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Sample records for 1-7 cell culture

  1. Analysis of functional differences between hepatitis C virus NS5A of genotypes 1-7 in infectious cell culture systems

    Scheel, Troels K H; Prentoe, Jannick; Carlsen, Thomas H R;

    2012-01-01

    directly-acting antiviral compounds. NS5A is important for replication and virus production, but has not been studied for most HCV genotypes. We studied the function of NS5A using infectious NS5A genotype 1-7 cell culture systems, and through reverse genetics demonstrated a universal importance of the...... amphipathic alpha-helix, domain I and II and the low-complexity sequence (LCS) I for HCV replication; the replicon-enhancing LCSI mutation S225P attenuated all genotypes. Mutation of conserved prolines in LCSII led to minor reductions in virus production for the JFH1(genotype 2a) NS5A recombinant, but had...... introduced changes in NS5A led to changes in p7 and vice versa. Finally, NS5A function depended on genotype-specific residues in domain I, as changing genotype 2a-specific residues to genotype 1a sequence and vice versa led to highly attenuated mutants. In conclusion, this study identified NS5A genetic...

  2. Analysis of functional differences between hepatitis C virus NS5A of genotypes 1-7 in infectious cell culture systems.

    Troels K H Scheel

    Full Text Available Hepatitis C virus (HCV is an important cause of chronic liver disease. Several highly diverse HCV genotypes exist with potential key functional differences. The HCV NS5A protein was associated with response to interferon (IFN-α based therapy, and is a primary target of currently developed directly-acting antiviral compounds. NS5A is important for replication and virus production, but has not been studied for most HCV genotypes. We studied the function of NS5A using infectious NS5A genotype 1-7 cell culture systems, and through reverse genetics demonstrated a universal importance of the amphipathic alpha-helix, domain I and II and the low-complexity sequence (LCS I for HCV replication; the replicon-enhancing LCSI mutation S225P attenuated all genotypes. Mutation of conserved prolines in LCSII led to minor reductions in virus production for the JFH1(genotype 2a NS5A recombinant, but had greater effects on other isolates; replication was highly attenuated for ED43(4a and QC69(7a recombinants. Deletion of the conserved residues 414-428 in domain III reduced virus production for most recombinants but not JFH1(2a. Reduced virus production was linked to attenuated replication in all cases, but ED43(4a and SA13(5a also displayed impaired particle assembly. Compared to the original H77C(1a NS5A recombinant, the changes in LCSII and domain III reduced the amounts of NS5A present. For H77C(1a and TN(1a NS5A recombinants, we observed a genetic linkage between NS5A and p7, since introduced changes in NS5A led to changes in p7 and vice versa. Finally, NS5A function depended on genotype-specific residues in domain I, as changing genotype 2a-specific residues to genotype 1a sequence and vice versa led to highly attenuated mutants. In conclusion, this study identified NS5A genetic elements essential for all major HCV genotypes in infectious cell culture systems. Genotype- or isolate- specific NS5A functional differences were identified, which will be important

  3. Angiotensin-(1-7) regulates Angiotensin II-induced VCAM-1 expression on vascular endothelial cells

    Zhang, Feng [Department of Cardiology, Peking University People' s Hospital, Beijing (China); William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London (United Kingdom); Ren, Jingyi [Department of Cardiology, Peking University People' s Hospital, Beijing (China); Chan, Kenneth [William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London (United Kingdom); Chen, Hong, E-mail: chenhongbj@medmail.com.cn [Department of Cardiology, Peking University People' s Hospital, Beijing (China)

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer We for the first time found that Ang-(1-7) inhibits Ang II-induced VCAM-1 expression. Black-Right-Pointing-Pointer The inhibitory effect of Ang-(1-7) on VCAM-1 is mediated by MAS receptor. Black-Right-Pointing-Pointer The effect of Ang-(1-7) is due to the suppression of NF-kappaB translocation. -- Abstract: Angiotensin II (Ang II) and Angiotensin-(1-7) (Ang-(1-7)) are key effector peptides in the renin-angiotensin system. Increased circulatory Ang II level is associated with the development of hypertension and atherosclerosis, whereas Ang-(1-7) is a counter-regulatory mediator of Ang II which appears to be protective against cardiovascular disease. However, whether Ang-(1-7) regulates the action of Ang II on vascular endothelial cells (EC) remains unclear. We investigated the effects of Ang II and Ang-(1-7) in the context of atherogenesis, specifically endothelial cell VCAM-1 expression that is implicated in early plaque formation. The results show that Ang II increased VCAM-1 mRNA expression and protein displayed on EC surface, while Ang-(1-7) alone exerted no effects. However, Ang-(1-7) significantly suppressed Ang II-induced VCAM-1 expression. Ang-(1-7) also inhibited the Ang II-induced VCAM-1 promoter activity driven by transcription factor NF-KappaB. Furthermore, immunofluorescence assay and ELISA showed that Ang II facilitated the nuclear translocation of NF-kappaB in ECs, and this was attenuated by the presence of Ang-(1-7). The inhibitory effects of Ang-(1-7) on Ang II-induced VCAM-1 promoter activity and NF-kappaB nuclear translocation were all reversed by the competitive antagonist of Ang-(1-7) at the Mas receptor. Our results suggest that Ang-(1-7) mediates its affects on ECs through the Mas receptor, and negatively regulates Ang II-induced VCAM-1 expression by attenuating nuclear translocation of NF-kappaB.

  4. Fish Stem Cell Cultures

    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...

  5. Optimizing stem cell culture.

    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

  6. Cell Culture Made Easy.

    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…

  7. Bacterial cell culture

    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...

  8. Mammalian Cell Culture Simplified.

    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)

  9. Fish Stem Cell Cultures

    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.

  10. Optimizing stem cell culture.

    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...

  11. Digital Microfluidic Cell Culture.

    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

  12. Mycoplasmas detection in cells cultures

    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...

  13. Perfusion based cell culture chips

    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...

  14. Oscillating Cell Culture Bioreactor

    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

  15. Microfluidic Cell Culture Device

    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.

  16. Huanglongbing and psyllid cell cultures

    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...

  17. Cell death mechanisms in GT1-7 GnRH cells exposed to polychlorinated biphenyls PCB74, PCB118, and PCB153

    Exposure to endocrine disrupting chemicals (EDCs) such as polychlorinated biphenyls (PCBs) causes functional deficits in neuroendocrine systems. We used an immortalized hypothalamic GT1-7 cell line, which synthesizes the neuroendocrine peptide gonadotropin-releasing hormone (GnRH), to examine the neurotoxic and endocrine disrupting effects of PCBs and their mechanisms of action. Cells were treated for 1, 4, 8, or 24 h with a range of doses of a representative PCB from each of three classes: coplanar (2,4,4',5-tetrachlorobiphenyl: PCB74), dioxin-like coplanar (2',3,4,4',5' pentachlorobiphenyl: PCB118), non-coplanar (2,2',4,4',5,5'-hexachlorobiphenyl: PCB153), or their combination. GnRH peptide concentrations, cell viability, apoptotic and necrotic cell death, and caspase activation were quantified. In general, GnRH peptide levels were suppressed by high doses and longer durations of PCBs, and elevated at low doses and shorter timepoints. The suppression of GnRH peptide levels was partially reversed in cultures co-treated with the estrogen receptor antagonist ICI 182,780. All PCBs reduced viability and increased both apoptotic and necrotic cell death. Although the effects for the three classes of PCBs were often similar, subtle differences in responses, together with evidence that the combination of PCBs acted slightly different from individual PCBs, suggest that the three tested PCB compounds may act via slightly different or more than one mechanism. These results provide evidence that PCB congeners have endocrine disrupting and/or neurotoxic effects on the hypothalamic GnRH cell line, a finding that has implications for environmental endocrine disruption in animals.

  18. High density cell culture system

    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.

  19. Polyphosphoinositides are present in plant tissue culture cells

    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

  20. Cell culture purity issues and DFAT cells

    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

  1. β1- and β3- voltage-gated sodium channel subunits modulate cell surface expression and glycosylation of Nav1.7 in HEK293 cells

    Ninda Syam

    2013-08-01

    Full Text Available Voltage-gated sodium channels (Navs are glycoproteins composed of a pore-forming α-subunit and associated β-subunits that regulate Nav α-subunit plasma membrane density and biophysical properties. Glycosylation of the Nav α-subunit also directly affects Navs gating. β-subunits and glycosylation thus comodulate Nav α-subunit gating. We hypothesized that β-subunits could directly influence α-subunit glycosylation. Whole-cell patch clamp of HEK293 cells revealed that both β1- and β3-subunits coexpression shifted V1/2 of steady-state activation and inactivation and increased Nav1.7-mediated INa density. Biotinylation of cell surface proteins, combined with the use of deglycosydases, confirmed that Nav1.7 α-subunits exist in multiple glycosylated states. The α-subunit intracellular fraction was found in a core-glycosylated state, migrating at approximately 250 kDa. At the plasma membrane, in addition to the core-glycosylated form, a fully glycosylated form of Nav1.7 (~280 kDa was observed. This higher band shifted to an intermediate band (~260 kDa when β1-subunits were coexpressed, suggesting that the β1-subunit promotes an alternative glycosylated form of Nav1.7. Furthermore, the β1-subunit increased the expression of this alternative glycosylated form and the β3-subunit increased the expression of the core-glycosylated form of Nav1.7. This study describes a novel role for β1- and β3-subunits in the modulation of Nav1.7 α-subunit glycosylation and cell surface expression.

  2. Aseptic technique for cell culture.

    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

  3. Cell culture compositions

    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.

  4. Dynamized Preparations in Cell Culture

    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 ...

  5. Insect Cell Culture and Biotechnology

    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.

  6. Techniques for mammalian cell tissue culture.

    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

  7. Dynamized Preparations in Cell Culture

    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.

  8. Expanding intestinal stem cells in culture

    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.

  9. Cell Culture as an Alternative in Education.

    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)

  10. Structure and organ specificity of an anionic peroxidase from Arabidopsis thaliana cell suspension culture

    Ostergaard, L; Abelskov, A K; Mattsson, O; Welinder, K G

    The predominant peroxidase (pI 3.5) (E.C. 1.11.1.7) of an Arabidopsis thaliana cell suspension culture was purified and partially sequenced. Oligonucleotides were designed and a specific probe was obtained. A cDNA clone was isolated from an Arabidopsis cell suspension cDNA library and completely ...

  11. In vitro culture of human thyroid cells

    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)

  12. Culture of Cells from Amphibian Embryos.

    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)

  13. Advantages of embryogenic cell cultures of Gramineae

    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)

  14. Cell Suspension Culture of Neem Tree

    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...

  15. Callus and cell suspension cultures of carnation

    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...

  16. 9 CFR 101.6 - Cell cultures.

    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...

  17. Plant cell cultures and their biotechnological potential

    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.

  18. Studying cell-cell communication in co-culture

    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...

  19. Cell Culture for Production of Insecticidal Viruses.

    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

  20. Emulsions Containing Perfluorocarbon Support Cell Cultures

    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.

  1. Constructing a High Density Cell Culture System

    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.

  2. 3D Cell Culture in Alginate Hydrogels

    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.

  3. An In Vitro System Comprising Immortalized Hypothalamic Neuronal Cells (GT1-7 Cells) for Evaluation of the Neuroendocrine Effects of Essential Oils.

    Mizuno, Dai; Konoha-Mizuno, Keiko; Mori, Miwako; Yamazaki, Kentaro; Haneda, Toshihiro; Koyama, Hironari; Kawahara, Masahiro

    2015-01-01

    Aromatherapy and plant-based essential oils are widely used as complementary and alternative therapies for symptoms including anxiety. Furthermore, it was reportedly effective for the care of several diseases such as Alzheimer's disease and depressive illness. To investigate the pharmacological effects of essential oils, we developed an in vitro assay system using immortalized hypothalamic neuronal cells (GT1-7 cells). In this study, we evaluated the effects of essential oils on neuronal death induced by hydrogen peroxide (H2O2), aluminum, zinc, or the antagonist of estrogen receptor (tamoxifen). Among tests of various essential oils, we found that H2O2-induced neuronal death was attenuated by the essential oils of damask rose, eucalyptus, fennel, geranium, ginger, kabosu, mandarin, myrrh, and neroli. Damask rose oil had protective effects against aluminum-induced neurotoxicity, while geranium and rosemary oil showed protective activity against zinc-induced neurotoxicity. In contrast, geranium oil and ginger oil enhanced the neurotoxicity of tamoxifen. Our in vitro assay system could be useful for the neuropharmacological and endocrine pharmacological studies of essential oils. PMID:26576190

  4. Regulation of MAPKs Signaling Contributes to the Growth Inhibition of 1,7-Dihydroxy-3,4-dimethoxyxanthone on Multidrug Resistance A549/Taxol Cells.

    Zuo, Jian; Jiang, Hui; Zhu, Yan-Hong; Wang, Ya-Qin; Zhang, Wen; Luan, Jia-Jie

    2016-01-01

    1,7-Dihydroxy-3,4-dimethoxyxanthone (XAN) is a bioactive compound isolated from Securidaca inappendiculata Hassk. and validated with antiproliferative activities on a panel of cancer cell lines. This study was designed to investigate its growth inhibitory effects on multidrug resistance (MDR) non-small cell lung carcinoma (NSCLC) cell line A549/Taxol and explore the possible linkage between modulation of MAPKs and the bioactivities. Its growth inhibitory potency on the cells was estimated by MTT assay, and flow cytometric analysis was employed to investigate its potential cell cycle arrest and proapoptosis effects. Expressions of hallmark proteins were assessed by Western-Blot method. The results showed A549/Taxol cells were sensitive to XAN. XAN inhibited the proliferation of A549/Taxol cells in the time and concentration dependent manners. It acted as a potent inducer of apoptosis and cell cycle arrest in the cells. Western-Blot investigation validated the proapoptosis and cell cycle arrest activities of XAN and the potential of MDR reversion. Upregulation of p38 by XAN, which accounted for the cell cycle arrest at G2 phase, and the downregulation of ERK associated with the proapoptosis activity were also revealed. Further analysis found p53 may be the central role mediated the bioactivities of MAPKs in A549/Taxol cells. Based on these evidences, a conclusion has been deduced that XAN could be a potential agent for MDR NSCLC therapy targeting specifically MAPKs. PMID:27403196

  5. Regulation of MAPKs Signaling Contributes to the Growth Inhibition of 1,7-Dihydroxy-3,4-dimethoxyxanthone on Multidrug Resistance A549/Taxol Cells

    Jian Zuo

    2016-01-01

    Full Text Available 1,7-Dihydroxy-3,4-dimethoxyxanthone (XAN is a bioactive compound isolated from Securidaca inappendiculata Hassk. and validated with antiproliferative activities on a panel of cancer cell lines. This study was designed to investigate its growth inhibitory effects on multidrug resistance (MDR non-small cell lung carcinoma (NSCLC cell line A549/Taxol and explore the possible linkage between modulation of MAPKs and the bioactivities. Its growth inhibitory potency on the cells was estimated by MTT assay, and flow cytometric analysis was employed to investigate its potential cell cycle arrest and proapoptosis effects. Expressions of hallmark proteins were assessed by Western-Blot method. The results showed A549/Taxol cells were sensitive to XAN. XAN inhibited the proliferation of A549/Taxol cells in the time and concentration dependent manners. It acted as a potent inducer of apoptosis and cell cycle arrest in the cells. Western-Blot investigation validated the proapoptosis and cell cycle arrest activities of XAN and the potential of MDR reversion. Upregulation of p38 by XAN, which accounted for the cell cycle arrest at G2 phase, and the downregulation of ERK associated with the proapoptosis activity were also revealed. Further analysis found p53 may be the central role mediated the bioactivities of MAPKs in A549/Taxol cells. Based on these evidences, a conclusion has been deduced that XAN could be a potential agent for MDR NSCLC therapy targeting specifically MAPKs.

  6. Human hematopoietic cell culture, transduction, and analyses

    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...

  7. Methods for Maintaining Insect Cell Cultures

    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.

  8. Calculations for a BWR Lattice with Adjacent Gadolinium Pins Using the Monte Carlo Cell Code Serpent v.1.1.7

    Diego Ferraro

    2011-01-01

    Full Text Available Monte Carlo neutron transport codes are usually used to perform criticality calculations and to solve shielding problems due to their capability to model complex systems without major approximations. However, these codes demand high computational resources. The improvement in computer capabilities leads to several new applications of Monte Carlo neutron transport codes. An interesting one is to use this method to perform cell-level fuel assembly calculations in order to obtain few group constants to be used on core calculations. In the present work the VTT recently developed Serpent v.1.1.7 cell-oriented neutronic calculation code is used to perform cell calculations of a theoretical BWR lattice benchmark with burnable poisons, and the main results are compared to reported ones and with calculations performed with Condor v.2.61, the INVAP's neutronic collision probability cell code.

  9. Callus and cell suspension cultures of carnation

    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...

  10. Cell culture models for study of differentiated adipose cells

    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.

  11. Human cell culture in a space bioreactor

    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.

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

    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.

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

    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.

  14. Melphalan metabolism in cultured cells

    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

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

    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

  16. Cell culture experiments planned for the space bioreactor

    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.

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

    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...

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

    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...

  19. Spectral analysis of the effects of 1.7 MeV electron irradiation on the current transfer characteristic of cadmium telluride solar cells.

    Tian, Jin-Xiu; Zeng, Guang-Gen; He, Xu-Lin; Zhang, Jing-Quan; Wu, Li-Li; Li, Wei; Li, Bing; Wang, Wen-Wu; Feng, Liang-Huan

    2014-04-01

    The effects of device performance of 1.7 MeV electron irradiation on cadmium telluride polycrystalline thin film solar cells with the structure of anti-radiation glass/ITO/ZnO/CdS/CdTe/ZnTe/ZnTe : Cu/Ni have been studied. Light and dark I-V characteristics, dark C-V characteristics, quantum efficiency (QE), admittance spectrum (AS) and other testing methods were used to analyze cells performance such as the open-circuit voltage (Voc), short-circuit current (Isc), fill factor (FF) and conversion efficiency (eta). It was explored to find out the effects of irradiation on the current transfer characteristic of solar cells combined with the dark current density (Jo), diode ideal factor (A), quantum efficiency, carrier concentration and the depletion layer width. The decline in short-circuit current was very large and the efficiency of solar cells decreased obviously after irradiation. Reverse saturation current density increased, which indicates that p-n junction characteristics of solar cells were damaged, and diode ideal factor was almost the same, so current transport mechanism of solar cells has not changed. Quantum efficiency curves proved that the damage of solar cells' p-n junction influenced the collection of photo-generated carriers. Irradiation made carrier concentration reduce to 40.6%. The analyses have shown that. A new defect was induced by electron irradiation, whose position is close to 0.58 eV above the valence band in the forbidden band, and capture cross section is 1.78 x 10(-16) cm2. These results indicate that irradiation influences the generation of photo-generated carriers, increases the risk of the carrier recombination and the reverse dark current, and eventually makes the short-circuit current of solar cells decay. PMID:25007593

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

    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

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

    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

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

    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.

  3. Integrated biosensors for cell culture monitoring

    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...

  4. Cell Culture Microfluidic Biochips: Experimental Throughput Maximization

    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....

  5. Cell culture from sponges: pluripotency and immortality

    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

  6. THE METHODS FOR MAINTAINING INSECT CELL CULTURES

    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...

  7. ANTHOCYANIN (ACN) STABILITY IN CELL CULTURE MEDIA

    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, ...

  8. Nanotechnology, Cell Culture and Tissue Engineering

    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

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

    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

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

    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

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

    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.

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

    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...

  13. Prevention and Detection of Mycoplasma Contamination in Cell Culture

    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.

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

    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.

  15. Mouse cell culture - Methods and protocols

    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........

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

    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.

  17. Degradation of TNT by plant cell cultures

    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

  18. Darwinian Evolution of Prions in Cell Culture*

    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, ...

  19. Bioactive sugar surfaces for hepatocyte cell culture

    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...

  20. Prevention and Detection of Mycoplasma Contamination in Cell Culture

    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...

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

    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

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

    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

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

    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.

  4. Effect of Angiotensin-(1-7) on Proliferation of Insulin-secreting Cell Line NIT-1%血管紧张素-(1-7)对胰岛素分泌细胞株NIT-1增殖的影响

    王晓云; 徐明彤; 林秀红; 宛彦; 任萌; 李焱; 严励

    2013-01-01

    [Objective] To investigate whether Angiotensin-(1-7) can affect the proliferation of mouse insulin-secreting cell line NIT-1.[Methods] NIT-1 cells were divided into different groups as follows and cell counting Kit-8 method (CCK-8) was performed to detect the proliferation of NIT-1 at different groups separately:(1) Glucose at concentration of 11.1,25.0,30.0,35.0,and 40.0 mmol/L,mannitol at concentration of 35 mmol/L for 24 h; (2) Ang-(1-7) at concentrations of 0,10-7,10-6,10-5,and 10-4 mol/L for 24 h; (3) High glucose(HG),HG+Ang-(1-7),HG+Ang-(1-7)+Mas receptor inhibitor(A-779),HG+A-779.[Results] (1)Compared with the 11.1 mmol/L group,the cell proliferation were inhibited in the 35.0 mmol/L and 40.0 mmol/L groups (1.02 ± 0.07 vs 1.21 ± 0.10; 0.90 ± 0.05 vs 1.21 ± 0.10,P < 0.05).(2) No significant difference of the proliferation in N1T-1 cells was found between Ang-(1-7) treatment groups (10-7 ~ 10-4 mol/L).(3) Compared to the HG group,the proliferation of the HG+Ang-(1-7) group was higher (1.14 ± 0.07 vs 1.44 ± 0.24,P < 0.05); Compared to the HG+Ang-(1-7) group,the proliferation of the HG+Ang-(1-7)+A-779 group was decreased (1.44 ± 0.24 vs 1.20 ± 0.02,P < 0.05).[Conclusion] By binding to Mas receptor,Ang-(1-7) could reverse the inhibitory effect of high glucose on proliferation of NIT-1 cells.%[目的]探讨血管紧张素[Ang-(1-7)]对小鼠胰岛素分泌细胞株NIT-1细胞增殖的影响.[方法]NIT-1细胞按以下分组分别处理24 h,采用CCK-8比色法检测细胞增殖.(1)11.1、25.0、30.0、35.0和40.0 mmol/L葡萄糖液,35.0 mmol/L甘露醇分别处理24 h;(2)0、10-7、10-6、10-5和10-4 mol/L Ang-(1-7)分别处理24 h;(3)高糖(HG,35.0 mmol/L)、HG+Ang-(1-7)(10-5 mol/L)、HG+Ang-(1-7)+Mas受体拮抗剂(A-779,10-5 mol/L)和HG+A-779组.[结果](1)较11.1 mmol/L组,葡萄糖浓度为35.0和40.0 mmol/L时,NIT-1细胞的增殖明显减低(1.02±0.07 vs 1.21±0.10;0.90±0.05 vs 1.21±0.10,P<0.05).(2)在11.1 mmol

  5. An Introductory Undergraduate Course Covering Animal Cell Culture Techniques

    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…

  6. Sodium 22+ washout from cultured rat cells

    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

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

    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.

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

    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

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

    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...

  10. PHYTOCHEMICAL STUDY OF CELL CULTURE JATROPHA CURCAS

    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.

  11. Rotating bio-reactor cell culture apparatus

    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.

  12. Renotropic stimulation in rat kidney cell culture

    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

  13. Optical Oxygen Sensors for Applications in Microfluidic Cell Culture

    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...

  14. Cardiac Cells Beating in Culture: A Laboratory Exercise

    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…

  15. Growth of cultured porcine retinal pigment epithelial cells

    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....

  16. Equipment for large-scale mammalian cell culture.

    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

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

    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...

  18. Metabolic flux rewiring in mammalian cell cultures.

    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

  19. Phosphatidylinositol species of suspension cultured plant cells

    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.

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

    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)

  1. Recombinant Protein Production and Insect Cell Culture and Process

    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.

  2. Glucagon-like peptide 1 (7-36) amide stimulates exocytosis in human pancreatic beta-cells by both proximal and distal regulatory steps in stimulus-secretion coupling

    Gromada, J; Bokvist, K; Ding, W G; Holst, J J; Nielsen, Jens Høiriis; Rorsman, P

    1998-01-01

    The effect of glucagon-like peptide 1(7-36) amide [GLP-1(7-36) amide] on membrane potential, whole-cell ATP-sensitive potassium channel (K[ATP]) and Ca2+ currents, cytoplasmic Ca2+ concentration, and exocytosis was explored in single human beta-cells. GLP-1(7-36) amide induced membrane...... depolarization that was associated with inhibition of whole-cell K(ATP) current. In addition, GLP-1(7-36) amide (and forskolin) produced greater than fourfold potentiation of Ca2+-dependent exocytosis. The latter effect resulted in part (40%) from acceleration of Ca2+ influx through voltage-dependent (L-type) Ca......2+ channels. More importantly, GLP-1(7-36) amide (via generation of cyclic AMP and activation of protein kinase A) potentiated exocytosis at a site distal to a rise in the cytoplasmic Ca2+ concentration. Photorelease of caged cAMP produced a two- to threefold potentiation of exocytosis when the...

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

    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...

  4. Protection of cultured mammalian cells by rebamipide

    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)

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

    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...

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

    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.

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

    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.

  8. Electrospinning of microbial polyester for cell culture

    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

  9. Particle Trajectories in Rotating Wall Cell Culture Devices

    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.

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

    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.

  11. Usability and Applicability of Microfluidic Cell Culture Systems

    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...

  12. Constrained H-Phe-Phe-NH2 analogues with high affinity to the substance P 1-7 binding site and with improved metabolic stability and cell permeability.

    Fransson, Rebecca; Sköld, Christian; Kratz, Jadel M; Svensson, Richard; Artursson, Per; Nyberg, Fred; Hallberg, Mathias; Sandström, Anja

    2013-06-27

    We recently reported the discovery of H-Phe-Phe-NH2 as a small and high affinity ligand for the substance P 1-7 (SP(1-7), H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH) specific binding site and its intriguing ability to reduce neuropathic pain. With the overall aim to develop stable and orally bioavailable SP(1-7) mimetics, the dipeptide was chosen as a lead compound. Herein the structure-activity relationship (SAR) of a set of modified H-Phe-Phe-NH2 analogues is presented together with their potential active uptake by PEPT1 transporter, intestinal permeability, and metabolic stability. Local constraints via peptide backbone methylation or preparation of cyclized analogues based on pyrrolidine were evaluated and were shown to significantly improve the in vitro pharmacokinetic properties. The SAR was rationalized by deriving a plausible binding pose for the high affinity ligands. Rigidification using a 3-phenylpyrrolidine moiety in the C-terminal of H-Phe-Phe-NH2 resulted in high affinity and improved intrinsic clearance and intestinal epithelial permeability. PMID:23735006

  13. Cytotoxicity of antimicrobial photodynamic inactivation on epithelial cells when co-cultured with Candida albicans.

    Pellissari, Claudia Viviane Guimarães; Pavarina, Ana Claudia; Bagnato, Vanderlei Salvador; Mima, Ewerton Garcia de Oliveira; Vergani, Carlos Eduardo; Jorge, Janaina Habib

    2016-05-11

    This study assessed the cytotoxicity of antimicrobial Photodynamic Inactivation (aPDI), mediated by curcumin, using human keratinocytes co-cultured with Candida albicans. Cells and microorganisms were grown separately for 24 hours and then kept in contact for an additional 24 hours. After this period, aPDI was applied. The conditions tested were: P+L+ (experimental group aPDI); P-L+ (light emitting diode [LED] group); P+L- (curcumin group); and P-L- (cells in co-culture without curcumin nor LED). In addition, keratinocytes and C. albicans were grown separately, were not placed in the co-culture and did not receive aPDI (control group). Cell proliferation was assessed using Alamar Blue, MTT, XTT and CFU tests. Qualitative and quantitative analyses were performed. Analysis of variance (ANOVA) was applied to the survival percentages of cells compared to the control group (considered as 100% viability), complemented by multiple comparisons using Tukey's test. A 5% significance level was adopted. The results of this study showed no interference in the metabolism of the cells in co-culture, since no differences were observed between the control group (cultured cells by themselves) and the P-L- group (co-culture cells without aPDI). The aPDI group reached the highest reduction (p = 0.009), which was equivalent to 1.7 log10 when compared to the control group. The P+L-, P-L+, P-L- and control groups were not statistically different (ρ > 0.05). aPDI inhibited the growth of keratinocytes and C. albicans in all tests, so the therapy was considered slightly (inhibition between 25 and 50% compared to the control group) to moderately (inhibition between 50 and 75% compared to the control group) cytotoxic. PMID:27110908

  14. THE ALKALOID CYTISINE IN THE CELL CULTURE

    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

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

    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)

  16. Systems Biology for Organotypic Cell Cultures

    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.

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

    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...

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

    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

  19. Enhancing growth of cultured human skin cells using low-energy CO2 laser

    Grossman, Nili; Reuveni, Haim; Halevy, Sima; Lubart, Rachel

    1997-12-01

    In view of the versatility and usage of the CO2 laser as a too. in surgery and dermatology, we have studied its effect on enhancing proliferation of cultured skin cells using an attenuated CO2 laser. Exposure of cultured keratinocytes or fibroblasts to continuous wave or pulse mode irradiation enhanced thymidine incorporation by 1.4 to 1.7 folds, and cell number by 1.25 to 1.4 folds, measured 24 and 48 hours later, depending on the fluency applied. As expected, these effects were not suppressed by added antioxidants, indicating that the mechanism involved in this newly observed effect, differ from photosensitization by low energy visible and near IR lasers.

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

    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...

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

    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.

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

    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

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

    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...

  4. 21 CFR 864.2280 - Cultured animal and human cells.

    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 §...

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

    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...

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

    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

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

    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.

  8. Biona-C Cell Culture pH Monitoring System

    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.

  9. PECULIARITIES OF SECONDARY METABOLITES BIOSYNTHESIS IN PLANT CELL CULTURES

    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

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

    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

  11. Expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in control of GnRH secretion

    Ying YANG; Li-bin ZHOU; Shang-quan LIU; Jing-feng TANG; Feng-yin LI; Rong-ying LI; Huai-dong SONG; Ming-dao CHEN

    2005-01-01

    Aim: To investigate the expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in the control of GnRH secretion.Methods: Receptors of bombesin3, cholecystokinin (CCK)-A, CCK-B, glucagonlike peptide (GLP)1, melanin-concentrating hormone (MCH)1, orexinl, orexin2,neuromedin-B, neuropeptide Y (NPY) 1 and NPY5, neurotensin (NT) 1, NT2, NT3,and leptin receptor long form mRNA in GT1-7 cells were detected by reversed transcriptase-polymerase chain reaction. GT1-7 cells were treated with leptin,orexin A and orexin B at a cohort of concentrations for different lengths of time,and GnRH in medium was determined by radioimmunoassay (RIA). Results:Receptors of bombesin 3, CCK-B, GLP1, MCH1, orexinl, neuromedin-B, NPY1,NPY5, NT1, NT3, and leptin receptor long form mRNA were expressed in GT1-7cells, of which, receptors of GLP1, neuromedin-B, NPY1, and NT3 were highly expressed. No amplified fragments of orexin2, NT2, and CCK-A receptor cDNA were generated with GT1-7 RNA, indicating that the GT1-7 cells did not express mRNA of them. Leptin induced a significant stimulation of GnRH release, the results being most significant at 0.1 nmol/L for 15 min. In contrast to other studies in hypothalamic explants, neither orexin A nor orexin B affected basal GnRH secretion over a wide range of concentrations ranging from 1 nmol/L to 500 nmol/Lat 15, 30, and 60 min. Conclusion: Feeding and reproductive function are closely linked. Many orexigenic and anorexigenic signals may control feeding behavior as well as alter GnRH secretion through their receptors on GnRH neurons.

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

    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...

  13. Microglial cells in astroglial cultures: a cautionary note

    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.

  14. SPONTANEOUS TRANSFORMATION OF CULTURED PORCINE BONE MARROW STROMAL CELLS

    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....

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

    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

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

    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.

  17. Multizone paper platform for 3D cell cultures.

    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.

  18. Chemotherapy in heterogeneous cultures of cancer cells with interconversion

    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)

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

    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.

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

    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.

  1. A practical guide to hydrogels for cell culture.

    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

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

    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

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

    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

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

    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.

  5. Surface modified alginate microcapsules for 3D cell culture

    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.

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

    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

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

    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.

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

    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...

  9. Air pollutant production by algal cell cultures

    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.

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

    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...

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

    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

  12. Detection and Treatment of Mycoplasma Contamination in Cultured Cells

    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.

  13. Novel culturing platform for brain slices and neuronal cells

    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...

  14. Cell/Tissue Culture Radiation Exposure Facility Project

    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...

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

    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....

  16. Inducing effects of macrophage stimulating protein on the expansion of early hematopoietic progenitor cells in liquid culture

    MA Li-xia; HUANG Yan-hong; CHENG La-mei; LEI Jun; WANG Qi-ru

    2007-01-01

    Background Macrophage stimulating protein (MSP) is produced by human bone marrow endothelial cells. In this study,we sought to observe its effects on inducing the expansion of early hematopoietic progenitor cells which were cultured in a liquid culture system in the presence of the combination of stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), granulocyte macrophage-colony stimulating factor (GM-CSF), erythropoietin (EPO) (Cys) and MSP or of Cys and bone marrow endothelial cell conditioned medium (EC-CM).Methods Human bone marrow CD34+ cells were separated and cultured in a liquid culture system for 6 days.Granulocyte-macrophage colony forming unit (CFU-GM) and colony forming unit-granulocyte, erythrocyte, macrophage,megakaryocyte (CFU-GEMM) were employed to assay the effects of different treatment on the proliferation of hematopoeitic stem/progenitor cells. The nitroblue tetrazolium (NBT) reductive test and hoechest 33258 staining were employed to reflect the differentiation and apoptosis of the cells respectively.Results MSP inhibited the proliferation of CFU-GM and CFU-GEMM in semi-solid culture and the inhibitory effect on CFU-GEMM was stronger than on CFU-GM. MSP inhibited the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators. Bone marrow (BM) CFU-GEMM was 2.3-fold or 1.7-fold increase or significantly decreased in either Cys+EC-CM, Cys+MSP or Cys compared with 0 hour control in liquid culture system after 6 days.Conclusion MSP, a hematopoietic inhibitor, inhibits the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators and makes the early hematopoietic progenitor cells expand in a liquid culture system.

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

    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

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

    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

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

    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

  20. Hydrofocusing Bioreactor for Three-Dimensional Cell Culture

    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.

  1. Qualitative study of three cell culture methods.

    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

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

    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

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

    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…

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

    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...

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

    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

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

    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.

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

    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...

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

    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.

  9. Magic clusters and (2x1) local structure formed in a half-unit cell of the Si(1 1 1)-(7x7) surface by Tl adsorption

    Formation of a (2x1) local structure is found at submonolayer growth of Tl on the Si(1 1 1)-(7x7) surface by means of scanning tunneling microscopy (STM). At low coverages, the (7x7) grid remains and the (2x1) structure is formed inside of the half-unit cells of the (7x7). The (2x1) coexists with other structures, such as of 'magic' clusters observed in the case of all other group III metals. Based on our STM observations we propose an atomic arrangement of the (2x1) structure

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

    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.

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

    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....

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

    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.

  13. Co-culturing with High-Charge and Energy Particle Irradiated Cells Increases Mutagenic Joining of Enzymatically Induced DNA Double-Strand Breaks in Nonirradiated Cells.

    Li, Zhentian; Doho, Gregory; Zheng, Xuan; Jella, Kishore Kumar; Li, Shuyi; Wang, Ya; Dynan, William S

    2015-09-01

    Cell populations that have been exposed to high-charge and energy (HZE) particle radiation, and then challenged by expression of a rare-cutting nuclease, show an increased frequency of deletions and translocations originating at the enzyme cut sites. Here, we examine whether this effect also occurs in nonirradiated cells that have been co-cultured with irradiated cells. Human cells were irradiated with 0.3-1.0 Gy of either 600 MeV/u (56)Fe or 1,000 MeV/u (48)Ti ions or with 0.3-3.0 Gy of 320 kV X rays. These were co-cultured with I-SceI-expressing reporter cells at intervals up to 21 days postirradiation. Co-culture with HZE-irradiated cells led to an increase in the frequency of I-SceI-stimulated translocations and deletions in the nonirradiated cells. The effect size was similar to that seen previously in directly irradiated populations (maximum effect in bystander cells of 1.7- to 4-fold depending on ion and end point). The effect was not observed when X-ray-irradiated cells were co-cultured with nonirradiated cells, but was correlated with an increase in γ-H2AX foci-positive cells in the nonirradiated population, suggesting the presence of genomic stress. Transcriptional profiling of a directly irradiated cell population showed that many genes for cytokines and other secretory proteins were persistently upregulated, but their induction was not well correlated with functional effects on repair in co-cultured cells, suggesting that this transcriptional response alone is not sufficient to evoke the effect. The finding that HZE-irradiated cells influence the DNA double-strand break repair fidelity in their nonirradiated neighbors has implications for risk in the space radiation environment. PMID:26284422

  14. Endothelial cell cultures as a tool in biomaterial research

    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)

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

    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...

  16. Cell cultures from the symbiotic soft coral Sinularia flexibilis

    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

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

    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-...

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

    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

  19. Schwann cell cultures from human fetal dorsal root ganglia

    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.

  20. Culture and immortalization of pancreatic ductal epithelial cells.

    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

  1. Miniature Bioreactor System for Long-Term Cell Culture

    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.

  2. Hydrodynamic effects on cells in agitated tissue culture reactors

    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.

  3. Microfluidic bioreactors for culture of non-adherent cells

    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....

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

    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)

  5. Radiosensitivity of cultured insect cells: I. Lepidoptera

    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

  6. Establishing a stem cell culture laboratory for clinical trials

    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.

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

    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

  8. Simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell cultures and in sub-regions of guinea pig brain

    Schou-Pedersen, Anne Marie Voigt; Hansen, Stine Normann; Tveden-Nyborg, Pernille;

    2016-01-01

    In the present paper, we describe a validated chromatographic method for the simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell culture and in sub-regions of the guinea pig brain. Electrochemical...... intracellular and extracellular amounts of monoamine neurotransmitters and their metabolites in guinea pig frontal cortex and hippocampal primary neuronal cell cultures. Noradrenaline, dopamine and serotonin were found to be in a range from 0.31 to 1.7 pmol per 2 million cells intracellularly, but only the...

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

    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

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

    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

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

    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...

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

    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...

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

    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.

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

    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 ...

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

    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.)

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

    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...

  17. Epithelial morphogenesis in three-dimensional cell culture system

    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...

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

    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

  19. Specimen Sample Preservation for Cell and Tissue Cultures

    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.

  20. Expression of CD44 in Cultured Human Trabecular Meshwork Cells

    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.

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

    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

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

    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....

  3. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    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...

  4. Hollow fiber clinostat for simulating microgravity in cell culture

    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.

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

    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

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

    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...

  7. Survival and DNA repair in ultraviolet-irradiated haploid and diploid cultured frog cells

    Survival and repair of DNA following ultraviolet (254-nm) radiation have been investigated in ICR 2A, a cultured cell line from haploid embryos of the grassfrog, Rana pipiens. Survival curves from cells recovering in the dark gave mean lethal dose value (D0) in the range 1.5-1.7 Jm-2 for both haploid and diploid cell stocks. The only significant difference observed between haploids and diploids was in the extent of the shoulder at low fluence (Dsub(q)), the value for exponentially multiplying diploid cells (3.0 Jm-2) being higher than that found for haploids (1.2 Jm-2). Irradiation of cultures reversibly blocked in the G1 phase of the cell cycle gave survival-curve coefficients indistinguishable between haploids and diploids. Post-irradiation exposure to visible light restored colony-forming capacity and removed chromatographically estimated pyrimidine dimers from DNA at the same rates. After fluences killing 90% of the cells, complete restoration of survival was obtained after 60-min exposure to 500 foot-candles, indicating that in this range lethality is entirely photoreversible and therefore attributable to pyrimidine dimers in DNA. Dimer removal required illumination following ultraviolet exposure, intact cells and physiological temperature, implying that the photoreversal involved DNA photolyase activity. Excision-repair capacity was slight, since no loss of dimers could be detected chromoatographically during up to 48 h incubation in the dark and since autoradiographically detected 'unscheduled DNA synthesis' was limited to a 2-fold increase saturated at 10 Jm-2. These properties make ICR 2A frog cells useful to explore how DNA-repair pathways influence mutant yield. (Auth.)

  8. Lingual Epithelial Stem Cells and Organoid Culture of Them

    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.

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

    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...

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

    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

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

    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.

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

    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

  13. Radiosensitizing effects of nitroimidazole nucleoside analogues on cultured mammalian cells at low radiation doses

    Radiosensitizing effects of nitroimidazole nucleoside analogues (RK-27, RK-28 and RK-29) were studied using cultured mouse leukemic L5178Y (L5178Y) cells and Chinese hamster V79 (CH V79) cells. There was no significant difference in sensitizer enhancement ratio among the three RK-compounds. The ratios were 1.7 for L5178Y cells and 1.6 for CH V79 cells at 1 mM concentration and were comparable to that of misonidazole. Sensitizer enhancement ratios of RK-28 for L5178Y cells irradiated with 1 to 4 Gy X-rays were, respectively, 1.5 to 1.6 at 0.5 mM concentration, which were nearly the same as those obtained at high doses. RK-28 was as effective for fractionated X-irradiation as for single X-irradiation, assuming complete repair of sublethal damage during incubation at 37degC between sessions of fractionated X-irradiation. The sensitizing effects of RK-28 were additive in combination with neocarzinostatin, an antitumor drug, which has different pharmacological and physiological properties, and enhances the radioresponse of cells. RK-28 is known to be rapidly metabolized and excreted in vivo and is thus expected to be less toxic than misonidazole. It was effective at low radiation doses routinely used in radiotherapy practice and for fractionated X-irradiation, suggesting its clinical utility. (author)

  14. Pedigree analysis of proliferation kinetics in cultured mammalian cells

    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.)

  15. Metabolic flux rewiring in mammalian cell cultures

    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...

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

    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...

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

    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

  18. 1.7 MeV电子辐照对CdTe太阳电池电流传输特性影响的图谱分析%Spectral Analysis of the Effects of 1.7 MeV Electron Irradiation on the Current Transfer Characteristic of Cadmium Telluride Solar Cells

    田金秀; 曾广根; 何绪林; 张静全; 武莉莉; 李卫; 黎兵; 王文武; 冯良桓

    2014-01-01

    研究了1.7 MeV的电子辐照对具有Anti-radiation glass/ITO/ZnO/CdS/CdTe/ZnTe/ZnTe∶Cu/Ni结构的碲化镉多晶薄膜太阳电池器件性能的影响。抗辐照玻璃的使用,有效防止了普通玻璃受辐照后性能变化对测试结果的影响。利用光、暗I-V ,C-V ,QE ,AS等测试手段,分析了包括开路电压、短路电流、转换效率在内的电池性能。通过对比研究暗电流密度、分析了辐照对电池电流传输特性的影响。辐照后短路电流下降很大,电池效率明显降低。反向饱和电流密度有所增加,表明太阳电池的pn结特性受到损伤,而二极管理想因子几乎不变,说明太阳电池电流的输运机制未发生了变化。量子效率曲线证明是由于太阳电池结区损伤影响了光生载流子的收集。辐照使载流子浓度下降为原来的40.6%。导纳谱研究最终发生辐照会引入Cd2+缺陷能级,其位置为 Et -Ev =(0.58±0.02)eV ,俘获截面为1.78×10-16 cm2,表明辐照会影响光生载流子的产生,增加了载流子复合的概率,使得反向暗电流增大,最终导致电池的短路电流衰减。%The effects of device performance of 1.7 MeV electron irradiation on cadmium telluride polycrystal-line thin film solar cells with the structure of anti-radiation glass/ITO/ZnO/CdS/CdTe/ZnTe/ZnTe∶Cu/Ni have been studied .Light and dark I-V characteristics ,dark C-V characteristics ,quantum efficiency (QE) ,ad-mittance spectrum (AS) and other testing methods were used to analyze cells performance such as the open-cir-cuit voltage (Voc) ,short-circuit current (Isc) ,fill factor (FF) and conversion efficiency (η) .It was explored to find out the effects of irradiation on the current transfer characteristic of solar cells combined with the dark current density (Jo) ,diode ideal factor (A) ,quantum efficiency ,carrier concentration and the depletion layer width .The decline in short-circuit current was very

  19. Cell culture systems for the hepatitis C virus

    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.

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

    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

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

    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

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

    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

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

    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

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

    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.

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

    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

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

    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.

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

    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...

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

    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...

  9. Effects of Visible Light on Cultured Bovine Trabecular Cells

    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.

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

    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.)

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

    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

  12. Cannabinoids induce incomplete maturation of cultured human leukemia cells

    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.

  13. Isolation and culture of umbilical vein mesenchymal stem cells

    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.

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

    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...

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

    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...

  16. Radiation transformation in differentiated human cells in culture

    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)

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

    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...

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

    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

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

    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

  20. Test chambers for cell culture in static magnetic field

    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

  1. Biodegradable Mg corrosion and osteoblast cell culture studies

    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.

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

    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...

  3. Plant Cell Cultures as Source of Cosmetic Active Ingredients

    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.

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

    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...

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

    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...

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

    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...

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

    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.

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

    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

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

    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.

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

    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)