated lymphocytes in unfoldment of the body mass distribution by radiation dose from the lymphocyte distribution by the number of dicentrics in case of nonuniform irradiation. Peripheral blood of 27 healthy donors nonirradiated and irradiated by doses 1-8 Gy was the material. Interphase death and mitotic delay of irradiated lymphocytes effect reduction of unfolding fraction of irradiated cells that require correction of computer method unfolding the body mass distribution by radiation dose from the lymphocyte distribution by the number of dicentrics in case of nonuniform irradiation. Reduction of unfold dose in mixed cultures apparently indicate computable interrelations of cells with different number of chromosome aberrations

  18. Thromboxane A2 receptors are influenced by cell density in cultured rat aortic smooth muscle cells

    International Nuclear Information System (INIS)

    The influence of cell density on the binding characteristics of thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors in rat aortic vascular smooth muscle cells in culture were determined using (1S- (1α, 2β (5Z), 3a (1E, 3R*), 4α)) - (3- (3-hydroxy-4- (4'-iodophenoxy)-1-butyenyl)-7-oxabicyclo-(2.2.1)heptan-2yl)-5-heptenoic acid (125I-BOP). The Bmax for 125I-BOP was 5,430 ± 139 sites/cell (26.9 ± 5.7 fmoles/mg protein) for cells cultured in 1% fetal calf serum and 2,809 ± 830 sites/cell (13.1 ± 2.2 fmoles/mg protein) for cells cultured in 10% fetal calf serum. Cells were allowed to grow to varying densities and then harvested for assay. There was a negative correlation between the Bmax and the cell density per flask. The Kd for I-BOP did not significantly vary in any of the studies. The results demonstrate that cell density plays an important role in influencing the expression of vascular TXA2/PGH2 receptors

  19. Biological effects of positron emitters in thyroid cell cultures

    International Nuclear Information System (INIS)

    Full text of publication follows. Aim: Today, the use of 124I- (β+, half-life 4.2 d) is an increasing field in positron emission tomography (PET). In principle, positrons deposit their energy in the surrounding material like electrons. Therefore, we investigated the biological effects of positron emitters in comparison to electrons in vitro. Materials and Methods: two different thyroid cell lines (Fischer Rat Thyroid Cell Line No. 5 (FRTL5) and human papillary thyroid cancer cell line BCPAP) were investigated in vitro. While FRTL5 has been described to express a high level of sodium iodine transporter (NIS), the NIS expression of BCPAP is known to be low. Parallel cultures were incubated with either 50 -400 kBq/ml 124I- (IBA) or 50-400 kBq/ml 131I- (GE Health care). Cell count and radioiodine uptake were determined 24 h to 144 h after isotope application. Additionally, 4',6-diamidino-2-phenylindole (DAPI) staining of ethanol fixed cells was performed and induced apoptosis was determined by morphological analysis of the cell nucleus (condensation, fragmentation) by fluorescence microscopy. Results: BCPAP showed no significant uptake (<0.1 % per one million cells). The proliferation of BCPAP cells was not significantly influenced by radioiodine incubation. The uptake of NIS-expressing FRTL5 cells ranged between 0.6 % and 4 % per one million cells, independently of the isotope. In FRTL5 cells the incubation with 131I- induced a significant dose-dependent inhibition of proliferation (p<0.05). The positron emitter 124I- induced analogue effects on proliferation compared to the electron emitter 131I- (30-40 % inhibition, 144 h incubation with 400 kBq/ml). In parallel, in FRTL5 cell lines an isotope-independent increase of morphological changes in the cell nuclei (up to 5 fold) could be determined. In contrast, no significant changes could be verified in BCPAP cell nuclei. Conclusions: As expected from the physical point of view, the biological effects of positrons

  20. A rapid bioassay for detecting saxitoxins using a Daphnia acute toxicity test

    Energy Technology Data Exchange (ETDEWEB)

    Ferrao-Filho, Aloysio da S., E-mail: aloysio@ioc.fiocruz.b [Laboratorio de Avaliacao e Promocao da Saude Ambiental, Departamento de Biologia, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, Manguinhos, Rio de Janeiro, RJ 21045-900 (Brazil); Soares, Maria Carolina S., E-mail: mcarolsoares@gmail.co [Departamento de Engenharia Sanitaria e Ambiental Faculdade de Engenharia, Universidade Federal de Juiz de Fora, Juiz de Fora, MG 36036-900 (Brazil); Freitas de Magalhaes, Valeria, E-mail: valeria@biof.ufrj.b [Laboratorio de Ecofisiologia e Toxicologia de Cianobacterias, Instituto de Biofisica Carlos Chagas Filho, CCS, Universidade Federal do Rio de Janeiro, Ilha do Fundao, Rio de Janeiro, RJ 21949-900 (Brazil); Azevedo, Sandra M.F.O., E-mail: sazevedo@biof.ufrj.b [Laboratorio de Ecofisiologia e Toxicologia de Cianobacterias, Instituto de Biofisica Carlos Chagas Filho, CCS, Universidade Federal do Rio de Janeiro, Ilha do Fundao, Rio de Janeiro, RJ 21949-900 (Brazil)

    2010-06-15

    Bioassays using Daphnia pulex and Moina micrura were designed to detect cyanobacterial neurotoxins in raw water samples. Phytoplankton and cyanotoxins from seston were analyzed during 15 months in a eutrophic reservoir. Effective time to immobilize 50% of the exposed individuals (ET{sub 50}) was adopted as the endpoint. Paralysis of swimming movements was observed between approx0.5-3 h of exposure to lake water containing toxic cyanobacteria, followed by an almost complete recovery of the swimming activity within 24 h after being placed in control water. The same effects were observed in bioassays with a saxitoxin-producer strain of Cylindrospermopsis raciborskii isolated from the reservoir. Regression analysis showed significant relationships between ET{sub 50}vs. cell density, biomass and saxitoxins content, suggesting that the paralysis of Daphnia in lake water samples was caused by saxitoxins found in C. raciborskii. Daphnia bioassay was found to be a sensitive method for detecting fast-acting neurotoxins in natural samples, with important advantages over mouse bioassays. - A new Daphnia bioassay, as an alternative to the mouse bioassay, is able to detect effects of fast-acting, potent neurotoxins in raw water.

  1. Effects of ultraviolet irradiation on cultured B-16 melanoma cells

    International Nuclear Information System (INIS)

    Changes of the plasma membrane during exposure to ultraviolet light were studied in cultured B-16 melanoma cells by the method of electron spin resonance with the spin labelling technique. The lipid peroxide content increased immediately after exposure to UV light and returned to the normal level, 3 hrs post-exposure. Plasma membrane fluidity increased significantly, 3 - 6 hrs after exposure, and remained at the increased level in the hydrophobic region of the membrane until 18 hrs. There were no significant differences in phospholipid content before and after exposure, except for cardiolipin. (author)

  2. Self-Assembled Peptide Gels for 3D Cell Culture

    OpenAIRE

    Tang, Claire

    2010-01-01

    Under specific conditions short peptides modified with an N-terminal fluorenyl-9-methoxycarbonyl (Fmoc) group can self-assemble into hydrogel scaffolds similar in properties to the natural extracellular matrix. Fmoc-diphenylalanine (Fmoc-FF) for instance, has been shown to form hydrogels at physiological pH that have the ability to support 2D and 3D cell culture. The aim of this investigation is to provide further understanding of the self-assembly mechanism of such systems in order to progre...

  3. Regulation of Taurine transporter activity in cultured rat retinal ganglion cells and rat retinal Muller Cells

    International Nuclear Information System (INIS)

    Diabetic retinopathy is one of the most common complications of diabetes. The amino acid taurine is believed to play an antioxidant protective role in diabetic retinopathy through the scavenging of the reactive species. It is not well established whether taurine uptake is altered in retina cells during diabetic conditions. Thus, the present study was designed to investigate the changes in taurine transport in cultures of rat retinal Muller cells and rat retinal ganglion cells under conditions associated with diabetes. Taurine was abundantly taken up by retinal Muller cells and rat retinal ganglion cells under normal glycemic condition. Taurine was actively transported to rat Muller cells and rat retinal ganglion cells in a Na and Cl dependant manner. Taurine uptake further significantly elevated in both type of cells after the incubation with high glucose concentration. This effect could be attributed to the increase in osmolarity. Because Nitric Oxide (NO) is a molecule implicated in the pathogenesis of diabetes, we also determined the activity of taurine transporter in cultured rat retinal Muller cells and rat retinal ganglion cells in the presence of the NO donors, SIN-1 and SNAP. Taurine uptake was elevated above control value after 24-h incubation with low concentration of NO donors. We finally investigated the ability of neurotoxic glutamate to change taurine transporter activity in both types of cells. Uptake of taurine was significantly increased in rat retinal ganglion cells when only incubated with high concentration of glutamate. Our data provide evidence that taurine transporter is present in cultured rat retinal ganglion and Muller cells and is regulated by hyperosmolarity. The data are relevant to disease such as diabetes and neuronal degeneration where retinal cell volume may dramatically change. (author)

  4. Cell and Molecular Biology of Ataxia Telangiectasia Heterozygous Human Mammary Epithelial Cells Irradiated in Culture

    Science.gov (United States)

    Richmond, Robert C.

    2001-01-01

    Autologous isolates of cell types from obligate heterozygotes with the autosomal disorder ataxia-telangiectasia (A-T)were used to begin a tissue culture model for assessing pathways of radiation-induced cancer formation in this target tissue. This was done by establishing cultures of stromal fibroblasts and long-term growth human mammary epithelial cells (HMEC) in standard 2-dimensional tissue culture in order to establish expression of markers detailing early steps of carcinogenesis. The presumptive breast cancer susceptibility of A-T heterozygotes as a sequel to damage caused by ionizing radiation provided reason to study expression of markers in irradiated HMEC. Findings from our study with HMEC have included determination of differences in specific protein expression amongst growth phase (e.g., log vs stationary) and growth progression (e.g., pass 7 vs pass 9), as well as differences in morphologic markers within populations of irradiated HMEC (e.g., development of multinucleated cells).

  5. The Effect of Primary Cancer Cell Culture Models on the Results of Drug Chemosensitivity Assays: The Application of Perfusion Microbioreactor System as Cell Culture Vessel

    OpenAIRE

    Chia-Hsun Hsieh; Yi-Dao Chen; Shiang-Fu Huang; Hung-Ming Wang; Min-Hsien Wu

    2015-01-01

    To precisely and faithfully perform cell-based drug chemosensitivity assays, a well-defined and biologically relevant culture condition is required. For the former, a perfusion microbioreactor system capable of providing a stable culture condition was adopted. For the latter, however, little is known about the impact of culture models on the physiology and chemosensitivity assay results of primary oral cavity cancer cells. To address the issues, experiments were performed. Results showed that...

  6. Studies on radiation transformation of cultured mammalian cells

    International Nuclear Information System (INIS)

    In an attempt to induce in vitro transformation by radiation, several cell lines and primary cultures derived from embryonal tissues of hamster, mouse and man were tested. Under various conditions favorable for transformation, none of these were successfully transformed except for C3H mouse embryo-derived 10Tl/2 cells. Normally the cells contact-inhibited were irradiated with single graded doses and dispersed 3 hours after, followed by inoculation and 8-week cultivation with repeated medium renewals. A few types of focus were identified according to the description of Reznikoff et al. The foci characterized by (i) high cell density, (ii) increased affinity to a basic dye, and (iii) piled-up structure, were taken as an indication of transformation. The frequency of transformation was 6.5 x 10-4 for 300 R which was 4 times higher than the frequency found in the untreated control. It increased dose-dependently until 500 R and then levelled off. Another type of experiment using TR cells derived from a leukemia-prone trisomy 21 human embryo, revealed that a single 300 R exposure to x-ray induced clones showing higher plating efficiency and plateau density than unirradiated control after 200 days of post-irradiation cultivation. However, the clones isolated did not show any particular transformational properties in vitro and tumorigenic activity on inoculation into nude mice. (author)

  7. Soil bioassays and the 129I problem

    International Nuclear Information System (INIS)

    Iodine-129 is a very long-lived radionuclide associated with spent nuclear fuel. Because 129I has a 107-year half-life, is very mobile in the environment and is a biologically essential element, it is the most limiting radionuclide affecting disposal of spent fuel. Traditionally, the potential impacts of 129I have been estimated for human receptors, with the implicit assumption that all other organisms are less at risk. Risk is the operative word, the objective for protection of humans is to protect individuals, whereas the objective for other biota is usually to protect populations. Here, 129I poses an interesting problem: the half-life is so long it is barely radioactive. Thus, the chemical toxicity may be more limiting than the radiological impact. A series of soil bioassays were employed, including a life-cycle plant (Brassica rapa) bioassay, a modified earthworm survival bioassay, a microarthropod colonization/survival bioassay, and a series of more common soil and aquatic bioassays. Chemical toxicity was indicated at soil concentrations as low as 5 mg kg-1. At these levels, radiological impact on non-human biota would not be expected, and therefore the chemical toxicity effects are more critical. However, human food-chain model estimates show these levels, as pure 129I, would be unacceptable for human radiological exposure, so that for 129I, protection of the human environment should also be protective of non-human biota

  8. From cells to organisms: Can we learn about aging from cells in culture?

    International Nuclear Information System (INIS)

    Can studying cultured cells inform us about the biology of aging? The idea that this may be was stimulated by the first formal description of replicative senescence. Replicative senescence limits the proliferation of normal human cells in culture, causing them to irreversibly arrest growth and adopt striking changes in cell function. We now know that telomere shortening, which occurs in most somatic cells as a consequence of DNA replication, drives replicative senescence in human cells. However, rodent cells also undergo replicative senescence, despite very long telomeres, and DNA damage,the action of certain oncogenes and changes in chromatin induce a phenotype similar to that of replicatively senescent cells. Thus,replicative senescence is an example of the more general process of cellular senescence, indicating that the telomere hypothesis of aging is a misnomer. Cellular senescence appears to be a response to potentially oncogenic insults, including oxidative stress. The growth arrest almost certainly suppresses tumorigenesis, at least in young organisms, whereas the functional changes may contribute to aging,although this has yet to be critically tested. Thus, cellular senescence may be an example of antagonistic pleiotropy.Cross-species comparisons suggest there is a relationship between the senescence of cells in culture and organismal life span, but the relationship is neither quantitative nor direct

  9. Investigation and Application Progress of Vero Cell Serum-free Culture

    OpenAIRE

    Tian Chen; Keping Chen

    2009-01-01

    Serum-free culture is now becoming the general trend of biological production. Its application in Vero cell culture issignificant. In the present paper, we reviewed factors affecting Vero cell serum-free culture and several culturetechniques. In light of these reviews, we outlined its extensive application prospect.

  10. Cell Monitoring and Manipulation Systems (CMMSs based on Glass Cell-Culture Chips (GC3s

    Directory of Open Access Journals (Sweden)

    Sebastian M. Buehler

    2016-06-01

    Full Text Available We developed different types of glass cell-culture chips (GC3s for culturing cells for microscopic observation in open media-containing troughs or in microfluidic structures. Platinum sensor and manipulation structures were used to monitor physiological parameters and to allocate and permeabilize cells. Electro-thermal micro pumps distributed chemical compounds in the microfluidic systems. The integrated temperature sensors showed a linear, Pt1000-like behavior. Cell adhesion and proliferation were monitored using interdigitated electrode structures (IDESs. The cell-doubling times of primary murine embryonic neuronal cells (PNCs were determined based on the IDES capacitance-peak shifts. The electrical activity of PNC networks was detected using multi-electrode arrays (MEAs. During seeding, the cells were dielectrophoretically allocated to individual MEAs to improve network structures. MEA pads with diameters of 15, 20, 25, and 35 µm were tested. After 3 weeks, the magnitudes of the determined action potentials were highest for pads of 25 µm in diameter and did not differ when the inter-pad distances were 100 or 170 µm. Using 25-µm diameter circular oxygen electrodes, the signal currents in the cell-culture media were found to range from approximately −0.08 nA (0% O2 to −2.35 nA (21% O2. It was observed that 60-nm thick silicon nitride-sensor layers were stable potentiometric pH sensors under cell-culture conditions for periods of days. Their sensitivity between pH 5 and 9 was as high as 45 mV per pH step. We concluded that sensorized GC3s are potential animal replacement systems for purposes such as toxicity pre-screening. For example, the effect of mefloquine, a medication used to treat malaria, on the electrical activity of neuronal cells was determined in this study using a GC3 system.

  11. Cell-Culture Reactor Having a Porous Organic Polymer Membrane

    Science.gov (United States)

    Koontz, Steven L. (Inventor)

    2000-01-01

    A method for making a biocompatible polymer article using a uniform atomic oxygen treatment is disclosed. The substrate may be subsequently optionally grated with a compatibilizing compound. Compatibilizing compounds may include proteins, phosphory1choline groups, platelet adhesion preventing polymers, albumin adhesion promoters, and the like. The compatibilized substrate may also have a living cell layer adhered thereto. The atomic oxygen is preferably produced by a flowing afterglow microwave discharge, wherein the substrate resides in a sidearm out of the plasma. Also, methods for culturing cells for various purposes using the various membranes are disclosed as well. Also disclosed are porous organic polymers having a distributed pore chemistry (DPC) comprising hydrophilic and hydrophobic regions, and a method for making the DPC by exposing the polymer to atomic oxygen wherein the rate of hydrophilization is greater than the rate of mass loss.

  12. CD34+ cells cultured in stem cell factor and interleukin-2 generate CD56+ cells with antiproliferative effects on tumor cell lines

    Directory of Open Access Journals (Sweden)

    Hensel Nancy

    2005-04-01

    Full Text Available Abstract In vitro stimulation of CD34+ cells with IL-2 induces NK cell differentiation. In order to define the stages of NK cell development, which influence their generation from CD34 cells, we cultured G-CSF mobilized peripheral blood CD34+ cells in the presence of stem cell factor and IL-2. After three weeks culture we found a diversity of CD56+ subsets which possessed granzyme A, but lacked the cytotoxic apparatus required for classical NK-like cytotoxicity. However, these CD56+ cells had the unusual property of inhibiting proliferation of K562 and P815 cell lines in a cell-contact dependent fashion.

  13. Development of 3-D Hydrogel Culture Systems With On-Demand Cell Separation

    OpenAIRE

    Hamilton, Sharon K.; Bloodworth, Nathaniel C.; Massad, Christopher S.; Hammoudi, Taymour M.; Suri, Shalu; Yang, Peter J.; Lu, Hang; Temenoff, Johnna S.

    2013-01-01

    Recently there has been an increased interest in the effects of paracrine signaling between groups of cells, particularly in the context of better understanding how stem cells contribute to tissue repair. Most current 3-D co-culture methods lack the ability to effectively separate 2 cell populations after the culture period, which is important for simultaneously analyzing the reciprocal effects of each cell type on the other. Here, we detail the development of a 3-D hydrogel co-culture system...

  14. A primary culture of guinea pig gallbladder epithelial cells that is responsive to secretagogues

    OpenAIRE

    Gunter-Smith, Pamela J.; Abdulkadir, Oluwakemi; Hammonds-Odie, Latanya; Scanlon, Mary; Terrell, Raquel

    2000-01-01

    We have developed a cell culture of guinea pig gallbladder epithelial cells with which to study ion transport. When grown on permeable supports, the cultured epithelia developed a transepithelial resistance (Rt) of ∼500 Ω·cm2. The epithelial cell origin of the cell culture was further confirmed by immunocytochemical localization of cytokeratin. Ionomycin and forskolin increased transepithelial voltage and short-circuit current (Isc) and decreased Rt. The response to ionomycin was transient, w...

  15. Hemopoietic cell precursor responses to erythropoietin in plasma clot cultures

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, W.L.

    1979-01-01

    The time dependence of the response of mouse bone marrow cells to erythropoietin (Ep) in vitro was studied. Experiments include studies on the Ep response of marrow cells from normal, plethoric, or bled mice. Results with normal marrow reveal: (1) Not all erythroid precursors (CFU-E) are alike in their response to Ep. A significant number of the precursors develop to a mature erythroid colony after very short Ep exposures, but they account for only approx. 13% of the total colonies generated when Ep is active for 48 hrs. If Ep is active more than 6 hrs, a second population of erythroid colonies emerges at a nearly constant rate until the end of the culture. Full erythroid colony production requires prolonged exposure to erythropoietin. (2) The longer erythropoietin is actively present, the larger the number of erythroid colonies that reach 17 cells or more. Two distinct populations of immediate erythroid precursors are also present in marrow from plethoric mice. In these mice, total colony numbers are equal to or below those obtained from normal mice. However, the population of fast-responding CFU-E is consistently decreased to 10 to 20% of that found in normal marrow. The remaining colonies are formed from plethoric marrow at a rate equal to normal marrow. With increasing Ep exposures, the number of large colonies produced increases. From the marrow of bled mice, total erythroid colony production is equal to or above that of normal marrow. Two populations of colony-forming cells are again evident, with the fast-responding CFU-E being below normal levels. The lack of colonies from this group was compensated in bled mice by rapid colony production in the second population. A real increase in numbers of precursors present in this pool increased the rate of colony production in culture to twice that of normal marrow. The number of large colonies obtained from bled mice was again increased as the Ep exposure was lengthened. (ERB)

  16. Degradation of high density lipoprotein in cultured rat luteal cells

    International Nuclear Information System (INIS)

    In rat ovary luteal cells, degradation of high density lipoprotein (HDL) to tricholoracetic acid (TCA)-soluble products accounts for only a fraction of the HDL-derived cholesterol used for steroidogenesis. In this study the authors have investigated the fate of 125I]HDL bound to cultured luteal cells using pulse-chase technique. Luteal cell cultures were pulse labeled with [125I]HDL3 and reincubated in the absence of HDL. By 24 h about 50% of the initallay bound radioactivity was released into the medium, of which 60-65% could be precipitated with 10% TCA. Gel filtration of the chase incubation medium on 10% agarose showed that the amount of TCA-soluble radioactivity was nearly completely accounted for by a sharp peak in the low molecular weight region which was identified as 96% monoiodotyrosine by paper chromatography. The TCA-precipitable radioactivity was nearly completely accounted for by a sharp peak in the low molecular weight region which was identified as 96% monoiodotyrosine by paper chromatography. The TCA-precipitable radioactivity eluted over a wide range of molecular weights (15,000-80,000), and there was very little intact HDL present. Electrophoresis of the chase medium showed that component of the TCA-precipitable portion had mobility similar to apo AI. Lysosomal inhibitors of receptor-mediated endocytosis had no effect on the composition or quantity of radioactivity released during chase incubation. The results show that HDL3 binding to luteal cells is followed by complete degradation of the lipoprotein, although the TCA-soluble part does not reflect the extent of degradation

  17. Cell-to-Cell Diversity in a Synchronized Chlamydomonas Culture As Revealed by Single-Cell Analyses

    OpenAIRE

    Garz, Andreas; Sandmann, Michael; Rading, Michael; Ramm, Sascha; Menzel, Ralf; Steup, Martin

    2012-01-01

    In a synchronized photoautotrophic culture of Chlamydomonas reinhardtii, cell size, cell number, and the averaged starch content were determined throughout the light-dark cycle. For single-cell analyses, the relative cellular starch was quantified by measuring the second harmonic generation (SHG). In destained cells, amylopectin essentially represents the only biophotonic structure. As revealed by various validation procedures, SHG signal intensities are a reliable relative measure of the cel...

  18. Phase-segregated model for plant cell culture: The effect of cell volume fraction

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, W. [Univ. of Adelaide, Adelaide (Australia). Dept. of Chemical Engineering]|[Tokyo Univ. (Japan)hinese Academy of Sciences, Dalian (China). Dalian Inst. of Chemical Physics; Furusaki, S. [Tokyo Univ. (Japan)] Middelberg, A. [Univ. of Adelaide, Adelaide (Australia). Dept. of Chemical Engineering

    1998-06-01

    Plant cells are characterized by low water content, so the fraction of cell volume (volume fraction) in a vessel is large compared with other cell systems, even if the cell concentrations are the same. Therefore, concentration of plant cells should preferably be expressed by the liquid volume basis rather than by the total vessel volume basis. In this paper, a new model is proposed to analyze behavior of a plant cell culture by dividing the cell suspension into the biotic- and abiotic-phases. Using this model, we analyzed the cell-growth and the alkaloid production by Catharanthus roseus. Large errors in the simulated results were observed if the phase-segregation was not considered. 12 refs., 3 figs.

  19. Efficacy of decoquinate against Sarcocystis neurona in cell cultures.

    Science.gov (United States)

    Lindsay, David S; Nazir, M Mudasser; Maqbool, Azhar; Ellison, Siobhan P; Strobl, Jeannine S

    2013-09-01

    Decoquinate is a quinolone anticoccidial approved for use in the prevention of intestinal coccidiosis in farm animals. This compound has good activity against Toxoplasma gondii and Neospora caninum in cell cultures. The drug acts on the parasites' mitochondria. The activity of decoquinate against developing merozoites of 2 isolates of Sarcocystis neurona was examined in cell culture. Merozoite production at 10 days was completely inhibited when decoquinate was used at 20 or 240 nM. The IC50 of decoquinate was 0.5 ± 0.09nM for the Sn6 isolate of S. neurona from a horse and 1.1 ± 0.6 nM for the SnOP15 isolate of S. neurona from an opossum. Levamisole was toxic at 5 μg/ml and no synergism was observed when decoquinate was combined with levamisole and tested against the Sn3YFP isolate of S. neurona. Decoquinate was cidal for developing schizonts of S. neurona at 240 nM. PMID:23523012

  20. Isolation, culture and intraportal transplantation of rat marrow stromal cell

    International Nuclear Information System (INIS)

    Objective: To observe the tracing and evolution of marrow stromal cell (MSC) after intraportal transplantation into the liver of homogenous rats, and to provide experimental data for MSC differentiation to hepatocyte in vivo. Methods: The MSC was isolated from the leg bone marrow of adult SD rats, and purified by culture-expanded in vitro. Before transplantation, MSC was labeled with DAPI. Then 105 MSC were intraportally transplanted into the homogenous rat liver. Rats were killed at 2 hours and 1, 2, 3 and 4 weeks after transplantation. The cryosection samples of liver and lung were observed under fluorescence microscopy. Results: MSC in vitro culture had high ability of proliferation. Except 4 rats were dead because of abdominal bleeding or infection, other recipients were healthy until sacrificed. The implantation cells were detected by identifying the DAPI labeled MSC in the host livers, but not in the host lungs. Conclusion: Intraportal transplanted MSC could immigrate and survive in the host livers at least for 4 weeks. They could immigrate from the small branches of portal veins to hepatic parenchyma

  1. Sulforaphane induces DNA single strand breaks in cultured human cells

    Energy Technology Data Exchange (ETDEWEB)

    Sestili, Piero, E-mail: piero.sestili@uniurb.it [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Paolillo, Marco [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Lenzi, Monia [Dipartimento di Farmacologia, Universita degli Studi di Bologna, Via Irnerio 48, 40126 Bologna (Italy); Colombo, Evelin; Vallorani, Luciana; Casadei, Lucia; Martinelli, Chiara [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Fimognari, Carmela [Dipartimento di Farmacologia, Universita degli Studi di Bologna, Via Irnerio 48, 40126 Bologna (Italy)

    2010-07-07

    Sulforaphane (SFR), an isothiocyanate from cruciferous vegetables, possesses growth-inhibiting and apoptosis-inducing activities in cancer cell lines. Recently, SFR has been shown to promote the mitochondrial formation of reactive oxygen species (ROS) in human cancer cell lines. The present study was undertaken to see whether SFR-derived ROS might cause DNA damage in cultured human cells, namely T limphoblastoid Jurkat and human umbilical vein endothelial cells (HUVEC). 1-3 h treatments with 10-30 {mu}M SFR elicited intracellular ROS formation (as assayed with dihydrorhodamine, DHR, oxidation) as well as DNA breakage (as assessed with fast halo assay, FHA). These effects lacked cell-type specificity, since could be observed in both Jurkat and HUVEC. Differential-pH FHA analysis of damaged DNA showed that SFR causes frank DNA single strand breaks (SSBs); no DNA double strand breaks (DSBs) were found within the considered treatment times (up to 3 h). SFR-derived ROS were formed at the mitochondrial respiratory chain (MRC) level: indeed rotenone or myxothiazol (MRC Complex I and III inhibitors, respectively) abrogated ROS formation. Furthermore ROS were not formed in Jurkat cells pharmacologically depleted of respiring mitochondria (MRC-/Jurkat). Formation of ROS was causally linked to the induction of SSBs: indeed all the experimental conditions capable of preventing ROS formation also prevented the damage of nuclear DNA from SFR-intoxicated cells. As to the toxicological relevance of SSBs, we found that their prevention slightly but significantly attenuated SFR cytotoxicity, suggesting that high-dose SFR toxicity is the result of a complex series of events among which GSH depletion seems to play a pivotal role. In conclusion, the present study identifies a novel mechanism contributing to SFR toxicity which - since DNA damage is a prominent mechanism underlying the cytotoxic activity of established antineoplastic agents - might help to exploit the therapeutic value

  2. Gelatin methacrylamide as coating material in cell culture.

    Science.gov (United States)

    Egger, Michael; Tovar, Günter E M; Hoch, Eva; Southan, Alexander

    2016-01-01

    Unmodified gelatin (uG) is widely used as a coating material in cell culture for improving surface properties. In this study, the authors investigated if gelatin methacrylamide (GM) with a medium degree of methacrylamide modification (GM1.5) and a high degree of methacrylamide modification (GM4) are equally suitable for this purpose. Therefore, gold surfaces were coated with uG, GM1.5, and GM4 by adsorption of the polymers on the surfaces. Coating success was confirmed by spectroscopic ellipsometry, contact angle measurements, surface plasmon resonance spectroscopy (SPRS), and atomic force microscopy (AFM). The authors found that upon adsorption of uG, GM1.5, a nd GM4 on gold, thin films with thicknesses of 2.95 nm, 2.50 nm, and 2.26 nm were formed. The coated surfaces showed advancing contact angles of 46° (uG and GM1.5) and 52° (GM4) without alteration of the surface roughness determined by AFM. Protein adsorption taking place on the coated surfaces was measured during contact of the surfaces with fetal calf serum by SPRS. Protein adsorption on the coated surfaces was reduced by the factor of 6.4 (uG), 5.4 (GM1.5), and 4.6 (GM4) compared to gold surfaces. Human fibroblasts cultured on the surfaces showed excellent viability shown by water soluble tetrazolium salt assay as well as live/dead staining with propidium iodide and fluorescein diacetate. No cytotoxic effects of the GM coated surfaces were observed, giving rise to the conclusion that GMs are suitable materials as coatings in cell culture. PMID:27177620

  3. Cell differentiation mediated by co-culture of human umbilical cord blood stem cells with murine hepatic cells.

    Science.gov (United States)

    Stecklum, Maria; Wulf-Goldenberg, Annika; Purfürst, Bettina; Siegert, Antje; Keil, Marlen; Eckert, Klaus; Fichtner, Iduna

    2015-02-01

    In the present study, purified human cord blood stem cells were co-cultivated with murine hepatic alpha mouse liver 12 (AML12) cells to compare the effect on endodermal stem cell differentiation by either direct cell-cell interaction or by soluble factors in conditioned hepatic cell medium. With that approach, we want to mimic in vitro the situation of preclinical transplantation experiments using human cells in mice. Cord blood stem cells, cultivated with hepatic conditioned medium, showed a low endodermal differentiation but an increased connexin 32 (Cx32) and Cx43, and cytokeratin 8 (CK8) and CK19 expression was monitored by reverse transcription polymerase chain reaction (RT-PCR). Microarray profiling indicated that in cultivated cord blood cells, 604 genes were upregulated 2-fold, with the highest expression for epithelial CK19 and epithelial cadherin (E-cadherin). On ultrastructural level, there were no major changes in the cellular morphology, except a higher presence of phago(ly)some-like structures observed. Direct co-culture of AML12 cells with cord blood cells led to less incisive differentiation with increased sex-determining region Y-box 17 (SOX17), Cx32 and Cx43, as well as epithelial CK8 and CK19 expressions. On ultrastructural level, tight cell contacts along the plasma membranes were revealed. FACS analysis in co-cultivated cells quantified dye exchange on low level, as also proved by time relapse video-imaging of labelled cells. Modulators of gap junction formation influenced dye transfer between the co-cultured cells, whereby retinoic acid increased and 3-heptanol reduced the dye transfer. The study indicated that the cell-co-cultured model of human umbilical cord blood cells and murine AML12 cells may be a suitable approach to study some aspects of endodermal/hepatic cell differentiation induction. PMID:25270685

  4. Forskolin and the meiosis inducing substance synergistically initiate meiosis in fetal male germ cells

    DEFF Research Database (Denmark)

    Byskov, A G; Fenger, M; Westergaard, L;

    1993-01-01

    We have shown that Meiosis Inducing Substance (MIS) and forskolin synergistically and dose dependently induce meiosis in germ cells of cultured fetal mouse testes. We used a bioassay which consists of fetal mouse testes and ovaries cultured for 6 days. In this study MIS media are spent culture...... male germ cells during culture. We found that MIS media as well as forskolin induced meiosis in fetal male germ cells in a dose-dependent manner. In addition, MIS media and forskolin acted synergistically by inducing meiosis. Female germ cells seem to be unaffected by the various culture media. These...

  5. Effects of cell concentrations on the survival and repopulation of haemopoietic stem cells in irradiated bone marrow cell culture in vitro

    International Nuclear Information System (INIS)

    Effects of cell concentrations on the survival and repopulation of haemopoietic stem cells after irradiation were studied in the long-term culture of mouse bone marrow cells in vitro. No difference was observed in the survival of the stem cells among cultures in which 0 - 107 cells were re-inoculated on the adherent cell colonies in the culture flask. Stem cells showed a significant proliferation within 1 week and the number of the stem cells exceeded the control in 3 weeks after irradiation in the cultures with less than 106 re-inoculated cells per flask. In contrast, there was a considerable delay in the onset of stem cell proliferation after irradiation in the culture with 107 cells per flask. Based on these results, a possibility that a stimulator of stem cell proliferation, released from irradiated stromal cells, is cancelled by an inhibitory factor produced by irradiated or unirradiated haemopoietic cells is postulated. (author)

  6. Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells

    Directory of Open Access Journals (Sweden)

    Anthony Finoli

    2016-01-01

    Full Text Available Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications.

  7. Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells

    Science.gov (United States)

    Schmelzer, Eva; Over, Patrick; Nettleship, Ian; Gerlach, Joerg C.

    2016-01-01

    Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications.

  8. WE-E-BRE-03: Biological Validation of a Novel High-Throughput Irradiator for Predictive Radiation Sensitivity Bioassays

    Energy Technology Data Exchange (ETDEWEB)

    Fowler, TL; Martin, JA; Shepard, AJ; Bailey, AM; Nickel, KP; Kimple, RJ; Bednarz, BP [University of Wisconsin, Madison, WI (United States)

    2014-06-15

    Purpose: The large dose-response variation in both tumor and normal cells between individual patients has led to the recent implementation of predictive bioassays of patient-specific radiation sensitivity in order to personalize radiation therapy. This exciting new clinical paradigm has led us to develop a novel high-throughput, variable dose-rate irradiator to accompany these efforts. Here we present the biological validation of this irradiator through the use of human cells as a relative dosimeter assessed by two metrics, DNA double-strand break repair pathway modulation and intercellular reactive oxygen species production. Methods: Immortalized human tonsilar epithelial cells were cultured in 96-well micro titer plates and irradiated in groups of eight wells to absorbed doses of 0, 0.5, 1, 2, 4, and 8 Gy. High-throughput immunofluorescent microscopy was used to detect γH2AX, a DNA double-strand break repair mechanism recruiter. The same analysis was performed with the cells stained with CM-H2DCFDA that produces a fluorescent adduct when exposed to reactive oxygen species during the irradiation cycle. Results: Irradiations of the immortalized human tonsilar epithelial cells at absorbed doses of 0, 0.5, 1, 2, 4, and 8 Gy produced excellent linearity in γH2AX and CM-H2DCFDA with R2 values of 0.9939 and 0.9595 respectively. Single cell gel electrophoresis experimentation for the detection of physical DNA double-strand breaks in ongoing. Conclusions: This work indicates significant potential for our high-throughput variable dose rate irradiator for patient-specific predictive radiation sensitivity bioassays. This irradiator provides a powerful tool by increasing the efficiency and number of assay techniques available to help personalize radiation therapy.

  9. Technology for cell cycle research with unstressed steady-state cultures

    OpenAIRE

    Lebleu, Valerie S.; Thornton, Maureen; Gonda, Steven R.; Helmstetter, Charles E.

    2006-01-01

    A culture system for performing cell cycle analyses on cells in undisturbed steady-state populations was designed and tested. In this system, newborn cells are shed continuously from an immobilized, perfused culture rotating about the horizontal axis. As a result of this arrangement, the number of newborn cells released into the effluent medium each generation is identical to the number of cells residing in the immobilized population, indicating that one of the two new daughter cells is shed ...

  10. Enhanced cell attachment using a novel cell culture surface presenting functional domains from extracellular matrix proteins

    OpenAIRE

    Cooke, M. J.; Phillips, S R; Shah, D. S. H.; Athey, D.; Lakey, J H; Przyborski, S A

    2008-01-01

    Many factors contribute to the creation and maintenance of a realistic environment for cell growth in vitro, e.g. the consistency of the growth medium, the addition of supplements, and the surface on which the cells grow. The nature of the surface on which cells are cultured plays an important role in their ability to attach, proliferate, migrate and function. Components of the extracellular matrix (ECM) are often used to coat glass or plastic surfaces to enhance cell attachment in vitro. Fra...

  11. Azo-polysiloxanes as new supports for cell cultures

    Energy Technology Data Exchange (ETDEWEB)

    Hurduc, Nicolae, E-mail: nhurduc@ch.tuiasi.ro [“Gheorghe Asachi” Technical University of Iasi, Department of Natural and Synthetic Polymers, Prof. Dimitrie, Mangeron Street, 73, 700050-Iasi (Romania); Macovei, Alina [Institute of Biochemistry of the Romanian Academy, Department of Viral Glycoproteins, Splaiul Independentei 296, Sector 6, 060041-Bucuresti (Romania); Paius, Cristina; Raicu, Alina [“Gheorghe Asachi” Technical University of Iasi, Department of Natural and Synthetic Polymers, Prof. Dimitrie, Mangeron Street, 73, 700050-Iasi (Romania); Moleavin, Ioana [CEA, LIST Saclay, Laboratoire Capteurs et Architectures Électroniques, F-91191 Gif-sur-Yvette, Cedex (France); Branza-Nichita, Norica, E-mail: nichita@biochim.ro [Institute of Biochemistry of the Romanian Academy, Department of Viral Glycoproteins, Splaiul Independentei 296, Sector 6, 060041-Bucuresti (Romania); Hamel, Matthieu [CEA, LIST Saclay, Laboratoire Capteurs et Architectures Électroniques, F-91191 Gif-sur-Yvette, Cedex (France); Rocha, Licinio, E-mail: Licinio.ROCHA@cea.fr [CEA, LIST Saclay, Laboratoire Capteurs et Architectures Électroniques, F-91191 Gif-sur-Yvette, Cedex (France)

    2013-05-01

    The paper introduces a new class of materials with azo-polysiloxanic structure bearing the property to generate nano-structured surfaces by laser irradiation. The ability to modulate the optical response of the film, through a modification of the polymer chemical structure, has been investigated. The azo-materials were tested for their ability to support cell adhesion and growth, with very promising results. A future use of these materials as growth support in cell cultures is of great interest, due to an easy, one step-method to generate the surface relief grating and to the possibility to introduce a large range of chemical modifications due to the presence of the chlorobenzyl groups in the polymeric side-chain. - Graphical abstract: Cell development on a nano-structured surface obtained from an azo-polysiloxanic film. Highlights: ► New azo-polysiloxanic films for biological applications were reported. ► Nanostructured surfaces with controllable geometry are obtained by laser irradiation. ► Cells are very sensitive to the chemical and physical properties of the polymeric substrate.

  12. Azo-polysiloxanes as new supports for cell cultures

    International Nuclear Information System (INIS)

    The paper introduces a new class of materials with azo-polysiloxanic structure bearing the property to generate nano-structured surfaces by laser irradiation. The ability to modulate the optical response of the film, through a modification of the polymer chemical structure, has been investigated. The azo-materials were tested for their ability to support cell adhesion and growth, with very promising results. A future use of these materials as growth support in cell cultures is of great interest, due to an easy, one step-method to generate the surface relief grating and to the possibility to introduce a large range of chemical modifications due to the presence of the chlorobenzyl groups in the polymeric side-chain. - Graphical abstract: Cell development on a nano-structured surface obtained from an azo-polysiloxanic film. Highlights: ► New azo-polysiloxanic films for biological applications were reported. ► Nanostructured surfaces with controllable geometry are obtained by laser irradiation. ► Cells are very sensitive to the chemical and physical properties of the polymeric substrate

  13. Culture and identification of stem cells of uterine junctional zone

    Directory of Open Access Journals (Sweden)

    Kang KANG

    2015-04-01

    Full Text Available Objective To establish a method for isolation, culture, and identification of stem cells in uterine junctional zone (uJZSCs. Methods Specimens of uterus muscle layer at uterine junctional zone (uJZ were harvested under aseptic condition, and they digested, cultivated and amplified using trypsogen and collagenaseⅠ. The morphology of uJZSCs was observed with inverted microscope, and cell viability and phenotype were analyzed by flow cytometry. The adipogenic, osteogenic and chondrogenic differentiation was induced in vitro, and their biological growth characteristic was identified by CCK-8 marking. Results The cells were adhered after passage, and they presented long spindle in shape. Flow cytometry showed that the expressions of CD90, CD73, CD105, CD29, CD44, CD13, CD166, and HLA-ABC marking was also positive, while they were negative for CD34, CD45, CD14, HLA-DR and CD19. The CCK-8 growth curve was S-shaped. The induced differentiation experiments indicated that uJZSCs could be induced into osteoblast, adipocyte and chondrocytes. Conclusion uJZSCs possess a strong proliferation capacity, and they might become a new source of mesenchymal stem cells. DOI: 10.11855/j.issn.0577-7402.2015.03.01

  14. Phospholipids of subcellular organelles isolated from cultured BHK cells.

    Science.gov (United States)

    Brotherus, J; Renkonen, O

    1977-02-23

    Mitochondrial and nuclei were purified from cultured hamster fibroblasts (BHK21 cells) by centrifugation in sucrose gradients. The phospholipid compositions of the preparations were compared to those of the previously purified plasma membranes, endoplasmic reticulum and lysosomes. The mitochondria had a characteristically high content (approx. 16% of lipid phosphorus) of cardiolipin, which was practically absent from the other purified organelles. The nuclei were enriched in phosphatidylcholine and phosphatidylinositol (approx. 68% and 5% of lipid phosphorus, respectively). Lysobisphosphatidic acid was almost absent from the mitochondria and nuclei, as well as from the plasma membrane and endoplasmic reticulum, which suggests that this phospholipid is confined to the lysosomes of the BHK cell. The nuclei and the mitochondria contained relatively little sphingomyelin, a characteristic lipid of the plasma membrane. The distributions of the total cellular phospholipid and protein between the various organelles were calculated and compared to the corresponding data estimated for the rat liver. The BHK cell contained relatively more phospholipids in the nucleus and the lysosomes than the liver. All the organelles of the BHK cell contained less protein per phospholipid than the equivalent organelles of the liver. PMID:836856

  15. Molecular and cellular mechanisms of cadmium resistance in cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Grady, D.L.; Moyzis, R.K.; Hildebrand, C.E.

    1985-01-01

    Heavy metal induction of the synthesis of metallothioneins (MTs) provides an ideal model system for basic mechanistic studies of gene expression. Cell lines varying in their resistance to heavy metals have been isolated through a regime of exposure to serially increasing levels of Cd followed by clonal isolation. These cell lines have been used to examine the role of methylation and amplification in the Cd-resistant (Cd/sup r/) phenotype. It is suggested that regulation of expression of the MT genes in Cd/sup r/ Chinese hamster cells is modulated at both the transcriptional and translational levels. An analysis of the MT2 gene sequence has uncovered a potential alternative splice site in the first intron. Usage of this site would insert 3 or 12 additional amino acids between amino acids 9 and 10. Analysis of the splicing pattern of the MT2 gene transcript in cultured cells has indicated that the second intron is preferentially removed prior to first intron excision. 34 refs., 2 figs., 1 tab.

  16. Bioassays for the toxicity of petroleum oils to birds

    International Nuclear Information System (INIS)

    A study was conducted to develop bioassays to predict the potential toxicity of petroleum oils to birds. Two bioassay systems were assessed. The first was the embryonated chicken egg. Mortality and pathological changes were examined in embryos treated with 1-30 μl of petroleum oils applied to the eggshell on day 9 of incubation and examined on day 13. Lesions seen consistently were extensive edema, superficial zones of hepatic necrosis, distension of the heart, and enlargement of the spleen. These morphological changes could not be reproduced by covering the eggshell with inert sealants. Liver necrosis and edema were found in embryos exposed to the 6 petroleum oils, suggesting a common mechanism of toxic action. The 96-h LD25 and ED25 (lethal and effective doses, respectively, affecting 25% of the population) for liver necrosis were determined for 6 petroleum oils. Estimates of the ED25 for liver necrosis could discriminate among the toxicities of the 6 oils better than could estimates of the LD25, and the ED25 was more reliable and consistent when bioassays were repeated on the same oil. The second system was the red blood cell exposed to petroleum oils in vitro, designed to model the Heinz body hemolytic anemia described in birds ingesting crude oil. Formation of methemoglobin and Heinz bodies was measured in rabbit erythrocytes exposed to naphthalene or extracts of petroleum oils after incubation. The assay could detect dose-responsive Heinz body formation in red cells treated with naphthalene, but Heinz body formation was not detected in red cells treated with 6 different petroleum oils. 326 refs., 19 figs., 46 tabs

  17. Study on in vitro Cultural Behaviors of Spermatogonium Stem Cells of New Born Calves

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jinyou; HU Pengfei; HUANG Zhijun; LV Zhonghua; ZHANG Guixue

    2008-01-01

    Three methods were adopted in culture spermatogoniums of newly born calf in vitro, such as enzymatic digestion and percoll density gradient centrifugation(Method I ), tubular fragments culture(Method Ⅱ ) and tissue culture(Method Ⅲ), and cultural behaviors of cells were observed. The results showed that typical spermatogonium colonies appeard at 144 h of culture by enzymatic digestion-percoll density gradient centrifugation method and tubular fragments culture method, 2.5% FBS kept the characteristics of spermatogonium stem cell better than others, produced more mass clones, and FBS of more than 2.5% concentration benefited spermatogonium differentiation and the number of colonies was significantly affected by FBS concentration. After 1 week of culture in method Ⅲ, the diameter of lumens and quantity of sertoli's cells in tubal wall increased obviously, lumen of seminiferous tubules appeared. Sertoli's cells kept constant and the number of spermatogoniums decreased obviously after 2 weeks of culture.

  18. Ghrelin regulates cell cycle-related gene expression in cultured hippocampal neural stem cells.

    Science.gov (United States)

    Chung, Hyunju; Park, Seungjoon

    2016-08-01

    We have previously demonstrated that ghrelin stimulates the cellular proliferation of cultured adult rat hippocampal neural stem cells (NSCs). However, little is known about the molecular mechanisms by which ghrelin regulates cell cycle progression. The purpose of this study was to investigate the potential effects of ghrelin on cell cycle regulatory molecules in cultured hippocampal NSCs. Ghrelin treatment increased proliferation assessed by CCK-8 proliferation assay. The expression levels of proliferating cell nuclear antigen and cell division control 2, well-known cell-proliferating markers, were also increased by ghrelin. Fluorescence-activated cell sorting analysis revealed that ghrelin promoted progression of cell cycle from G0/G1 to S phase, whereas this progression was attenuated by the pretreatment with specific inhibitors of MEK/extracellular signal-regulated kinase 1/2, phosphoinositide 3-kinase/Akt, mammalian target of rapamycin, and janus kinase 2/signal transducer and activator of transcription 3. Ghrelin-induced proliferative effect was associated with increased expression of E2F1 transcription factor in the nucleus, as determined by Western blotting and immunofluorescence. We also found that ghrelin caused an increase in protein levels of positive regulators of cell cycle, such as cyclin A and cyclin-dependent kinase (CDK) 2. Moreover, p27(KIP1) and p57(KIP2) protein levels were reduced when cell were exposed to ghrelin, suggesting downregulation of CDK inhibitors may contribute to proliferative effect of ghrelin. Our data suggest that ghrelin targets both cell cycle positive and negative regulators to stimulate proliferation of cultured hippocampal NSCs. PMID:27325242

  19. Physiological properties of vertebrate nerve cells in tissue culture.

    Science.gov (United States)

    Dichter, M A

    1975-01-01

    Vertebrate neurons in tissue culture are providing us with a new model system for studying the complex events which occur during neuronal differentiation, synaptogenesis, and neural network formation. It is already apparent that dissociated embryo neurons are capable of differentiating both morphologically and physiologically along predetermined lines in the absence of external influences. These neurons can form new connections with one another but retain some specificity in their selections. Both simple and complex neural networks can be seen. At the present time, the development of the invitro model system is just being explored. The potential value of a system of this kind at a variety of investigative levels should be appreciated. Questions of a fundamental nature in neurobiology, such as how synapses form, what rules govern such interaction, how cells recognize one another, and the nature of the basic two-, three-, or four-cell circuits that comprise the more complex neurons tissue can be approached with this system. Studies of the neurons and synapses themselves can lead to a more basic understanding of vertebrate nervous system functioning. The development of certain pathophysiological processes and the effects of neuroactive drugs on vertebrate neurons may be studied at the cellular level. Finally, the basic mechanism of some genetic abnormalities which produce abnormal nervous structure and function may be more easily determined in a simplified in vitro model than in the intact central nervous system. The value of any model is not inherent in the elegance of the model itseld, but only in its ability to suggest answers to fundamental questions about the system being modeled. Many fundamental questions about brain mechanisms in mental retardation remain unanswered. Perhaps some day the model of nerve cells in tissue culture will bring us closer to the answers to these questions. PMID:173059

  20. Co-culture of neuroepithelial stem cells with interstitial cells of Cajal results in neuron differentiation

    OpenAIRE

    Zhao, Bin; Liu, Wei; Wu, Rongde

    2015-01-01

    Objective: The interstitial cells of Cajal (ICCs) interact morphologically and functionally with the elements of the enteric nervous system in the digestive tract. However, direct evidence that ICCs participate in the differentiation of the enteric nervous system is lacking. In this work, we examined in co-culture experiments whether ICCs could stimulate the differentiation of neuroepithelial stem cells (NESCs) to neurons. Methods: NESCs were harvested from the neural tube of embryonic (E11.5...

  1. CELL SHAPE AND HEXOSE TRANSPORT IN NORMAL AND VIRUS-TRANSFORMED CELLS IN CULTURE

    Energy Technology Data Exchange (ETDEWEB)

    Bissell, M.J.; Farson, D.; Tung, A.S.C.

    1976-07-01

    The rate of hexose transport was compared in normal and virus-transformed cells on a monolayer and in suspension. It was shown that: (1) Both trypsin-removed cells and those suspended for an additional day in methyl cellulose had decreased rates of transport and lower available water space when compared with cells on a monolayer. Thus, cell shape affects the overall rate of hexose transport, especially at higher sugar concentrations. (2) Even in suspension, the initial transport rates remained higher in transformed cells with reference to normal cells. Scanning electron micrographs of normal and transformed chick cells revealed morphological differences only in the flat state. This indicates that the increased rate of hexose transport after transformation is not due to a difference in the shape of these cells on a monolayer. The relation between the geometry of cells, transport rates, and growth regulation is undoubtedly very complex, and our knowledge of these relationships is still very elementary. In a recent review on the influence of geometry on control of cell growth, Folkman and Greenspan (1) pointed out that the permeability of cells in a flat versus a spherical state may indeed be very different. The growth properties of cells on a surface and in suspension have been compared often (1-5). However, with one exception. little is known about the changes in transport properties when cell shape is changed. Foster and Pardee (6) demonstrated that the active transport of a-aminoisobutyric acid was reduced 2.5 times in suspension cultures of Chinese hamster cells with respect to the cells grown on a coverslip. They attributed this to the smaller surface area of suspended cells. While it is not clear why active transport should be dependent on the surface area available, it is possible that once the cells assume a spherical configuration, the carrier proteins are redistributed in such a way as to make them less accessible to the substrate. What happens to

  2. The usefulness of three-dimensional cell culture in induction of cancer stem cells from esophageal squamous cell carcinoma cell lines

    International Nuclear Information System (INIS)

    Highlights: •Spheroids were created from esophageal carcinoma cells using NanoCulture® Plates. •The proportion of strongly ALDH-positive cells increased in 3-D culture. •Expression of cancer stem cell-related genes was enhanced in 3-D culture. •CA-9 expression was enhanced, suggesting hypoxia had been induced in 3-D culture. •Drug resistance was increased. 3-D culture is useful for inducing cancer stem cells. -- Abstract: In recent years, research on resistance to chemotherapy and radiotherapy in cancer treatment has come under the spotlight, and researchers have also begun investigating the relationship between resistance and cancer stem cells. Cancer stem cells are assumed to be present in esophageal cancer, but experimental methods for identification and culture of these cells have not yet been established. To solve this problem, we created spheroids using a NanoCulture® Plate (NCP) for 3-dimensional (3-D) cell culture, which was designed as a means for experimentally reproducing the 3-D structures found in the body. We investigated the potential for induction of cancer stem cells from esophageal cancer cells. Using flow cytometry we analyzed the expression of surface antigen markers CD44, CD133, CD338 (ABCG2), CD318 (CDCP1), and CD326 (EpCAM), which are known cancer stem cell markers. None of these surface antigen markers showed enhanced expression in 3-D cultured cells. We then analyzed aldehyde dehydrogenase (ALDH) enzymatic activity using the ALDEFLUOR reagent, which can identify immature cells such as stem cells and precursor cells. 3-D-cultured cells were strongly positive for ALDH enzyme activity. We also analyzed the expression of the stem cell-related genes Sox-2, Nanog, Oct3/4, and Lin28 using RT-PCR. Expression of Sox-2, Nanog, and Lin28 was enhanced. Analysis of expression of the hypoxic surface antigen marker carbonic anhydrase-9 (CA-9), which is an indicator of cancer stem cell induction and maintenance, revealed that CA-9 expression

  3. The usefulness of three-dimensional cell culture in induction of cancer stem cells from esophageal squamous cell carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Fujiwara, Daisuke [Department of Esophageal and Gastroenterological Surgery, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Kato, Kazunori, E-mail: kzkatou@juntendo.ac.jp [Department of Biomedical Engineering, Toyo University, 2100 Kujirai, Kawagoe, Saitama 350-8585 (Japan); Department of Atopy Research Center, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Nohara, Shigeo; Iwanuma, Yoshimi; Kajiyama, Yoshiaki [Department of Esophageal and Gastroenterological Surgery, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan)

    2013-05-17

    Highlights: •Spheroids were created from esophageal carcinoma cells using NanoCulture® Plates. •The proportion of strongly ALDH-positive cells increased in 3-D culture. •Expression of cancer stem cell-related genes was enhanced in 3-D culture. •CA-9 expression was enhanced, suggesting hypoxia had been induced in 3-D culture. •Drug resistance was increased. 3-D culture is useful for inducing cancer stem cells. -- Abstract: In recent years, research on resistance to chemotherapy and radiotherapy in cancer treatment has come under the spotlight, and researchers have also begun investigating the relationship between resistance and cancer stem cells. Cancer stem cells are assumed to be present in esophageal cancer, but experimental methods for identification and culture of these cells have not yet been established. To solve this problem, we created spheroids using a NanoCulture® Plate (NCP) for 3-dimensional (3-D) cell culture, which was designed as a means for experimentally reproducing the 3-D structures found in the body. We investigated the potential for induction of cancer stem cells from esophageal cancer cells. Using flow cytometry we analyzed the expression of surface antigen markers CD44, CD133, CD338 (ABCG2), CD318 (CDCP1), and CD326 (EpCAM), which are known cancer stem cell markers. None of these surface antigen markers showed enhanced expression in 3-D cultured cells. We then analyzed aldehyde dehydrogenase (ALDH) enzymatic activity using the ALDEFLUOR reagent, which can identify immature cells such as stem cells and precursor cells. 3-D-cultured cells were strongly positive for ALDH enzyme activity. We also analyzed the expression of the stem cell-related genes Sox-2, Nanog, Oct3/4, and Lin28 using RT-PCR. Expression of Sox-2, Nanog, and Lin28 was enhanced. Analysis of expression of the hypoxic surface antigen marker carbonic anhydrase-9 (CA-9), which is an indicator of cancer stem cell induction and maintenance, revealed that CA-9 expression

  4. Assessment of the Pathogenic Potential of Asbestiform vs. Nonasbestiform Particulates (Cleavage Fragments) in In Vitro (Cell or Organ Culture) Models and Bioassays

    OpenAIRE

    MOSSMAN, BROOKE T.

    2007-01-01

    Asbestos fibers are highly fibrous silicate fibers that are distinguished by having a large aspect (length to diameter) ratio and are crystallized in an asbestiform habit that causes them to separate into very thin fibers or fibrils. These fibers are distinct from nonasbestiform cleavage fragments and may appear as thick, short fibers which break along cleavage planes without the high strength and flexibility of asbestiform fibers. Since cleavage fragments of respirable dimensions have genera...

  5. Development of an Integrated Microfluidic Perfusion Cell Culture System for Real-Time Microscopic Observation of Biological Cells

    OpenAIRE

    Chih-Chin Oh-Yang; Min-Hsien Wu; Jr-Lung Lin; Shih-Siou Wang

    2011-01-01

    This study reports an integrated microfluidic perfusion cell culture system consisting of a microfluidic cell culture chip, and an indium tin oxide (ITO) glass-based microheater chip for micro-scale perfusion cell culture, and its real-time microscopic observation. The system features in maintaining both uniform, and stable chemical or thermal environments, and providing a backflow-free medium pumping, and a precise thermal control functions. In this work, the performance of the medium pumpin...

  6. Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

    Science.gov (United States)

    A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus th...

  7. Establishment and Molecular Cytogenetic Characterization of a Cell Culture Model of Head and Neck Squamous Cell Carcinoma (HNSCC)

    OpenAIRE

    Horst Zitzelsberger; Axel Walch; Johannes Weber; Herbert Braselmann; Reinhard Huber; Ludwig Hieber; Quirin Schaeffner; Bauer, Verena L.

    2010-01-01

    Cytogenetic analysis of head and neck squamous cell carcinoma (HNSCC) established several biomarkers that have been correlated to clinical parameters during the past years. Adequate cell culture model systems are required for functional studies investigating those potential prognostic markers in HNSCC. We have used a cell line, CAL 33, for the establishment of a cell culture model in order to perform functional analyses of interesting candidate genes and proteins. The cell line was cytogeneti...

  8. Nerve cells culture from lumbar spinal cord on surfaces modified by plasma pyrrole polymerization.

    Science.gov (United States)

    Zuñiga-Aguilar, E; Olayo, R; Ramírez-Fernández, O; Morales, J; Godínez, R

    2014-01-01

    Currently, there are several techniques for modified cell culture surfaces under research to improve cell growth and adhesion. Recently, different methods have been used for surface coating, using biomolecules that enhance cell attachment and growth of nerve cells from spinal cord, such as the use of Poly-DL-Ornithine/Laminin. Plasma-polymerized pyrrole (PPy)-treated surfaces have showed improvement on surfaces biocompatibility with the cells in culture since they do not interfere with any of the biological cell functions. In the present work, we present a novel mouse nerve cell culture technique, using PPy-treated cell culture surfaces. A comparative study of cell survival using Poly-DL-Ornithine/Laminin-treated surfaces was performed. Our results of cell survival when compared with data already reported by other investigators, show that cells cultured on the PPy-modified surface increased survival up to 21 days when compared with Poly-DL-Ornithine/Laminin-coated culture, where 8 days cell survival was obtained. There were electrical and morphological differences in the nerve cells grown in the different surfaces. By comparing the peak ion currents of Poly-DL-Ornithine/Laminin-seeded cells for 8 days with cells grown for 21 days on PPy, an increase of 516% in the Na(+) current and 127% in K(+) currents in cells seeded on PPy were observed. Immunofluorescence techniques showed the presence of cell synapses and culture viability after 21 days. Our results then showed that PPy-modified surfaces are an alternative culture method that increases nerve cells survival from lumbar spinal cord cell culture by preserving its electrical and morphological features. PMID:24650203

  9. Approaches to Optimizing Animal Cell Culture Process: Substrate Metabolism Regulation and Protein Expression Improvement

    Science.gov (United States)

    Zhang, Yuanxing

    Some high value proteins and vaccines for medical and veterinary applications by animal cell culture have an increasing market in China. In order to meet the demands of large-scale productions of proteins and vaccines, animal cell culture technology has been widely developed. In general, an animal cell culture process can be divided into two stages in a batch culture. In cell growth stage a high specific growth rate is expected to achieve a high cell density. In production stage a high specific production rate is stressed for the expression and secretion of qualified protein or replication of virus. It is always critical to maintain high cell viability in fed-batch and perfusion cultures. More concern has been focused on two points by the researchers in China. First, the cell metabolism of substrates is analyzed and the accumulation of toxic by-products is decreased through regulating cell metabolism in the culture process. Second, some important factors effecting protein expression are understood at the molecular level and the production ability of protein is improved. In pace with the rapid development of large-scale cell culture for the production of vaccines, antibodies and other recombinant proteins in China, the medium design and process optimization based on cell metabolism regulation and protein expression improvement will play an important role. The chapter outlines the main advances in metabolic regulation of cell and expression improvement of protein in animal cell culture in recent years.

  10. Mesenchymal Stromal Cell Phenotype is not Influenced by Confluence during Culture Expansion

    DEFF Research Database (Denmark)

    Haack-Sørensen, Mandana; Hansen, Susanne Kofoed; Hansen, Louise;

    2013-01-01

    cell quantity must not affect quality, but it is also a fact that in vitro culture conditions affect MSC phenotype. One possible variable is the degree of cell confluence during expansion. METHODS: We investigate the influence of cell density on homogeneity and differentiation during culture expansion...

  11. Development of Cell Culture Microdevice Actuated by Piezoelectric Thin Films for Delivering Mechanical Vibratory Stimuli to Cells

    International Nuclear Information System (INIS)

    In order to realize a cell culture microdevice actuated by piezoelectric thin films for on-chip regulation of cell functions, this paper reported on a feasibility study by using the microdevice with KOH-etched cavities surrounded by four (111) sidewalls as microchambers in order to introduce cells to be cultured. As a result, the vibration characteristic of the PZT actuator was improved by using an electric field -150 kV/cm at 70 C for 30 min in poling process. A feasibility study on cell culture for delivering mechanical vibratory stimuli to cells revealed the microdevice could be applicable to the culture with actual biological cells. In addition, it was found that O2-plasma treated parylene-C process could be applicable for obtaining homogeneous surface of cell culture microdevice.

  12. Culture models of human mammary epithelial cell transformation

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R.; Yaswen, Paul

    2000-11-10

    Human pre-malignant breast diseases, particularly ductal carcinoma in situ (DCIS)3 already display several of the aberrant phenotypes found in primary breast cancers, including chromosomal abnormalities, telomerase activity, inactivation of the p53 gene and overexpression of some oncogenes. Efforts to model early breast carcinogenesis in human cell cultures have largely involved studies in vitro transformation of normal finite lifespan human mammary epithelial cells (HMEC) to immortality and malignancy. We present a model of HMEC immortal transformation consistent with the know in vivo data. This model includes a recently described, presumably epigenetic process, termed conversion, which occurs in cells that have overcome stringent replicative senescence and are thus able to maintain proliferation with critically short telomeres. The conversion process involves reactivation of telomerase activity, and acquisition of good uniform growth in the absence and presence of TFGB. We propose th at overcoming the proliferative constraints set by senescence, and undergoing conversion, represent key rate-limiting steps in human breast carcinogenesis, and occur during early stage breast cancer progression.

  13. Proliferation of cultured mouse choroid plexus epithelial cells.

    Directory of Open Access Journals (Sweden)

    Basam Z Barkho

    Full Text Available The choroid plexus (ChP epithelium is a multifunctional tissue found in the ventricles of the brain. The major function of the ChP epithelium is to produce cerebrospinal fluid (CSF that bathes and nourishes the central nervous system (CNS. In addition to the CSF, ChP epithelial cells (CPECs produce and secrete numerous neurotrophic factors that support brain homeostasis, such as adult hippocampal neurogenesis. Accordingly, damage and dysfunction to CPECs are thought to accelerate and intensify multiple disease phenotypes, and CPEC regeneration would represent a potential therapeutic approach for these diseases. However, previous reports suggest that CPECs rarely divide, although this has not been extensively studied in response to extrinsic factors. Utilizing a cell-cycle reporter mouse line and live cell imaging, we identified scratch injury and the growth factors insulin-like growth factor 1 (IGF-1 and epidermal growth factor (EGF as extrinsic cues that promote increased CPEC expansion in vitro. Furthermore, we found that IGF-1 and EGF treatment enhances scratch injury-induced proliferation. Finally, we established whole tissue explant cultures and observed that IGF-1 and EGF promote CPEC division within the intact ChP epithelium. We conclude that although CPECs normally have a slow turnover rate, they expand in response to external stimuli such as injury and/or growth factors, which provides a potential avenue for enhancing ChP function after brain injury or neurodegeneration.

  14. Thioredoxin and NADP-thioredoxin reductase from cultured carrot cells

    Science.gov (United States)

    Johnson, T. C.; Cao, R. Q.; Kung, J. E.; Buchanan, B. B.

    1987-01-01

    Dark-grown carrot (Daucus carota L.) tissue cultures were found to contain both protein components of the NADP/thioredoxin system--NADP-thioredoxin reductase and the thioredoxin characteristic of heterotrophic systems, thioredoxin h. Thioredoxin h was purified to apparent homogeneity and, like typical bacterial counterparts, was a 12-kdalton (kDa) acidic protein capable of activating chloroplast NADP-malate dehydrogenase (EC 1.1.1.82) more effectively than fructose-1,6-bisphosphatase (EC 3.1.3.11). NADP-thioredoxin reductase (EC 1.6.4.5) was partially purified and found to be an arsenite-sensitive enzyme composed of two 34-kDa subunits. Carrot NADP-thioredoxin reductase resembled more closely its counterpart from bacteria rather than animal cells in acceptor (thioredoxin) specificity. Upon greening of the cells, the content of NADP-thioredoxin-reductase activity, and, to a lesser extent, thioredoxin h decreased. The results confirm the presence of a heterotrophic-type thioredoxin system in plant cells and raise the question of its physiological function.

  15. Cell culture monitoring by impedance mapping using a multielectrode scanning impedance spectroscopy system (CellMap)

    International Nuclear Information System (INIS)

    We report on the impedance mapping of in vitro cellular morphology by electrical impedance spectroscopy, using microelectrodes. A micro multielectrode system was designed, fabricated, assembled, tested and demonstrated for the monitoring of anchorage-dependent cell behavior and morphology. This system allowed continuous, label-free, quantitative monitoring and visualization of cell adhesion, spreading, proliferation and detachment due to cell cycle processes as well as cell–drug interaction, with spatio-temporal resolution. OvCa429 ovarian cancer cells were monitored in vitro over a period of 70 hours by inoculating the cell suspension directly on the multielectrode device. The phase angle of impedance was observed to develop a distinctive shape as a result of cell attachment and proliferation. The shape of the phase angle curve reverted back to the pre-attachment shape upon detachment of cells from the substrate, caused by the addition of trypsin to the cell culture medium. The impedance data of the cell culture were then successfully modeled as a multi-parametric equivalent circuit. The model incorporated both interfacial and cell-layer impedance parameters. Upon addition of trypsin, the cell-layer parameters showed a marked decline and were eventually eliminated from the multi-parametric model, confirming the correlation of the model to the electrode–cell–electrolyte system. These experiments demonstrate the applicability of the impedance mapping technique in visualizing and quantifying physiological changes in the cell layer due to cellular processes as well as the effect of external chemical stimulus on cells (cell–drug interaction)

  16. Polyamines and the Cell Cycle of Catharanthus roseus Cells in Culture 1

    Science.gov (United States)

    Maki, Hisae; Ando, Satoshi; Kodama, Hiroaki; Komamine, Atsushi

    1991-01-01

    Investigation was made on the effect of partial depletion of polyamines (PAs), induced by treatment with inhibitors of the biosynthesis of PAs, on the distribution of cells at each phase of the cell cycle in Catharanthus roseus (L.) G. Don. cells in suspension cultures, using flow cytometry. More cells treated with inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) were accumulated in the G1 phase than those in the control, while the treatment with an inhibitor of spermidine (SPD) synthase showed no effect on the distribution of cells. The endogenous levels of the PAs, putrescine (PUT), SPD, and spermine (SPM), were determined during the cell cycle in synchronous cultures of C. roseus. Two peaks of endogenous level of PAs, in particular, of PUT and SPD, were observed during the cell cycle. Levels of PAs increased markedly prior to synthesis of DNA in the S phase and prior to cytokinesis. Activities of ADC and ODC were also assayed during the cell cycle. Activities of ADC was much higher than that of ODC throughout the cell cycle, but both activities of ODC and ADC changed in concert with changes in levels of PAs. Therefore, it is suggested that these enzymes may regulate PA levels during the cell cycle. These results indicate that inhibitors of PUT biosynthesis caused the suppression of cell proliferation by prevention of the progression of the cell cycle, probably from the G1 to the S phase, and PUT may play more important roles in the progression of the cell cycle than other PAs. PMID:16668290

  17. Regulation of human renin expression in chorion cell primary cultures

    International Nuclear Information System (INIS)

    The human renin gene is expressed in the kidney, placenta, and several other sites. The release of renin or its precursor, prorenin, can be affected by several regulatory agents. In this study, primary cultures of human placental cells were used to examine the regulation of prorenin release and renin mRNA levels and of the transfected human renin promoter linked to chloramphenicol acetyltransferase reporter sequences. Treatment of the cultures with a calcium ionophore alone, calcium ionophore plus forskolin (that activates adenylate cyclase), or forskolin plus a phorbol ester increased prorenin release and renin mRNA levels 1.3 endash to 6 endash fold, but several classes of steroids did not affect prorenin secretion or renin RNA levels. These results suggest that (i) the first 584 base pairs of the renin gene 5'endash flanking DNA do not contain functional glucocorticoid or estrogen response elements, (ii) placental prorenin release and renin mRNA are regulated by calcium ion and by the combinations of cAMP with either C kinase or calcium ion, and (iii) the first 100 base pairs of the human renin 5'endash flanking DNA direct accurate initiation of transcription and can be regulated by cAMP. Thus, some control of renin release in the placenta (and by inference in other tissues) occurs via transcriptional influences on its promoter

  18. Maintenance of neural stem cell regional identity in culture.

    Science.gov (United States)

    Delgado, Ryan N; Lu, Changqing; Lim, Daniel A

    2016-01-01

    Neural stem cells (NSCs) are distributed throughout the ventricular-subventricular zone (V-SVZ) in the adult mouse brain. NSCs located in spatially distinct regions of the V-SVZ generate different types of olfactory bulb (OB) neurons, and the regional expression of specific transcription factors correlates with these differences in NSC developmental potential. In a recent article, we show that Nkx2.1-expressing embryonic precursors give rise to NKX2.1+ NSCs located in the ventral V-SVZ of adult mice. Here we characterize a V-SVZ monolayer culture system that retains regional gene expression and neurogenic potential of NSCs from the dorsal and ventral V-SVZ. In particular, we find that Nkx2.1-lineage V-SVZ NSCs maintain Nkx2.1 expression through serial passage and can generate new neurons in vitro. Thus, V-SVZ NSCs retain key aspects of their in vivo regional identity in culture, providing new experimental opportunities for understanding how such developmental patterns are established and maintained during development. PMID:27606338

  19. Lysine hydroxylation of collagen in a fibroblast cell culture system

    Science.gov (United States)

    Uzawa, Katsuhiro; Yeowell, Heather N.; Yamamoto, Kazushi; Mochida, Yoshiyuki; Tanzawa, Hideki; Yamauchi, Mitsuo

    2003-01-01

    The lysine (Lys) hydroxylation pattern of type I collagen produced by human fibroblasts in culture was analyzed and compared. Fibroblasts were cultured from normal human skin (NSF), keloid (KDF), fetal skin (FDF), and skin tissues of Ehlers-Danlos syndrome type VIA and VIB patients (EDS-VIA and -VIB). The type I collagen alpha chains with or without non-helical telopeptides were purified from the insoluble matrix and analyzed. In comparison with NSFs, KDF and FDF showed significantly higher Lys hydroxylation, particularly in the telopeptide domains of both alpha chains. Both EDS-VIA and -VIB showed markedly lower Lys hydroxylation in the helical domains of both alpha chains whereas that in the telopeptides was comparable with those of NSFs. A similar profile was observed in the tissue sample of the EDS-VIB patient. These results demonstrate that the Lys hydroxylation pattern is domain-specific within the collagen molecule and that this method is useful to characterize the cell phenotypes in normal/pathological connective tissues.

  20. Voltage effects on cells cultured on metallic biomedical implants

    Science.gov (United States)

    Haerihosseini, Seyed Morteza

    Electrochemical voltage shifts in metallic biomedical implants occur in-vivo due to a number of processes including mechanically assisted corrosion. Surface potential of biomedical implants and excursions from resting open circuit potential (OCP), which is the voltage they attain while in contact with an electrolyte, can significantly change the interfacial properties of the metallic surfaces and alter the behavior of the surrounding cells, compromising the biocompatibility of metallic implants. Voltages can also be controlled to modulate cell function and fate. To date, the details of the physico-chemical phenomena and the role of different biomaterial parameters involved in the interaction between cells and metallic surfaces under cathodic bias have not been fully elucidated. In this work, changes in the interfacial properties of a CoCrMo biomedical alloy (ASTM F-1537) in phosphate-buffered saline (PBS) (pH 7.4) at different voltages was studied. Step polarization impedance spectroscopy technique was used to apply 50 mV voltage steps to samples, and the time-based current transients were recorded. A new equation was derived based on capacitive discharge through a Tafel element and generalized to deal with non-ideal impedance behavior. The new function compared to the KWW-Randles function, better matched the time-transient response. The results also showed a voltage dependent oxide resistance and capacitance behavior. Additionally, the in-vitro effect of static voltages on the behavior of MC3T3-E1 pre-osteoblasts cultured on CoCrMo alloy (ASTM-1537) was studied to determine the range of cell viability and mode of cell death beyond the viable range. Cell viability and morphology, changes in actin cytoskeleton, adhesion complexes and nucleus, and mode of cell death (necrosis, or intrinsic or extrinsic apoptosis) were characterized at different voltages ranging from -1000 to +500 mV (Ag/AgCl). Moreover, electrochemical currents and metal ion concentrations at each

  1. Pigment cell differentiation in sea urchin blastula-derived primary cell cultures.

    Science.gov (United States)

    Ageenko, Natalya V; Kiselev, Konstantin V; Dmitrenok, Pavel S; Odintsova, Nelly A

    2014-07-01

    The quinone pigments of sea urchins, specifically echinochrome and spinochromes, are known for their effective antioxidant, antibacterial, antifungal, and antitumor activities. We developed in vitro technology for inducing pigment differentiation in cell culture. The intensification of the pigment differentiation was accompanied by a simultaneous decrease in cell proliferation. The number of pigment cells was two-fold higher in the cells cultivated in the coelomic fluids of injured sea urchins than in those intact. The possible roles of the specific components of the coelomic fluids in the pigment differentiation process and the quantitative measurement of the production of naphthoquinone pigments during cultivation were examined by MALDI and electrospray ionization mass spectrometry. Echinochrome A and spinochrome E were produced by the cultivated cells of the sand dollar Scaphechinus mirabilis in all tested media, while only spinochromes were found in the cultivated cells of another sea urchin, Strongylocentrotus intermedius. The expression of genes associated with the induction of pigment differentiation was increased in cells cultivated in the presence of shikimic acid, a precursor of naphthoquinone pigments. Our results should contribute to the development of new techniques in marine biotechnology, including the generation of cell cultures producing complex bioactive compounds with therapeutic potential. PMID:24979272

  2. Pigment Cell Differentiation in Sea Urchin Blastula-Derived Primary Cell Cultures

    Directory of Open Access Journals (Sweden)

    Natalya V. Ageenko

    2014-06-01

    Full Text Available The quinone pigments of sea urchins, specifically echinochrome and spinochromes, are known for their effective antioxidant, antibacterial, antifungal, and antitumor activities. We developed in vitro technology for inducing pigment differentiation in cell culture. The intensification of the pigment differentiation was accompanied by a simultaneous decrease in cell proliferation. The number of pigment cells was two-fold higher in the cells cultivated in the coelomic fluids of injured sea urchins than in those intact. The possible roles of the specific components of the coelomic fluids in the pigment differentiation process and the quantitative measurement of the production of naphthoquinone pigments during cultivation were examined by MALDI and electrospray ionization mass spectrometry. Echinochrome A and spinochrome E were produced by the cultivated cells of the sand dollar Scaphechinus mirabilis in all tested media, while only spinochromes were found in the cultivated cells of another sea urchin, Strongylocentrotus intermedius. The expression of genes associated with the induction of pigment differentiation was increased in cells cultivated in the presence of shikimic acid, a precursor of naphthoquinone pigments. Our results should contribute to the development of new techniques in marine biotechnology, including the generation of cell cultures producing complex bioactive compounds with therapeutic potential.

  3. Extraction and Assembly of Tissue-Derived Gels for Cell Culture and Tissue Engineering

    OpenAIRE

    Uriel, Shiri; Labay, Edwardine; Francis-Sedlak, Megan; Moya, Monica L.; Weichselbaum, Ralph R.; Ervin, Natalia; Cankova, Zdravka; Eric M Brey

    2008-01-01

    Interactions with the extracellular matrix (ECM) play an important role in regulating cell function. Cells cultured in, or on, three-dimensional ECM recapitulate similar features to those found in vivo that are not present in traditional two-dimensional culture. In addition, both natural and synthetic materials containing ECM components have shown promise in a number of tissue engineering applications. Current materials available for cell culture and tissue engineering do not adequately refle...

  4. The bio-oscillator: A circuit for cell-culture assays

    OpenAIRE

    Huertas, Gloria; Maldonado, Andrés; Yúfera, A.; Rueda, Adoración; Huertas-Díaz, J. L.

    2015-01-01

    A system for cell-culture real-time monitoring using an oscillation-based approach is proposed. The system transforms a cell culture under test into a suitable “biological” oscillator, without needing complex circuitry for excitation and measurement. The obtained oscillation parameters are directly related to biological test, owed to an empirically extracted cell–electrode electrical model. A discrete prototype is proposed and experimental results with living cell culture are presented, achie...

  5. Pneumocystis jirovecii Can Be Productively Cultured in Differentiated CuFi-8 Airway Cells

    OpenAIRE

    Schildgen, Verena; Mai, Stephanie; Khalfaoui, Soumaya; Lüsebrink, Jessica; Pieper, Monika; Tillmann, Ramona L.; Brockmann, Michael; Schildgen, Oliver

    2014-01-01

    ABSTRACT Although Pneumocystis jirovecii is a well-known and serious pathogen, all previous attempts to isolate, cultivate, and propagate this fungus have failed. This serious challenge in microbiology was addressed in the present study. We examined whether P. jirovecii could be cultured in a permanent three-dimensional air-liquid interface culture system formed by CuFi-8 cells, a differentiated pseudostratified airway epithelial cell line. Cultured pseudostratified cells were inoculated with...

  6. Induction of phytic acid synthesis by abscisic acid in suspension-cultured cells of rice

    OpenAIRE

    Matsuno, Koya; Fujimura, Tatsuhito

    2014-01-01

    A pathway of phytic acid (PA) synthesis in plants has been revealed via investigations of low phytic acid mutants. However, the regulation of this pathway is not well understood because it is difficult to control the environments of cells in the seeds, where PA is mainly synthesized. We modified a rice suspension culture system in order to study the regulation of PA synthesis. Rice cells cultured with abscisic acid (ABA) accumulate PA at higher levels than cells cultured without ABA, and PA a...

  7. Hsp90 inhibitors reduce influenza virus replication in cell culture

    International Nuclear Information System (INIS)

    The viral RNA polymerase complex of influenza A virus consists of three subunits PB1, PB2 and PA. Recently, the cellular chaperone Hsp90 was shown to play a role in nuclear import and assembly of the trimeric polymerase complex by binding to PB1 and PB2. Here we show that Hsp90 inhibitors, geldanamycin or its derivative 17-AAG, delay the growth of influenza virus in cell culture resulting in a 1-2 log reduction in viral titre early in infection. We suggest that this is caused by the reduced half-life of PB1 and PB2 and inhibition of nuclear import of PB1 and PA which lead to reduction in viral RNP assembly. Hsp90 inhibitors may represent a new class of antiviral compounds against influenza viruses

  8. Stem cells from human exfoliated deciduous teeth differentiate toward neural cells in a medium dynamically cultured with Schwann cells in a series of polydimethylsiloxanes scaffolds

    Science.gov (United States)

    Su, Wen-Ta; Pan, Yu-Jing

    2016-08-01

    Objective. Schwann cells (SCs) are primary structural and functional cells in the peripheral nervous system. These cells play a crucial role in peripheral nerve regeneration by releasing neurotrophic factors. This study evaluated the neural differentiation potential effects of stem cells from human exfoliated deciduous teeth (SHEDs) in a rat Schwann cell (RSC) culture medium. Approach. SHEDs and RSCs were individually cultured on a polydimethylsiloxane (PDMS) scaffold, and the effects of the RSC medium on the SHEDs differentiation between static and dynamic cultures were compared. Main results. Results demonstrated that the SHED cells differentiated by the RSC cultured medium in the static culture formed neurospheres after 7 days at the earliest, and SHED cells formed neurospheres within 3 days in the dynamic culture. These results confirm that the RSC culture medium can induce neurospheres formation, the speed of formation and the number of neurospheres (19.16 folds high) in a dynamic culture was superior to the static culture for 3 days culture. The SHED-derived spheres were further incubated in the RSCs culture medium, these neurospheres continuously differentiated into neurons and neuroglial cells. Immunofluorescent staining and RT-PCR revealed nestin, β-III tubulin, GFAP, and γ-enolase of neural markers on the differentiated cells. Significance. These results indicated that the RSC culture medium can induce the neural differentiation of SHED cells, and can be used as a new therapeutic tool to repair nerve damage.

  9. Multifunction Co-culture Model for Evaluating Cell–Cell Interactions

    OpenAIRE

    Bogdanowicz, Danielle R.; Lu, Helen H.

    2014-01-01

    Interactions within the same cell population (homotypic) and between different cell types (heterotypic) are essential for tissue development, repair, and homeostasis. To elucidate the underlying mechanisms of these cellular interactions, co-culture models have been used extensively to investigate the role of cell–cell physical contact, autocrine and/or paracrine interactions on cell function, as well as stem cell differentiation. Specifically, the mixed co-culture model is often optimal for i...

  10. Derivation of Myogenic Progenitors Directly From Human Pluripotent Stem Cells Using a Sphere-Based Culture

    OpenAIRE

    Hosoyama, Tohru; McGivern, Jered V.; Van Dyke, Jonathan M.; Allison D Ebert; Suzuki, Masatoshi

    2014-01-01

    The authors present a novel protocol for deriving myogenic progenitors from human embryonic stem cells and induced pluripotent stem cells using free-floating spherical culture. Results show that sphere-based cultures of human pluripotent stem cells, expanded in medium containing high concentrations of fibroblast growth factor and epidermal growth factor, can propagate myogenic progenitors from human embryonic stem cells and healthy and disease-specific induced pluripotent stem cells.

  11. Self-assembling Fmoc dipeptide hydrogel for in situ 3D cell culturing

    OpenAIRE

    Akpe Victor; Rydholm Susanna; Liebmann Thomas; Brismar Hjalmar

    2007-01-01

    Abstract Background Conventional cell culture studies have been performed on 2D surfaces, resulting in flat, extended cell growth. More relevant studies are desired to better mimic 3D in vivo tissue growth. Such realistic environments should be the aim of any cell growth study, requiring new methods for culturing cells in vitro. Cell biology is also tending toward miniaturization for increased efficiency and specificity. This paper discusses the application of a self-assembling peptide-derive...

  12. Study of Pluripotency Markers in Zebrafish Embryos and Transient Embryonic Stem Cell Cultures

    OpenAIRE

    Robles, Vanesa; Martí, Mercé; Belmonte, Juan Carlos Izpisua

    2011-01-01

    Targeted genomic manipulation using embryonic stem (ES) cells has not yet been achieved in zebrafish, although methods for zebrafish ES cell culture has been described in literature. The knowledge of pluripotency markers in this species is almost nonexistent and this is a very limiting factor in the definition of the ideal culture conditions for ES cells. Here, we studied the expression of several genes associated with pluripotency in zebrafish embryonic cells versus differentiated cells and ...

  13. Systematic evaluation of sericin protein as a substitute for fetal bovine serum in cell culture.

    Science.gov (United States)

    Liu, Liyuan; Wang, Jinhuan; Duan, Shengchang; Chen, Lei; Xiang, Hui; Dong, Yang; Wang, Wen

    2016-01-01

    Fetal bovine serum (FBS) shows obvious deficiencies in cell culture, such as low batch to batch consistency, adventitious biological contaminant risk, and high cost, which severely limit the development of the cell culture industry. Sericin protein derived from the silkworm cocoon has become increasingly popular due to its diverse and beneficial cell culture characteristics. However, systematic evaluation of sericin as a substitute for FBS in cell culture medium remains limited. In this study, we conducted cellular morphological, physiological, and transcriptomic evaluation on three widely used mammalian cells. Compared with cells cultured in the control, those cultured in sericin-substitute medium showed similar cellular morphology, similar or higher cellular overall survival, lower population doubling time (PDT), and a higher percentage of S-phase with similar G2/G1 ratio, indicating comparable or better cell growth and proliferation. At the transcriptomic level, differentially expressed genes between cells in the two media were mainly enriched in function and biological processes related to cell growth and proliferation, reflecting that genes were activated to facilitate cell growth and proliferation. The results of this study suggest that cells cultured in sericin-substituted medium perform as well as, or even better than, those cultured in FBS-containing medium. PMID:27531556

  14. Long-Term Oocyte-Like Cell Development in Cultures Derived from Neonatal Marmoset Monkey Ovary

    Directory of Open Access Journals (Sweden)

    Bentolhoda Fereydouni

    2016-01-01

    Full Text Available We use the common marmoset monkey (Callithrix jacchus as a preclinical nonhuman primate model to study reproductive and stem cell biology. The neonatal marmoset monkey ovary contains numerous primitive premeiotic germ cells (oogonia expressing pluripotent stem cell markers including OCT4A (POU5F1. This is a peculiarity compared to neonatal human and rodent ovaries. Here, we aimed at culturing marmoset oogonia from neonatal ovaries. We established a culture system being stable for more than 20 passages and 5 months. Importantly, comparative transcriptome analysis of the cultured cells with neonatal ovary, embryonic stem cells, and fibroblasts revealed a lack of germ cell and pluripotency genes indicating the complete loss of oogonia upon initiation of the culture. From passage 4 onwards, however, the cultured cells produced large spherical, free-floating cells resembling oocyte-like cells (OLCs. OLCs strongly expressed several germ cell genes and may derive from the ovarian surface epithelium. In summary, our novel primate ovarian cell culture initially lacked detectable germ cells but then produced OLCs over a long period of time. This culture system may allow a deeper analysis of early phases of female primate germ cell development and—after significant refinement—possibly also the production of monkey oocytes.

  15. Human skeletal muscle-derived stem cells retain stem cell properties after expansion in myosphere culture

    International Nuclear Information System (INIS)

    Human skeletal muscle contains an accessible adult stem-cell compartment in which differentiated myofibers are maintained and replaced by a self-renewing stem cell pool. Previously, studies using mouse models have established a critical role for resident stem cells in skeletal muscle, but little is known about this paradigm in human muscle. Here, we report the reproducible isolation of a population of cells from human skeletal muscle that is able to proliferate for extended periods of time as floating clusters of rounded cells, termed 'myospheres' or myosphere-derived progenitor cells (MDPCs). The phenotypic characteristics and functional properties of these cells were determined using reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry and immunocytochemistry. Our results showed that these cells are clonogenic, express skeletal progenitor cell markers Pax7, ALDH1, Myod, and Desmin and the stem cell markers Nanog, Sox2, and Oct3/4 significantly elevated over controls. They could be maintained proliferatively active in vitro for more than 20 weeks and passaged at least 18 times, despite an average donor-age of 63 years. Individual clones (4.2%) derived from single cells were successfully expanded showing clonogenic potential and sustained proliferation of a subpopulation in the myospheres. Myosphere-derived cells were capable of spontaneous differentiation into myotubes in differentiation media and into other mesodermal cell lineages in induction media. We demonstrate here that direct culture and expansion of stem cells from human skeletal muscle is straightforward and reproducible with the appropriate technique. These cells may provide a viable resource of adult stem cells for future therapies of disease affecting skeletal muscle or mesenchymal lineage derived cell types.

  16. Human skeletal muscle-derived stem cells retain stem cell properties after expansion in myosphere culture

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Yan [Department of Otolaryngology, Head and Neck Surgery Charite-Universitaetsmedizin Berlin, Berlin (Germany); Department of Otolaryngology, Head and Neck Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guang Zhou (China); Li, Yuan [Department of Otolaryngology, Head and Neck Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guang Zhou (China); Chen, Chao; Stoelzel, Katharina [Department of Otolaryngology, Head and Neck Surgery Charite-Universitaetsmedizin Berlin, Berlin (Germany); Kaufmann, Andreas M. [Clinic for Gynecology CCM/CBF, Charite-Universitaetsmedizin Berlin, Berlin (Germany); Albers, Andreas E., E-mail: andreas.albers@charite.de [Department of Otolaryngology, Head and Neck Surgery Charite-Universitaetsmedizin Berlin, Berlin (Germany)

    2011-04-15

    Human skeletal muscle contains an accessible adult stem-cell compartment in which differentiated myofibers are maintained and replaced by a self-renewing stem cell pool. Previously, studies using mouse models have established a critical role for resident stem cells in skeletal muscle, but little is known about this paradigm in human muscle. Here, we report the reproducible isolation of a population of cells from human skeletal muscle that is able to proliferate for extended periods of time as floating clusters of rounded cells, termed 'myospheres' or myosphere-derived progenitor cells (MDPCs). The phenotypic characteristics and functional properties of these cells were determined using reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry and immunocytochemistry. Our results showed that these cells are clonogenic, express skeletal progenitor cell markers Pax7, ALDH1, Myod, and Desmin and the stem cell markers Nanog, Sox2, and Oct3/4 significantly elevated over controls. They could be maintained proliferatively active in vitro for more than 20 weeks and passaged at least 18 times, despite an average donor-age of 63 years. Individual clones (4.2%) derived from single cells were successfully expanded showing clonogenic potential and sustained proliferation of a subpopulation in the myospheres. Myosphere-derived cells were capable of spontaneous differentiation into myotubes in differentiation media and into other mesodermal cell lineages in induction media. We demonstrate here that direct culture and expansion of stem cells from human skeletal muscle is straightforward and reproducible with the appropriate technique. These cells may provide a viable resource of adult stem cells for future therapies of disease affecting skeletal muscle or mesenchymal lineage derived cell types.

  17. Modification of Isolation and Culture of Human Retinal Pigment Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    ZhengJL; GuoY

    1999-01-01

    Purpose:To modify the isolation of human retinal pigment pithelial(RPE)cells and to increase the purification and production of cultured RPE cells.Methods:The human eyecups were fixed on a fubber holder.After digestion by trypsin,RPE cells were collected,then cultured and identified by morphology,immunohistochemistry and electron microscopy.Results:The cultured RPE cells grew actively in the early stage with transparent nucleus and abundant melanin particles in cytoplasm.These cells were positive in DOPA oxidase reaction and in anti-pancytokeratin antibody staining.Cellular microvilli and tight junctions could be seen through transmission electrom microscopy.Conclusion:We developed a rubber holder to fix the eyecup.Using this holder,more and purer cultured RPE cells can be obtained.These cultured REP cells are similar to those in vivo in morphology and immunohistochemical staining.

  18. Brine Shrimp Bioassays: A Useful Technique in Biological Investigations

    Science.gov (United States)

    Rice, Stanley A.; Maness, Ian B.

    2004-01-01

    A technique to measure the potency of leaf compounds against herbivores with the use of a bioassay is described. Bioassays are useful in classes where students have career plans like medicine in which bioassays can be used as tools for screening plants for possible medicinal potency.

  19. Flow perfusion culture of human mesenchymal stem cells on coralline hydroxyapatite scaffolds with various pore sizes

    DEFF Research Database (Denmark)

    Bjerre, Lea; Bünger, Cody; Baatrup, Anette;

    2011-01-01

    study was to obtain a clinically relevant substitute size using a direct perfusion culture system. Human bone marrowderived mesenchymal stem cells were seeded on coralline hydroxyapatite scaffolds with 200 μm or 500 μm pores, and resulting constructs were cultured in a perfusion bioreactor or in static...... μm ones. Adhesion and proliferation of the cells was seen on both scaffold sizes, but the vitality and morphology of cells changed unfavorably during perfusion culture. In contrast to previous studies using spinner flask that show increased cellularity and osteogenic properties of cells when cultured...... scaffolds. Our conclusion is that the specific scaffold surface microstructure and culturing system flow dynamics has a great impact on cell distribution and proliferation and on osteogenic differentiation, and the data presented warrant careful selection of in vitro culture settings to meet the specific...

  20. Production of putative virulence factors by Renibacterium salmoninarum grown in cell culture.

    Science.gov (United States)

    McIntosh, D; Flaño, E; Grayson, T H; Gilpin, M L; Austin, B; Villena, A J

    1997-10-01

    A cell culture system, employing the fish cell line Epithelioma papillosum cyprini (EPC), was developed to study the synthesis of intracellular antigen and the expression of putative virulence factors by Renibacterium salmoninarum. EPC cultures infected with R. salmoninarum could be maintained for 7 weeks, during which the pathogen multiplied intracellularly. Immunohistochemical examination of infected cultures revealed the production of the p57 antigen, haemolysin and cytolysin. The intracellular nature of the infection was confirmed by transmission electron microscopic examination of EPC monolayers. A comparison of the relative virulence of bacterial cells cultured in EPC cells and on agar plates revealed that the former were markedly more virulent in challenge experiments with juvenile rainbow trout (Oncorhynchus mykiss Walbaum). The EPC cell culture model provided a system for the study of R. salmoninarum under more natural conditions than those achieved with plate culture techniques. PMID:9353936

  1. Dopaminergic differentiation of human neural stem cells mediated by co-cultured rat striatal brain slices

    DEFF Research Database (Denmark)

    Anwar, Mohammad Raffaqat; Andreasen, Christian Maaløv; Lippert, Solvej Kølvraa;

    2008-01-01

    differentiation, we co-cultured cells from a human neural forebrain-derived stem cell line (hNS1) with rat striatal brain slices. In brief, coronal slices of neonatal rat striatum were cultured on semiporous membrane inserts placed in six-well trays overlying monolayers of hNS1 cells. After 12 days of co......-induced areas. The presence of dopamine in the conditioned culture medium was confirmed by HPLC analysis. Interestingly, not all striatal slice cultures induced TH-expression in underlying hNS1 cells. Common to TH-inductive cultures was, however, the presence of degenerating, necrotic areas, suggesting that...... factors released during striatal degeneration were responsible for the dopaminergic induction of the hNS1 cells. Ongoing experiments aim to identify such factors by comparing protein profiles of media conditioned by degenerating (necrotic) versus healthy striatal slice cultures....

  2. Cytoskeletal binding proteins distinguish cultured dental follicle cells and periodontal ligament cells.

    Science.gov (United States)

    Li, Jie; Li, Hui; Tian, Ye; Yang, Yaling; Chen, Guoqing; Guo, Weihua; Tian, Weidong

    2016-07-01

    Human dental follicle cells (DFCs) and periodontal ligament cells (PDLCs) derived from the ectomesenchymal tissue, have been shown to exhibit stem/progenitor cell properties and the ability to induce tissue regeneration. Stem cells in dental follicle differentiate into cementoblasts, periodontal ligament fibroblasts and osteoblasts, these cells form cementum, periodontal ligament and alveolar bone, respectively. While stem cells in dental follicle are a precursor to periodontal ligament fibroblasts, the molecular changes that distinguish cultured DFCs from PDLCs are still unknown. In this study, we have compared the immunophenotypic features and cell cycle status of the two cell lines. The results suggest that DFCs and PDLCs displayed similar features related to immunophenotype and cell cycle. Then we employed an isobaric tag for relative and absolute quantitation (iTRAQ) proteomics strategy to reveal the molecular differences between the two cell types. A total of 2138 proteins were identified and 39 of these proteins were consistently differentially expressed between DFCs and PDLCs. Gene ontology analyses revealed that the protein subsets expressed higher in PDLCs were related to actin binding, cytoskeletal protein binding, and structural constituent of muscle. Upon validation by real-time PCR, western blotting, and immunofluorescence staining. Tropomyosin 1 (TPM1) and caldesmon 1 (CALD1) were expressed higher in PDLCs than in DFCs. Our results suggested that PDLCs display enhanced actin cytoskeletal dynamics relative to DFCs while DFCs may exhibit a more robust antioxidant defense ability relative to PDLCs. This study expands our knowledge of the cultured DFCs and PDLCs proteome and provides new insights into possible mechanisms responsible for the different biological features observed in each cell type. PMID:26708290

  3. Inhibition of mast cell-dependent conversion of cultured macrophages into foam cells with antiallergic drugs.

    Science.gov (United States)

    Ma, H; Kovanen, P T

    2000-12-01

    Degranulation of isolated, rat peritoneal mast cells in the presence of low density lipoprotein (LDL) induces cholesteryl ester accumulation in cocultured macrophages with ensuing foam cell formation. This event occurs when the macrophages phagocytose LDL particles that have been bound to the heparin proteoglycans of exocytosed granules. In an attempt to inhibit such foam cell formation pharmacologically, rat peritoneal mast cells that had been passively sensitized with anti-ovalbumin-IgE were treated with 2 mast cell-stabilizing antianaphylactic drugs, MY-1250 or disodium cromoglycate (DSCG). Both drugs were found to inhibit antigen (ovalbumin)-triggered release of histamine from the mast cells, revealing mast cell stabilization. In cocultures of rat peritoneal macrophages and passively sensitized mast cells, addition of MY-1250 before addition of the antigen resulted in parallel reductions in histamine release from mast cells, uptake of [(14)C]sucrose-LDL, and accumulation of LDL-derived cholesteryl esters in the cocultured macrophages. Similarly, when passively sensitized mast cells were stimulated with antigen in the presence of DSCG and the preconditioned media containing all substances released from the drug-treated mast cells were collected and added to macrophages cultured in LDL-containing medium, uptake and esterification of LDL cholesterol by the macrophages were inhibited. The inhibitory effects of both drugs were mast cell-specific because neither drug inhibited the ability of macrophages to take up and esterify LDL cholesterol. Analysis of heparin proteoglycan contents of the incubation media revealed that both drugs had inhibited mast cells from expelling their granule remnants. Thus, both MY-1250 and DSCG prevent mast cells from releasing the heparin proteoglycan-containing vehicles that bind LDL and carry it into macrophages. This study suggests that antiallergic pharmacological agents could be used in animal models to prevent mast cell

  4. Somaclonal variation: a morphogenetic and biochemical analysis of Mandevilla velutina cultured cells

    Directory of Open Access Journals (Sweden)

    M. Maraschin

    2002-06-01

    Full Text Available Cell cultures of Mandevilla velutina have proved to be an interesting production system for biomass and secondary metabolites able to inhibit the hypotensive activity of bradykinin, a nonapeptide generated in plasma during tissue trauma. The crude ethyl acetate extract of cultured cells contains about 31- to 79-fold more potent anti-bradykinin compounds (e.g., velutinol A than that obtained with equivalent extracts of tubers. Somaclonal variation may be an explanation for the wide range of inhibitor activity found in the cell cultures. The heterogeneity concerning morphology, differentiation, carbon dissimilation, and velutinol A production in M. velutina cell cultures is reported. Cell cultures showed an asynchronous growth and cells in distinct developmental stages. Meristematic cells were found as the major type, with several morphological variations. Cell aggregates consisting only of meristematic cells, differentiated cells containing specialized cell structures such as functional chloroplasts (cytodifferentiation and cells with embryogenetic characteristics were observed. The time course for sucrose metabolism indicated cell populations with significant differences in growth and metabolic rates, with the highest biomass-producing cell line showing a cell cycle 60% shorter and a metabolic rate 33.6% higher than the control (F2 cell population. MALDI-TOF mass spectrometric analysis of velutinol A in selected cell lines demonstrated the existence of velutinol A producing and nonproducing somaclones. These results point to a high genetic heterogeneity in general and also in terms of secondary metabolite content.

  5. Optimization of an in vitro bioassay to monitor growth and formation of myotubes in real time.

    Science.gov (United States)

    Murphy, Sylvia M; Kiely, Maeve; Jakeman, Philip M; Kiely, Patrick A; Carson, Brian P

    2016-06-01

    The importance of growth and maintenance of skeletal muscle is vital for long term health and quality of life. Appropriate nutrition with specific bioactivities relevant to the functionalities of tissues such as skeletal muscle, can assist in maintaining and promoting adaptive responses to biological and environmental stresses which prevent muscle atrophy and promote hypertrophy. The aim of this investigation was to develop a novel in vitro cell-based electric impedance assay to study myoblast to myotube formation on the real time cell analysis (RTCA) platform (xCELLigence™, ACEA) and to validate the system by testing myotube responses to hypertrophic stimuli. C2C12 myoblasts were proliferated until 70% confluent in Dulbecco's Modified Eagles Medium (DMEM) (10% FBS) and subsequently differentiated to myotubes over 8 days in DMEM [2% horse serum (HS)]. Changes in cell behaviour and adhesion properties were monitored by measuring impedance via interdigitated microelectrodes in the base of E-16 cell culture dishes. To establish the suitability of this assay to monitor nutrient regulation of muscle hypertrophy, leucine, a known potent regulator of MPS was then supplemented to the fully formed myotubes in physiologically relevant conditions-0.20 mM, 0.40 mM, 0.6 mM, 0.8 mM and above 1.0 mM, 1.5 mM, 2.0 mM and impedance subsequently monitored. Parallel experiments highlighting alterations in myotube thickness, muscle protein synthesis (MPS) (mammalian target of rapamycin; mTOR) and differentiation (myogenin) were conducted to support RTCA bioassay findings. This in vitro bioassay can be used to monitor skeletal muscle behaviour and identify nutrient compounds with bioactivities promoting skeletal muscle hypertrophy, reducing muscle atrophy and thus inform the development of novel nutrient formulations for the maintenance of skeletal muscle. PMID:27009307

  6. Commercially Available Gas-Permeable Cell Culture Bags May Not Prevent Anoxia in Cultured or Shipped Islets

    OpenAIRE

    Avgoustiniatos, E.S.; Hering, B.J.; Rozak, P.R.; Wilson, J.R.; Tempelman, L.A.; Balamurugan, A.N.; Welch, D.P.; Weegman, B. P.; Suszynski, T.M.; Papas, K.K.

    2008-01-01

    Prolonged anoxia has deleterious effects on islets. Gas-permeable cell culture devices can be used to minimize anoxia during islet culture and especially during shipment when elimination of gas-liquid interfaces is required to prevent the formation of damaging gas bubbles. Gas-permeable bags may have several drawbacks, such as propensity for puncture and contamination, difficult islet retrieval, and significantly lower oxygen permeability than silicone rubber membranes (SRM). We hypothesized ...

  7. A cell culture assay for the detection of cardiotoxicity

    International Nuclear Information System (INIS)

    An important step in minimizing the number of animal experiments in medical research is the study of in vitro model systems. The authors propose the use of shock protein formation, which is a cellular response to cell-damaging stress as an assay to monitor cardiotoxicity. Isolated and cultured cardiac myocytes were prepared by a trypsin digestion method from 18-day-old fetal mice. These cells respond to typical substances inducing shock protein formation in other cellular systems as well as to known cardiotoxins with the de novo synthesis of shock proteins. Pharmaceuticals relevant in transplant medicine were tested for possible cardiotoxic effects: Cyclosporine A evokes shock protein formation at subtherapeutic concentrations. Azathioprine and methyl-prednisolone exert the same effect but at concentration ranges highly above the therapeutic level. The ability to induce shock protein synthesis obviously seems to be restricted to toxic drugs. The data presented demonstrate that the proposed in vitro model system for cardiotoxicity is animal saving and sensitive

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

    International Nuclear Information System (INIS)

    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.

  9. Nitrogen metabolism in Lignifying Pinus taeda cell cultures

    Science.gov (United States)

    van Heerden, P. S.; Towers, G. H.; Lewis, N. G.

    1996-01-01

    The primary metabolic fate of phyenylalanine, following its deamination in plants, is conscription of its carbon skeleton for lignin, suberin, flavonoid, and related metabolite formation. Since this accounts for approximately 30-40% of all organic carbon, an effective means of recycling the liberated ammonium ion must be operative. In order to establish how this occurs, the uptake and metabolism of various 15N-labeled precursors (15N-Phe, 15NH4Cl, 15N-Gln, and 15N-Glu) in lignifying Pinus taeda cell cultures was investigated, using a combination of high performance liquid chromatography, 15N NMR, and gas chromatograph-mass spectrometry analyses. It was found that the ammonium ion released during active phenylpropanoid metabolism was not made available for general amino acid/protein synthesis. Rather it was rapidly recycled back to regenerate phenylalanine, thereby providing an effective means of maintaining active phenylpropanoid metabolism with no additional nitrogen requirement. These results strongly suggest that, in lignifying cells, ammonium ion reassimilation is tightly compartmentalized.

  10. A cell culture assay for the detection of cardiotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Loew-Friedrich, Iv.; von Bredow, F.; Schoeppe, W. (Department of Nephrology, Hospital of the Johann Wolfgang Goethe-University, Frankfurt am Main (Germany))

    1991-04-01

    An important step in minimizing the number of animal experiments in medical research is the study of in vitro model systems. The authors propose the use of shock protein formation, which is a cellular response to cell-damaging stress as an assay to monitor cardiotoxicity. Isolated and cultured cardiac myocytes were prepared by a trypsin digestion method from 18-day-old fetal mice. These cells respond to typical substances inducing shock protein formation in other cellular systems as well as to known cardiotoxins with the de novo synthesis of shock proteins. Pharmaceuticals relevant in transplant medicine were tested for possible cardiotoxic effects: Cyclosporine A evokes shock protein formation at subtherapeutic concentrations. Azathioprine and methyl-prednisolone exert the same effect but at concentration ranges highly above the therapeutic level. The ability to induce shock protein synthesis obviously seems to be restricted to toxic drugs. The data presented demonstrate that the proposed in vitro model system for cardiotoxicity is animal saving and sensitive.

  11. Silver nanoparticle protein corona composition in cell culture media.

    Directory of Open Access Journals (Sweden)

    Jonathan H Shannahan

    Full Text Available The potential applications of nanomaterials as drug delivery systems and in other products continue to expand. Upon introduction into physiological environments and driven by energetics, nanomaterials readily associate proteins forming a protein corona (PC on their surface. This PC influences the nanomaterial's surface characteristics and may impact their interaction with cells. To determine the biological impact of nanomaterial exposure as well as nanotherapeutic applications, it is necessary to understand PC formation. Utilizing a label-free mass spectrometry-based proteomics approach, we examined the composition of the PC for a set of four silver nanoparticles (AgNPs including citrate-stabilized and polyvinlypyrrolidone-stabilized (PVP colloidal silver (20 or 110 nm diameter. To simulate cell culture conditions, AgNPs were incubated for 1 h in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, washed, coronal proteins solubilized, and proteins identified and quantified by label-free LC-MS/MS. To determine which attributes influence PC formation, the AgNPs were characterized in both water and cell culture media with 10% FBS. All AgNPs associated a common subset of 11 proteins including albumin, apolipoproteins, keratins, and other serum proteins. 110 nm citrate- and PVP-stabilized AgNPs were found to bind the greatest number of proteins (79 and 85 respectively compared to 20 nm citrate- and PVP-stabilized AgNPs (45 and 48 respectively, suggesting a difference in PC formation based on surface curvature. While no relationships were found for other protein parameters (isoelectric point or aliphatic index, the PC on 20 nm AgNPs (PVP and citrate consisted of more hydrophobic proteins compared to 110 nm AgNPs implying that this class of proteins are more receptive to curvature-induced folding and crowding in exchange for an increased hydration in the aqueous environment. These observations demonstrate the significance of

  12. Feeder-independent continuous culture of the PICM-19 pig liver stem cell line

    Science.gov (United States)

    The PICM-19 pig liver stem cell line is a bipotent cell line, i.e., capable of forming either bile ductules or hepatocyte monolayers in vitro, that was derived from the primary culture of pig embryonic stem cells. The cell line has been strictly feeder-dependent in that cell replication morphology,...

  13. An Engineered Approach to Stem Cell Culture: Automating the Decision Process for Real-Time Adaptive Subculture of Stem Cells

    OpenAIRE

    Ker, Dai Fei Elmer; Weiss, Lee E.; Junkers, Silvina N.; Chen, Mei; Yin, Zhaozheng; Sandbothe, Michael F.; Huh, Seung-il; Eom, Sungeun; Bise, Ryoma; Osuna-Highley, Elvira; Kanade, Takeo; Campbell, Phil G.

    2011-01-01

    Current cell culture practices are dependent upon human operators and remain laborious and highly subjective, resulting in large variations and inconsistent outcomes, especially when using visual assessments of cell confluency to determine the appropriate time to subculture cells. Although efforts to automate cell culture with robotic systems are underway, the majority of such systems still require human intervention to determine when to subculture. Thus, it is necessary to accurately and obj...

  14. Urokinase Separation from Cell Culture Broth of a Human Kidney Cell Line

    Directory of Open Access Journals (Sweden)

    Vibha Bansal, Pradip K. Roychoudhury, Ashok Kumar

    2007-01-01

    Full Text Available A single step ion-exchange chromatography on a sulfo-propyl (SP- Sepharose column was performed to separate both the high molecular weight (HMW- and low molecular weight (LMW- forms of enzymatically active urokinase type plasminogen activator from human kidney (HT1080 cell culture media. The level of urokinase secreted by the cell line reached to about 145 Plough units/ml culture broth within 48 h of cultivation. The conditioned cell culture media was applied directly to the column without any prior concentration steps. Polyacrylamide gel electrophoresis of the column eluates in the presence of sodium dodecyl sulphate showed that the cell line secretes three forms of two-chain high molecular weight (HMW urokinase of molecular weights (Mr 64,000, 60,900 and 55,000. In addition, two low molecular weight (LMW forms of Mr 22,000 and 20,000; proteolytic cleavage products of HMW, were also found. The HMW and LMW forms had intrinsic plasminogen dependent proteolytic activity as judged by zymographic analysis. The specific activity of the pooled peak fractions increased (approximately 93-fold to values as high as 1481 Plough units/ mg protein. Both HMW as well as LMW forms were obtained in significantly high yields.

  15. METHYLCELLULOSE CELL-CULTURE AS A NEW CYTOTOXICITY TEST SYSTEM FOR BIOMATERIALS

    NARCIS (Netherlands)

    VANLUYN, MJA; VANWACHEM, PB; NIEUWENHUIS, P; DAMINK, LO; TENHOOPEN, H; FEIJEN, J

    1991-01-01

    The cytotoxicity of biomaterials can be tested in vitro using various culture systems. Liquid culture systems may detect cytotoxicity of a material either by culture of cells with extracts or with the material itself. In the latter instance, renewing the medium will remove possible released cytotoxi

  16. Foaming and cell flotation in suspended plant cell cultures and the effect of chemical antifoams.

    Science.gov (United States)

    Wongsamuth, R; Doran, P M

    1994-08-01

    Foam development and stability in Atropa belladonna suspensions were investigated as a function of culture conditions. Foaming was due mainly to properties of the cell-free broth and was correlated with protein content; effects due to presence of cells increased towards the end of batch culture. Highest foam levels were measured 11 days after inoculation. Air flow rate was of major importance in determining foam volume; foam volume and stability were also strongly dependent on pH. Foam flotation of plant cells was very effective. After 30 min foaming, ca. 55% of cells were found in the foam; this increased to ca. 75% after 90 min. Polypropylene glycol 1025 and 2025, Pluronic PE 6100, and Antifoam-C emulsion were tested as chemical antifoams. Polypropylene glycol 1025 and Antifoam C at concentrations up to 600 ppm had no adverse effect on growth in shake flasks; Pluronic PE 6100 has an inhibitory effect at all levels tested. Concentrations of polypropylene glycol 2025 and Pluronic PE 6100 as low as 20 ppm reduced foam volumes by a factor of ca. 10. Addition of antifoam reduced k(L)a values in bubble-column and stirred-tank bioreactors. After operation of a stirred reactor for 2 days using Antifoam C for foam control, cell production was limited by oxygen due to the effect of antifoam on mass transfer. Theoretical analysis showed that maximum cell concentrations and biomass levels decline with increasing reactors working volume due to greater consumption of antifoam to prevent foam overflow. The results indicate that when chemical foam control is used in plant cell cultures, head-space volume and tolerable foam levels must be considered to optimize biomass production. (c) 1994 John Wiley & Sons, Inc. PMID:18618782

  17. Culture of normal human blood cells in a diffusion chamber system II. Lymphocyte and plasma cell kinetics

    International Nuclear Information System (INIS)

    Normal human blood leukocytes were cultured in Millipore diffusion chambers implanted into the peritoneal cavities of irradiated mice. The evaluation of survival and proliferation kinetics of cells in lymphyocytic series suggested that the lymphoid cells are formed from transition of small and/or large lymphocytes, and the lymphoblasts from the lymphoid cells. There was also evidence indicating that some of the cells in these two compartments are formed by proliferation. The evaluation of plasmacytic series suggested that the plasma cells are formed from plasmacytoid-lymphocytes by transition, and the latter from the transition of lymphocytes. In addition, relatively a small fraction of cells in these two compartments are formed by proliferation. mature plasma cells do not and immature plasma cells do proliferate. Estimation of magnitude of plasma cells formed in the cultures at day 18 indicated that at least one plasma cell is formed for every 6 normal human blood lymphocytes introduced into the culture

  18. Design and Performance of an Automated Bioreactor for Cell Culture Experiments in a Microgravity Environment

    Science.gov (United States)

    Kim, Youn-Kyu; Park, Seul-Hyun; Lee, Joo-Hee; Choi, Gi-Hyuk

    2015-03-01

    In this paper, we describe the development of a bioreactor for a cell-culture experiment on the International Space Station (ISS). The bioreactor is an experimental device for culturing mouse muscle cells in a microgravity environment. The purpose of the experiment was to assess the impact of microgravity on the muscles to address the possibility of longterm human residence in space. After investigation of previously developed bioreactors, and analysis of the requirements for microgravity cell culture experiments, a bioreactor design is herein proposed that is able to automatically culture 32 samples simultaneously. This reactor design is capable of automatic control of temperature, humidity, and culture-medium injection rate; and satisfies the interface requirements of the ISS. Since bioreactors are vulnerable to cell contamination, the medium-circulation modules were designed to be a completely replaceable, in order to reuse the bioreactor after each experiment. The bioreactor control system is designed to circulate culture media to 32 culture chambers at a maximum speed of 1 ml/min, to maintain the temperature of the reactor at 36°C, and to keep the relative humidity of the reactor above 70%. Because bubbles in the culture media negatively affect cell culture, a de-bubbler unit was provided to eliminate such bubbles. A working model of the reactor was built according to the new design, to verify its performance, and was used to perform a cell culture experiment that confirmed the feasibility of this device.

  19. Design of 3D printed insert for hanging culture of Caco-2 cells

    International Nuclear Information System (INIS)

    A Caco-2 cell culture on Transwell, an alternative testing to animal or human testing used in evaluating drug intestinal permeability, incorrectly estimated the absorption of actively transported drugs due to the low expression of membrane transporters. Similarly, three-dimensional (3D) cultures of Caco-2 cells, which have been recommended to be more physiological relevant, were not superior to the Transwell culture in either accuracy or convenience in drug permeability testing. Using rapid 3D printing prototyping techniques, this study proposed a hanging culture of Caco-2 cells that performed with high accuracy in predicting drug permeability in humans. As found, hanging cultured Caco-2 cells formed a confluent monolayer and maintained high cell viability on the 3D printed insert. Compared with the normal culture on Transwell, the Caco-2 cells on the 3D printed insert presented ∼30–100% higher brush border enzyme activity and ∼2–7 folds higher activity of P-glycoprotein/multidrug resistance-associated protein 2 during 21 days of incubation. For the eight membrane transporter substrates, the predictive curve of the 3D printing culture exhibited better linearity (R2 = 0.92) to the human oral adsorption than that of the Transwell culture (R2 = 0.84), indicating better prediction by the 3D printing culture. In this regard, the 3D printed insert for hanging culture could be potentially developed as a convenient and low-cost tool for testing drug oral absorption. (paper)

  20. Regulation of Cytoplasmic and Vacuolar Volumes by Plant Cells in Suspension Culture

    DEFF Research Database (Denmark)

    Owens, Trevor; Poole, Ronald J

    1979-01-01

    Quantitative microscopical measurements have been made of the proportion of cell volume occupied by cytoplasm in a cell suspension culture derived from cotyledons of bush bean (cv. Contender). On a 7-day culture cycle, the content of cytoplasm varies from 25% at the time of transfer to 45% at the...... start of the phase of rapid cell division. If the culture is continued beyond 7 days, the vacuole volume reaches 90% of cell volume by day 12.Attempts to measure relative cytoplasmic volumes by compartmental analysis of nonelectrolyte efflux were unsuccessful. The proportion of cell volume occupied by...... cytoplasm is roughly correlated with protein content, but shows no correlation with cell size or with intracellular concentrations of K or Na. The most striking observation is that the growth of cytoplasmic volume for the culture as a whole appears to be constant throughout the culture cycle, despite...

  1. Differences in the primary culture, purification and biological characteristics between endothelial cells and smooth muscle cells from rat aorta

    Institute of Scientific and Technical Information of China (English)

    Shaobo Hu; Zifang Song; Qichang Zheng; Jun Nie

    2009-01-01

    Objective: To investigate the differences of primary culture, purification and biological characteristics between endothelial cells and smooth muscle cells from rat aorta. Methods: Endothelial cells were obtained using the vascular ring adherence, collagenase digestion method and an improved vascular ring adherence method, while smooth muscle cells were separated from tissue sections of rat aorta. Clones of endothelial cells were selected by limiting dilution assay. Both cell types were identified using specific cell immunofluorescent markers,and phase contrast microscopy was used to observe the morphological disparity between endothelial cells and smooth muscle cells at the single cell and colony level. Cell proliferation was determined by the cell counting kit-8. Differences between endothelial cells and smooth muscle cells were evaluated in trypsin digestion 6me, attachment time and recovery after cryopreservation. Results: Endothelial cells were obtained by all three methods. The improved vascular ring method provided the most reproducible results. Cells were in good condition, and of high purity. Smooth muscle cells were cultured successfully by the tissue fragment culture method. Clonal expansion of singleendothelial cells was attained. The two cell types expressed their respective specific markers, and the rate of proliferation of smooth muscle cells exceeded that of endothelial cells. Endothelial cells were more sensitive to trypsin digestion than smooth muscle cells. In addition, they had a shorter adherence time and better recovery following cryopreservation than smooth muscle cells. Conclusion: The improved vascular ring method was optimal for yielding endothelial cells. Limiting dilution is a novel and valid method for purifying primary endothelial cells from rat aorta. Primary rat endothelial cell and vascular smooth muscle cell cultures exhibited different morphological characteristics, proliferation rate, adherence time, susceptibility to trypsin

  2. Characterization of proteins by in-cell NMR spectroscopy in cultured mammalian cells.

    Science.gov (United States)

    Barbieri, Letizia; Luchinat, Enrico; Banci, Lucia

    2016-06-01

    In-cell NMR spectroscopy is a unique tool for characterizing biological macromolecules in their physiological environment at atomic resolution. Recent progress in NMR instruments and sample preparation methods allows functional processes, such as metal uptake, disulfide-bond formation and protein folding, to be analyzed by NMR in living, cultured human cells. This protocol describes the necessary steps to overexpress one or more proteins of interest inside human embryonic kidney 293T (HEK293T) cells, and it explains how to set up in-cell NMR experiments. The cDNA is transiently transfected as a complex with a cationic polymer (DNA:PEI (polyethylenimine)), and protein expression is carried on for 2-3 d, after which the NMR sample is prepared. (1)H and (1)H-(15)N correlation NMR experiments (for example, using band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence (SOFAST-HMQC)) can be carried out in <2 h, ensuring cell viability. Uniform (15)N labeling and amino-acid-specific (e.g., cysteine, methionine) labeling schemes are possible. The entire procedure takes 4 d from cell culture seeding to NMR data collection. PMID:27196722

  3. Growth factor regulation of sugar uptake in cultured cells

    International Nuclear Information System (INIS)

    Mouse EGF stimulates the uptake of 2-deoxygluclose (dGIc), a non-metabolized glucose analogue, into cultured mouse 3T3 fibroblasts (Clone 1) 2 to 4 fold, and EGF dependent Balb/MK-1 epidermal kerotinocytes, 5 to 8 fold. Initial stimulation is detected at 15 minutes. Maximal effects are seen at 2 hours with 10 ng/ml EGF. Binding of 125I-labeled EGF to cells is rapid and complete by 2 hours at 370C. Antibodies which specifically inhibit 125I-labeled EGF binding to cells inhibit EGF stimulation as much as 85%. Human platelet derived TGF-β stimulates dGlc uptake up to 5 fold. Maximum effects are seen with 1 ng/ml TGF-β within 2 hours and stimulation is detected 30 minutes after exposure to 0.1 ng/ml, the minimum effective concentration. TGF-β, like EGF, stimulates sugar transport into Balb/MK-1 cells without additional factors. However, neither stimulates uptake in a 3T3 variant, NR-6, which is EGF-receptor negative. The co-addition of EGF and/or PDGF enhances TGF-β stimulation. Binding of 125I-labeled TGF-β is nearly complete by 1 hour at 370C, but continues to increase for as long as 4 hours after addition. Antibodies which inhibit EGF binding have no effect on TGF-β binding, but they block TGF-β stimulation of hexose uptake. It is concluded from these results that the TGF-β receptor is distinct from the EGF receptor, and that although TGF-β stimulation of dGLc uptake does not require exogenously added EGF, it does require an active or available EGF receptor kinase system

  4. Microdroplet photobioreactor for the photoautotrophic culture of microalgal cells.

    Science.gov (United States)

    Sung, Young Joon; Kim, Jaoon Young Hwan; Bong, Ki Wan; Sim, Sang Jun

    2016-02-01

    Microalgae, unicellular photoautotrophic microorganisms, have attracted great attention for the production of biofuel and high-value products, but the commercial use of microalgae has been limited by low photosynthetic productivity. To overcome this limitation, it is required to develop an efficient platform for the rapid evaluation of photoautotrophic growth performance and productivity of microalgal strains. Here we describe a droplet-based photobioreactor for high-throughput analysis of the photoautotrophic growth of microalgal cells. By integrating micropillar arrays and adjusting the height of the microchamber, we could accurately monitor the growth kinetics of microalgae in an immobilized microdroplet and improve the transfer rate of CO2 into the microdroplet photobioreactor with an increased contact area between the microdroplet and PDMS surface. The improvement of CO2 transfer into the microdroplet was also confirmed by improved microalgal cell growth and a decrease in pH measured using colorimetric and fluorescence-based assays. The photoautotrophic growth kinetics of Chlorella vulgaris were measured under different CO2 concentrations (ambient, 1%, 2.5%, 5% and 7.5%) and light intensity (35, 55, 100, 150, and 200 μmol photons per m(2) per s) conditions, which are key factors for photoautotrophic growth. Chlorella vulgaris in a microdroplet showed better cell growth performance compared to a flask culture due to the reduced shading effects and improved mass transfer. Finally, we could evaluate the photoautotrophic growth performance of four microalgal strains (Chlorella vulgaris, Chlorella protothecoides, Chlorella sorokiniana and Neochloris oleoabundans) for 120 hours. These results demonstrate that our microdroplet system can be used as an efficient photobioreactor for the rapid evaluation of the photoautotrophic growth of microalgal strains under various conditions. PMID:26673975

  5. Statistical prediction of nanoparticle delivery: from culture media to cell.

    Science.gov (United States)

    Brown, M Rowan; Hondow, Nicole; Brydson, Rik; Rees, Paul; Brown, Andrew P; Summers, Huw D

    2015-04-17

    The application of nanoparticles (NPs) within medicine is of great interest; their innate physicochemical characteristics provide the potential to enhance current technology, diagnostics and therapeutics. Recently a number of NP-based diagnostic and therapeutic agents have been developed for treatment of various diseases, where judicious surface functionalization is exploited to increase efficacy of administered therapeutic dose. However, quantification of heterogeneity associated with absolute dose of a nanotherapeutic (NP number), how this is trafficked across biological barriers has proven difficult to achieve. The main issue being the quantitative assessment of NP number at the spatial scale of the individual NP, data which is essential for the continued growth and development of the next generation of nanotherapeutics. Recent advances in sample preparation and the imaging fidelity of transmission electron microscopy (TEM) platforms provide information at the required spatial scale, where individual NPs can be individually identified. High spatial resolution however reduces the sample frequency and as a result dynamic biological features or processes become opaque. However, the combination of TEM data with appropriate probabilistic models provide a means to extract biophysical information that imaging alone cannot. Previously, we demonstrated that limited cell sampling via TEM can be statistically coupled to large population flow cytometry measurements to quantify exact NP dose. Here we extended this concept to link TEM measurements of NP agglomerates in cell culture media to that encapsulated within vesicles in human osteosarcoma cells. By construction and validation of a data-driven transfer function, we are able to investigate the dynamic properties of NP agglomeration through endocytosis. In particular, we statistically predict how NP agglomerates may traverse a biological barrier, detailing inter-agglomerate merging events providing the basis for

  6. The compartmentation of phosphorylated thiamine derivatives in cultured neuroblastoma cells.

    Science.gov (United States)

    Bettendorff, L

    1994-05-26

    Thiamine transport in cultured neuroblastoma cells is mediated by a high-affinity carrier (KM = 40 nM). In contrast, the uptake of the more hydrophobic sulbutiamine (isobutyrylthiamine disulfide) is unsaturable and its initial transport rate is 20-times faster than for thiamine. In the cytoplasm, sulbutiamine is rapidly hydrolyzed and reduced to free thiamine, the overall process resulting in a rapid and concentrative thiamine accumulation. Incorporation of radioactivity from [14C]thiamine or [14C]sulbutiamine into intracellular thiamine diphosphate is slow in both cases. Despite the fact that the diphosphate is probably the direct precursor for both thiamine monophosphate and triphosphate, the specific radioactivity increased much faster for the latter two compounds than for thiamine diphosphate. This suggests the existence of two pools of thiamine diphosphate, the larger one having a very slow turnover (about 17 h); a much smaller, rapidly turning over pool would be the precursor of thiamine mono- and triphosphate. The turnover time for thiamine triphosphate could be estimated to be 1-2 h. When preloading the cells with [14C]sulbutiamine was followed by a chase with the same concentration of the unlabeled compound, the specific radioactivities of thiamine and thiamine monophosphate decreased exponentially as expected, but labeling of the diphosphate continued to increase slowly. Specific radioactivity of thiamine triphosphate increased first, but after 30 min it began to slowly decrease. These results show for the first time the existence of distinct thiamine diphosphate pools in the same homogeneous cell population. They also suggest a complex compartmentation of thiamine metabolism. PMID:8186267

  7. Induction of chromosome aberrations in two lines of cultured cells using different types of radiation

    International Nuclear Information System (INIS)

    The induction of chromosome aberrations has been investigated in two lines of cultured cells for different types of radiation. The obtained results are compared with information on induction of cell reproductive death and malignant transformation. (Auth.)

  8. Fabrication and optimization of alginate hydrogel constructs for use in 3D neural cell culture

    International Nuclear Information System (INIS)

    Two-dimensional (2D) culture systems provide useful information about many biological processes. However, some applications including tissue engineering, drug transport studies, and analysis of cell growth and dynamics are better studied using three-dimensional (3D) culture systems. 3D culture systems can potentially offer higher degrees of organization and control of cell growth environments, more physiologically relevant diffusion characteristics, and permit the formation of more extensive 3D networks of cell-cell interactions. A 3D culture system has been developed using alginate as a cell scaffold, capable of maintaining the viability and function of a variety of neural cell types. Alginate was functionalized by the covalent attachment of a variety of whole proteins and peptide epitopes selected to provide sites for cell attachment. Alginate constructs were used to entrap a variety of neural cell types including astroglioma cells, astrocytes, microglia and neurons. Neural cells displayed process outgrowth over time in culture. Cell-seeded scaffolds were characterized in terms of their biochemical and biomechanical properties, effects on seeded neural cells, and suitability for use as 3D neural cell culture models.

  9. Fabrication and optimization of alginate hydrogel constructs for use in 3D neural cell culture

    Energy Technology Data Exchange (ETDEWEB)

    Frampton, J P; Hynd, M R; Shain, W [Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, NY 12210 (United States); Shuler, M L, E-mail: jf7674@albany.edu [Department of Biomedical Engineering, 270 Olin Hall, Cornell University, Ithaca, NY 14850 (United States)

    2011-02-15

    Two-dimensional (2D) culture systems provide useful information about many biological processes. However, some applications including tissue engineering, drug transport studies, and analysis of cell growth and dynamics are better studied using three-dimensional (3D) culture systems. 3D culture systems can potentially offer higher degrees of organization and control of cell growth environments, more physiologically relevant diffusion characteristics, and permit the formation of more extensive 3D networks of cell-cell interactions. A 3D culture system has been developed using alginate as a cell scaffold, capable of maintaining the viability and function of a variety of neural cell types. Alginate was functionalized by the covalent attachment of a variety of whole proteins and peptide epitopes selected to provide sites for cell attachment. Alginate constructs were used to entrap a variety of neural cell types including astroglioma cells, astrocytes, microglia and neurons. Neural cells displayed process outgrowth over time in culture. Cell-seeded scaffolds were characterized in terms of their biochemical and biomechanical properties, effects on seeded neural cells, and suitability for use as 3D neural cell culture models.

  10. Breast Cancer Stem Cell Culture and Enrichment Using Poly(ε-Caprolactone) Scaffolds.

    Science.gov (United States)

    Palomeras, Sònia; Rabionet, Marc; Ferrer, Inés; Sarrats, Ariadna; Garcia-Romeu, Maria Luisa; Puig, Teresa; Ciurana, Joaquim

    2016-01-01

    The cancer stem cell (CSC) population displays self-renewal capabilities, resistance to conventional therapies, and a tendency to post-treatment recurrence. Increasing knowledge about CSCs' phenotype and functions is needed to investigate new therapeutic strategies against the CSC population. Here, poly(ε-caprolactone) (PCL), a biocompatible polymer free of toxic dye, has been used to fabricate scaffolds, solid structures suitable for 3D cancer cell culture. It has been reported that scaffold cell culture enhances the CSCs population. A RepRap BCN3D+ printer and 3 mm PCL wire were used to fabricate circular scaffolds. PCL design and fabrication parameters were first determined and then optimized considering several measurable variables of the resulting scaffolds. MCF7 breast carcinoma cell line was used to assess scaffolds adequacy for 3D cell culture. To evaluate CSC enrichment, the Mammosphere Forming Index (MFI) was performed in 2D and 3D MCF7 cultures. Results showed that the 60° scaffolds were more suitable for 3D culture than the 45° and 90° ones. Moreover, 3D culture experiments, in adherent and non-adherent conditions, showed a significant increase in MFI compared to 2D cultures (control). Thus, 3D cell culture with PCL scaffolds could be useful to improve cancer cell culture and enrich the CSCs population. PMID:27120585

  11. Breast Cancer Stem Cell Culture and Enrichment Using Poly(ε-Caprolactone Scaffolds

    Directory of Open Access Journals (Sweden)

    Sònia Palomeras

    2016-04-01

    Full Text Available The cancer stem cell (CSC population displays self-renewal capabilities, resistance to conventional therapies, and a tendency to post-treatment recurrence. Increasing knowledge about CSCs’ phenotype and functions is needed to investigate new therapeutic strategies against the CSC population. Here, poly(ε-caprolactone (PCL, a biocompatible polymer free of toxic dye, has been used to fabricate scaffolds, solid structures suitable for 3D cancer cell culture. It has been reported that scaffold cell culture enhances the CSCs population. A RepRap BCN3D+ printer and 3 mm PCL wire were used to fabricate circular scaffolds. PCL design and fabrication parameters were first determined and then optimized considering several measurable variables of the resulting scaffolds. MCF7 breast carcinoma cell line was used to assess scaffolds adequacy for 3D cell culture. To evaluate CSC enrichment, the Mammosphere Forming Index (MFI was performed in 2D and 3D MCF7 cultures. Results showed that the 60° scaffolds were more suitable for 3D culture than the 45° and 90° ones. Moreover, 3D culture experiments, in adherent and non-adherent conditions, showed a significant increase in MFI compared to 2D cultures (control. Thus, 3D cell culture with PCL scaffolds could be useful to improve cancer cell culture and enrich the CSCs population.

  12. Human autologous serum as a substitute for fetal bovine serum in human Schwann cell culture.

    Directory of Open Access Journals (Sweden)

    Parisa Goodarzi

    2014-04-01

    Full Text Available Nowadays, cell -based and tissue engineered products have opened new horizons in treatment of incurable nervous system disorders. The number of studies on the role of Schwann cells (SC in treating nervous disorders is higher than other cell types. Different protocols have been suggested for isolation and expansion of SC which most of them have used multiple growth factors, mitogens and fetal bovine sera (FBS in culture medium. Because of potential hazards of animal-derived reagents, this study was designed to evaluate the effect of replacing FBS with human autologous serum (HAS on SC's yield and culture parameters. Samples from 10 peripheral nerve biopsies were retrieved and processed under aseptic condition. The isolated cells cultured in FBS (1st group or autologous serum (2nd group. After primary culture the cells were seeded at 10000 cell/cm2 in a 12 wells cell culture plate for each group. At 100% confluency, the cell culture parameters (count, viability, purity and culture duration of 2 groups were compared using paired t-test. The average donors' age was 35.80 (SD=13.35 and except for 1 sample the others cultured successfully. In first group, the averages of cell purity, viability and culture duration were 97% (SD=1.32, 97/33% (SD=1.22 and 11.77 (SD=2.58 days respectively. This parameters were 97.33% (SD=1.00, 97.55% (SD=1.33 and 10.33 days (SD=1.65 in second group. The difference of cell count, purity and viability were not significant between 2 groups (P>0.05. The cells of second group reached to 100% confluency in shorter period of time (P=0.03. The results of this study showed that autologous serum can be a good substitute for FBS in human SC culture. This can reduce the costs and improve the safety of cell product for clinical application.

  13. Strategies for Transferring Mixtures of Organic Contaminants from Aquatic Environments into Bioassays.

    Science.gov (United States)

    Jahnke, Annika; Mayer, Philipp; Schäfer, Sabine; Witt, Gesine; Haase, Nora; Escher, Beate I

    2016-06-01

    Mixtures of organic contaminants are ubiquitous in the environment. Depending on their persistence and physicochemical properties, individual chemicals that make up the mixture partition and distribute within the environment and might then jointly elicit toxicological effects. For the assessment and monitoring of such mixtures, a variety of cell-based in vitro and low-complexity in vivo bioassays based on algae, daphnids or fish embryos are available. A very important and sometimes unrecognized challenge is how to combine sampling, extraction and dosing to transfer the mixtures from the environment into bioassays, while conserving (or re-establishing) their chemical composition at adjustable levels for concentration-effect assessment. This article outlines various strategies for quantifiable transfer from environmental samples including water, sediment, and biota into bioassays using total extraction or polymer-based passive sampling combined with either solvent spiking or passive dosing. PMID:26804122

  14. Strategies for Transferring Mixtures of Organic Contaminants from Aquatic Environments into Bioassays

    DEFF Research Database (Denmark)

    Jahnke, Annika; Mayer, Philipp; Schäfer, Sabine;

    2016-01-01

    Mixtures of organic contaminants are ubiquitous in the environment. Depending on their persistence and physicochemical properties, individual chemicals that make up the mixture partition and distribute within the environment and might then jointly elicit toxicological effects. For the assessment...... and monitoring of such mixtures, a variety of cell-based in vitro and low-complexity in vivo bioassays based on algae, daphnids or fish embryos are available. A very important and sometimes unrecognized challenge is how to combine sampling, extraction and dosing to transfer the mixtures from the...... environment into bioassays, while conserving (or re-establishing) their chemical composition at adjustable levels for concentration-effect assessment. This article outlines various strategies for quantifiable transfer from environmental samples including water, sediment, and biota into bioassays using total...

  15. Suspension Culture Alters Insulin Secretion in Induced Human Umbilical Cord Matrix-Derived Mesenchymal Cells

    OpenAIRE

    Fatemeh Seyedi; Alireza Farsinejad; Seyed Amirmahdi Nematollahi-Mahani; Touba Eslaminejad; Seyed Noureddin Nematollahi-Mahani

    2016-01-01

    Objective: Worldwide, diabetes mellitus (DM) is an ever-increasing metabolic disorder. A promising approach to the treatment of DM is the implantation of insulin producing cells (IPC) that have been derived from various stem cells. Culture conditions play a pivotal role in the quality and quantity of the differentiated cells. In this experimental study, we have applied various culture conditions to differentiate human umbilical cord matrix-derived mesenchymal cells (hUCMs) into...

  16. A miniaturized bioreactor system for the evaluation of cell interaction with designed substrates in perfusion culture.

    Science.gov (United States)

    Sun, T; Donoghue, P S; Higginson, J R; Gadegaard, N; Barnett, S C; Riehle, M O

    2012-12-01

    In tissue engineering, chemical and topographical cues are normally developed using static cell cultures but then applied directly to tissue cultures in three dimensions (3D) and under perfusion. As human cells are very sensitive to changes in the culture environment, it is essential to evaluate the performance of any such cues in a perfused environment before they are applied to tissue engineering. Thus, the aim of this research was to bridge the gap between static and perfusion cultures by addressing the effect of perfusion on cell cultures within 3D scaffolds. For this we developed a scaled-down bioreactor system, which allows evaluation of the effectiveness of various chemical and topographical cues incorporated into our previously developed tubular ε-polycaprolactone scaffold under perfused conditions. Investigation of two exemplary cell types (fibroblasts and cortical astrocytes) using the miniaturized bioreactor indicated that: (a) quick and firm cell adhesion in the 3D scaffold was critical for cell survival in perfusion culture compared with static culture; thus, cell-seeding procedures for static cultures might not be applicable, therefore it was necessary to re-evaluate cell attachment on different surfaces under perfused conditions before a 3D scaffold was applied for tissue cultures; (b) continuous medium perfusion adversely influenced cell spread and survival, which could be balanced by intermittent perfusion; (c) micro-grooves still maintained their influences on cell alignment under perfused conditions, while medium perfusion demonstrated additional influence on fibroblast alignment but not on astrocyte alignment on grooved substrates. This research demonstrated that the mini-bioreactor system is crucial for the development of functional scaffolds with suitable chemical and topographical cues by bridging the gap between static culture and perfusion culture. PMID:22170765

  17. Biosynthesis and biotransformation of lipids in plant cell cultures and algae

    International Nuclear Information System (INIS)

    The biosynthesis and metabolism of lipids in plant cell cultures grown photoautotrophically, has been studied since 1970. The most prominently occuring lipids in cell cultures and whole plants are phospholipids, glycolipids, triglycerides and glycosides. Radioactively labelled lipids have been produced from soybean cell cultures incubated with 14C-linoleic acid, and the fate of the phospholipid formed was investigated. Freshwater and marine algae cultured under different conditions of light, temperature and nutrient media have also been investigated for their lipid and fatty acid content. The exploitation of biotechnological processes for producing valuable lipids is encouraged. (U.K.)

  18. Spontaneous Electrical Activity of Cultured Interstitial Cells of Cajal from Mouse Urinary Bladder

    OpenAIRE

    Kim, Sun-Ouck; Jeong, Han-Seong; Jang, Sujeong; Wu, Mei-Jin; Park, Jong Kyu; Jiao, Han-Yi; Jun, Jae Yeoul; Park, Jong-Seong

    2013-01-01

    Interstitial cells of Cajal (ICCs) from the urinary bladder regulate detrusor smooth muscle activities. We cultured ICCs from the urinary bladder of mice and performed patch clamp and intracellular Ca2+ ([Ca2+]i) imaging to investigate whether cultured ICCs can be a valuable tool for cellular functional studies. The cultured ICCs displayed two types of spontaneous electrical activities which are similar to those recorded in intact bladder tissues. Spontaneous electrical activities of cultured...

  19. AUDITORY HAIR CELL EXPLANT CO-CULTURES PROMOTE THE DIFFERENTIATION OF STEM CELLS INTO BIPOLAR NEURONS

    OpenAIRE

    Coleman, B.; Fallon, J. B.; Gillespie, L.N.; Silva, M.G.; Shepherd, R.K.

    2006-01-01

    Auditory neurons, the target neurons of the cochlear implant, degenerate following a sensorineural hearing loss. The goal of this research is to direct the differentiation of embryonic stem cells (SCs) into bipolar auditory neurons that can be used to replace degenerating neurons in the deafened mammalian cochlea. Successful replacement of auditory neurons is likely to result in improved clinical outcomes for cochlear implant recipients. We examined two post-natal auditory co-culture models w...

  20. Ectopic lignification in primary cellulose-deficient cell walls of maize cell suspension cultures

    Institute of Scientific and Technical Information of China (English)

    Hugo Melida; Antonio Encina; Asier Largo-Gosens; Esther Novo-Uzal; Rogelio Santiago; Federico Pomar; Pedro Garca; Penelope Garca-Angulo; Jose Luis Acebes; Jesus Alvarez

    2015-01-01

    Maize (Zea mays L.) suspension-cultured cells with up to 70% less cellulose were obtained by stepwise habituation to dichlobenil (DCB), a cellulose biosynthesis inhibitor. Cellulose deficiency was accompanied by marked changes in cell wall matrix polysaccharides and phenolics as revealed by Fourier transform infrared (FTIR) spectroscopy. Cell wall compositional analysis indicated that the cellulose-deficient cell walls showed an enhancement of highly branched and cross-linked arabinoxylans, as well as an increased content in ferulic acid, diferulates and p-coumaric acid, and the presence of a polymer that stained positive for phloroglucinol. In accordance with this, cellulose-deficient cell walls showed a fivefold increase in Klason-type lignin. Thioacidolysis/GC-MS analysis of cellulose-deficient cell walls indicated the presence of a lignin-like polymer with a Syringyl/Guaiacyl ratio of 1.45, which differed from the sensu stricto stress-related lignin that arose in response to short-term DCB-treatments. Gene expression analysis of these cells indicated an overexpression of genes specific for the biosynthesis of monolignol units of lignin. A study of stress signaling pathways revealed an overexpression of some of the jasmonate signaling pathway genes, which might trigger ectopic lignification in response to cell wall integrity disruptions. In summary, the structural plasticity of primary cell walls is proven, since a lignification process is possible in response to cellulose impoverishment.