Sample records for cell culture studies

  1. Insect Cell Culture

    Microsoft Academic Search

    Oers van M. M; D. E. Lynn

    2010-01-01

    Insect cell cultures are widely used in studies on insect cell physiology, developmental biology and microbial pathology. In particular, insect cell culture is an indispensable tool for the study of insect viruses. The first continuously growing insect cell cultures were established from lepidopteran insects around 1960. Since then, more than 600 insect cell lines have been described from over 100

  2. Chalcone dimethylallyltransferase from Morus nigra cell cultures. Substrate specificity studies.

    PubMed

    Vitali, Alberto; Giardina, Bruno; Delle Monache, Giuliano; Rocca, Filippo; Silvestrini, Andrea; Tafi, Andrea; Botta, Bruno

    2004-01-16

    A new prenyltransferase (PT) enzyme derived from the microsomal fractions of cell cultures of Morus nigra was shown to be able to prenylate exclusively chalcones with a 2',4'-dihydroxy substitution and the isoflavone genistein. Computational studies were performed to shed some light on the relationship between the structure of the substrate and the enzymatic activity. PT requires divalent cations, particularly Mg(2+), to be effective. The apparent K(m) values for gamma,gamma-dimethylallyldiphosphate and 2',4'-dihydroxychalcone were 63 and 142 microM, respectively. The maximum activity of the enzyme was expressed during the first 10 days of cell growth. PMID:14741337

  3. Microfluidic devices for studying heterotypic cell-cell interactions and tissue specimen cultures under controlled microenvironments

    PubMed Central

    Zervantonakis, Ioannis K.; Kothapalli, Chandrasekhar R.; Chung, Seok; Sudo, Ryo; Kamm, Roger D.

    2011-01-01

    Microfluidic devices allow for precise control of the cellular and noncellular microenvironment at physiologically relevant length- and time-scales. These devices have been shown to mimic the complex in vivo microenvironment better than conventional in vitro assays, and allow real-time monitoring of homotypic or heterotypic cellular interactions. Microfluidic culture platforms enable new assay designs for culturing multiple different cell populations and?or tissue specimens under controlled user-defined conditions. Applications include fundamental studies of cell population behaviors, high-throughput drug screening, and tissue engineering. In this review, we summarize recent developments in this field along with studies of heterotypic cell-cell interactions and tissue specimen culture in microfluidic devices from our own laboratory. PMID:21522496

  4. Double-layer agar cell cultures as a method of studying cell growth factors

    SciTech Connect

    Cherepantseva, E.A.; Shevlyagin, V.Ya.; Al'tshtein, A.D.

    1986-12-01

    This paper describes the development of a method of double-layer agar cell cultures which can be conveniently used to study the ability of some donor cells to produce tumor growth factors for other recipient or test cells. The donor and recipient cells are placed in different layers of agar, separated by an intermediate layer. Under a microscope, the two cell layers can be distinguished and assessed in relation to colony formation. Hamster cells were used in the experiments. The role of proliferation in manifestation of the donor properties of the cells was assessed, using donor cells seeded in different amounts of agar and also cells irradiated with gamma-rays and which had lost their ability to divide. The cells were compared and results are presented.

  5. Bovine oviductal epithelial cells: their cell culture and applications in studies for reproductive biology

    Microsoft Academic Search

    Hiroyuki Abe; Hiroyoshi Hoshi

    1997-01-01

    Epithelial cells of the mammalian oviduct play an important role in reproductive and developmental events that occur there. Oviductal epithelial cells from several mammalian species can be isolated and cultured in serum or serum-free medium in vitro and cell culture of bovine oviductal epithelial cells (BOEC) has been described by many investigators. Cultured BOEC show a wide variety of secretory

  6. Proteomic Analysis of Grape Berry Cell Cultures Reveals that Developmentally Regulated Ripening Related Processes Can Be Studied Using Cultured Cells

    PubMed Central

    Sharathchandra, Ramaschandra G.; Stander, Charmaine; Jacobson, Dan; Ndimba, Bongani; Vivier, Melané A.

    2011-01-01

    Background This work describes a proteomics profiling method, optimized and applied to berry cell suspensions to evaluate organ-specific cultures as a platform to study grape berry ripening. Variations in berry ripening within a cluster(s) on a vine and in a vineyard are a major impediment towards complete understanding of the functional processes that control ripening, specifically when a characterized and homogenous sample is required. Berry cell suspensions could overcome some of these problems, but their suitability as a model system for berry development and ripening needs to be established first. Methodology/Principal Findings In this study we report on the proteomic evaluation of the cytosolic proteins obtained from synchronized cell suspension cultures that were established from callus lines originating from green, véraison and ripe Vitis vinifera berry explants. The proteins were separated using liquid phase IEF in a Microrotofor cell and SDS PAGE. This method proved superior to gel-based 2DE. Principal component analysis confirmed that biological and technical repeats grouped tightly and importantly, showed that the proteomes of berry cultures originating from the different growth/ripening stages were distinct. A total of twenty six common bands were selected after band matching between different growth stages and twenty two of these bands were positively identified. Thirty two % of the identified proteins are currently annotated as hypothetical. The differential expression profile of the identified proteins, when compared with published literature on grape berry ripening, suggested common trends in terms of relative abundance in the different developmental stages between real berries and cell suspensions. Conclusions The advantages of having suspension cultures that accurately mimic specific developmental stages are profound and could significantly contribute to the study of the intricate regulatory and signaling networks responsible for berry development and ripening. PMID:21379583

  7. Studies on tissue cultures of the genus Cinchona L. alkaloid production in cell suspension cultures

    Microsoft Academic Search

    H. Koblitz; D. Koblitz; H. P. Schmauder; D. Gröger

    1983-01-01

    Initiation and culture of callus and cell suspensions of Cinchona ledgeriana and C. succirubra as well as the successful isolation and selection of a high-yielding alkaloid-forming strain derived from the leaf rachis of a C. succirubra plant are described. Results of feeding experiments with L-tryptophan using two different culture procedures are presented and discussed. Maximum alkaloid yields of up to

  8. Biosynthesis of cellulose: studies with tobacco protoplasts and cultured cells

    SciTech Connect

    Franz, G.; Blaschek, W.; Haass, D.; Koehler, H.

    1983-01-01

    The cell wall of regenerating tobacco protoplasts was shown to be mainly composed of noncellulosic ..beta..-1,3- and ..beta..-1,4-linked glucans with a cellulose content of only about 5%. Some pectic and hemicellulosic material is released by these protoplasts into the culture medium. The DP distribution of the ..cap alpha..-cellulose in regenerating protoplasts as well as in suspension-cultured cells, callus, or tobacco mesophyll revealed the existence of mainly two DP fractions with low (DP<500) and higher (DP 2000-3000) molecular weight, both of which contribute to the cellulosic network of the primary cell wall. The alkali-soluble and alkali-insoluble products of glucan synthetase assays with particulate enzyme fractions were analyzed in detail. By prelabeling with (/sup 14/C)glucose, the existence of primer glucans, which are elongated in the appropriate in vitro assay, could be substantiated. Alkali-soluble glucans consisted of a very short, if any, primer glucan, to which about 40 glucose units were added in vitro. The glucans in the alkali-insoluble fraction have an average DP of 200-250 and are synthesized in vitro by chain elongation via addition of about 30 new glucose units to a 1,4-linked primer glucan of DPapprox.200. 27 references, 6 figures, 2 tables.

  9. Molluscan cells in culture: primary cell cultures and cell lines.

    PubMed

    Yoshino, T P; Bickham, U; Bayne, C J

    2013-06-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

  10. Keratin Classes as Molecular Markers Epithelia: Cell Culture Studies

    Microsoft Academic Search

    WILLIAM G. NELSON; TUNG-TIEN SUN

    1983-01-01

    The keratins are a highly heterogeneous group of proteins that form intermediate filaments in a wide variety of epithelial cells. These proteins can be divided into at least seven major classes according to their molecular weight and their immunological reactivity with monoclonal antibodies. Tissue-distribution studies have revealed a correlation between the expression of specific keratin classes and different morphological features

  11. Isolation and primary culture of rat cerebral microvascular endothelial cells for studying drug transport in vitro

    Microsoft Academic Search

    Nobuhiro Ichikawa; Kohji Naora; Hidenari Hirano; Michio Hashimoto; Sumio Masumura; Kikuo Iwamoto

    1996-01-01

    To establish the cerebral microvascular endothelial cell (CMEC) culture system for animals commonly utilized in in vivo studies, we developed a method for isolation and culture of rat CMECs, and the model system was used in preliminary in vitro transport experiments. The isolated rat brains were minced. After an incubation with dispase, a fraction of microvessels was obtained by the

  12. Fatty acid metabolism studies of human epidermal cell cultures

    Microsoft Academic Search

    Cynthia L. Marcelo; William R. Dunham

    Adult human epidermal keratinocytes grow rapidly in medium that is essential fatty acid (EFA)-deficient. In this medium they exhibit decreased amounts of the fatty acids, 18:2, 20:3, 20:4, and contain increased amounts of monounsaturated fatty acids. (I%)- and (SHIacetate and radiolabeled fatty acids, 16:0, 18:2, and 20:4 were used to study the fatty acid metabolism of these cells. Label from

  13. Biotransformations of Antidiabetic Vanadium Prodrugs in Mammalian Cells and Cell Culture Media: A XANES Spectroscopic Study.

    PubMed

    Levina, Aviva; McLeod, Andrew I; Pulte, Anna; Aitken, Jade B; Lay, Peter A

    2015-07-20

    The antidiabetic activities of vanadium(V) and -(IV) prodrugs are determined by their ability to release active species upon interactions with components of biological media. The first X-ray absorption spectroscopic study of the reactivity of typical vanadium (V) antidiabetics, vanadate ([V(V)O4](3-), A) and a vanadium(IV) bis(maltolato) complex (B), with mammalian cell cultures has been performed using HepG2 (human hepatoma), A549 (human lung carcinoma), and 3T3-L1 (mouse adipocytes and preadipocytes) cell lines, as well as the corresponding cell culture media. X-ray absorption near-edge structure data were analyzed using empirical correlations with a library of model vanadium(V), -(IV), and -(III) complexes. Both A and B ([V] = 1.0 mM) gradually converged into similar mixtures of predominantly five- and six-coordinate V(V) species (?75% total V) in a cell culture medium within 24 h at 310 K. Speciation of V in intact HepG2 cells also changed with the incubation time (from ?20% to ?70% V(IV) of total V), but it was largely independent of the prodrug used (A or B) or of the predominant V oxidation state in the medium. Subcellular fractionation of A549 cells suggested that V(V) reduction to V(IV) occurred predominantly in the cytoplasm, while accumulation of V(V) in the nucleus was likely to have been facilitated by noncovalent bonding to histone proteins. The nuclear V(V) is likely to modulate the transcription process and to be ultimately related to cell death at high concentrations of V, which may be important in anticancer activities. Mature 3T3-L1 adipocytes (unlike for preadipocytes) showed a higher propensity to form V(IV) species, despite the prevalence of V(V) in the medium. The distinct V biochemistry in these cells is consistent with their crucial role in insulin-dependent glucose and fat metabolism and may also point to an endogenous role of V in adipocytes. PMID:25906315

  14. Studying the drift of in line pH measurements in cell culture.

    PubMed

    Saucedo, V; Wolk, B; Arroyo, A; Feng, C D

    2011-01-01

    The culture of Chinese hamster ovary (CHO) cells to produce monoclonal antibodies (MAb) requires accurate measurement and control of pH. Unwanted pH drifts in cell culture can adversely affect process performance, product quality, and product yield. To measure and control pH throughout the length of a culture, most cell culture processes use traditional glass pH probes. Several variables can affect the design and performance of glass pH electrodes and lead to drift in the measurement. Understanding these variables and their effects on pH performance can lead to design improvements and potentially reduce the drift. In this study, a set of Rosemount Analytical glass pH probes was investigated in cell culture operations. Electrochemical properties of the probes were monitored throughout the experiments. Experimental results show that the glass membrane potential experiences the biggest change during cell culture operations. Changes in the reference electrode potential are small compared with the changes in glass membrane potential. The glass membranes are affected by the steam sterilization process and this is the main cause for drift in the probe sensing signal during cell culture operations. Steam sterilization can cause the potential of glass membranes to change by up to 15 mV (? 0.25 pH units). This change in membrane potential can be observed as an undesirable pH drift in bioreactors. PMID:21567988

  15. Nanopillar sheets as a new type of cell culture dish: detailed study of HeLa cells cultured on nanopillar sheets

    Microsoft Academic Search

    Shinobu Nomura; Hiroko Kojima; Yoshimi Ohyabu; Kosuke Kuwabara; Akihiro Miyauchi; Toshimasa Uemura

    2006-01-01

    Nanopillar sheets were fabricated with high-aspect ratio structures with a diameter of 160–1000?nm and a height of 1??m by\\u000a nanoimprinting. The suitability of nanopillar sheets as a new type of cell culture dish was examined by studying the behavior\\u000a of HeLa cells cultured on the sheets using light microscopy, scanning electron microscopy, and fluorescence microscopy observing\\u000a actin and vinculin molecules.

  16. Study of pluripotency markers in zebrafish embryos and transient embryonic stem cell cultures.

    PubMed

    Robles, Vanesa; Martí, Mercé; Izpisua Belmonte, Juan Carlos

    2011-06-01

    Targeted genomic manipulation using embryonic stem (ES) cells has not yet been achieved in zebrafish, although methods for zebrafish ES cell culture has been described in literature. The knowledge of pluripotency markers in this species is almost nonexistent and this is a very limiting factor in the definition of the ideal culture conditions for ES cells. Here, we studied the expression of several genes associated with pluripotency in zebrafish embryonic cells versus differentiated cells and the expression of some of these genes is recorded throughout embryonic development. Some of the commonly accepted pluripotency markers are also tested in embryonic cells, transient embryonic cell cultures, and differentiated cells. Our results support the hypothesis that stage-specific embryonic antigen 1 (SSEA1) is a marker that precedes the expression of pluripotency genes in a zebrafish embryonic cell colony, in the same way that SOX2 precedes nestin expression in those colonies that have already started differentiation toward neurons. We consider this study a step forward in the knowledge of zebrafish pluripotency markers and, therefore, an important tool for the monitoring of zebrafish embryonic cell cultures. PMID:21563922

  17. Study of Pluripotency Markers in Zebrafish Embryos and Transient Embryonic Stem Cell Cultures

    PubMed Central

    Robles, Vanesa; Martí, Mercé

    2011-01-01

    Abstract Targeted genomic manipulation using embryonic stem (ES) cells has not yet been achieved in zebrafish, although methods for zebrafish ES cell culture has been described in literature. The knowledge of pluripotency markers in this species is almost nonexistent and this is a very limiting factor in the definition of the ideal culture conditions for ES cells. Here, we studied the expression of several genes associated with pluripotency in zebrafish embryonic cells versus differentiated cells and the expression of some of these genes is recorded throughout embryonic development. Some of the commonly accepted pluripotency markers are also tested in embryonic cells, transient embryonic cell cultures, and differentiated cells. Our results support the hypothesis that stage-specific embryonic antigen 1 (SSEA1) is a marker that precedes the expression of pluripotency genes in a zebrafish embryonic cell colony, in the same way that SOX2 precedes nestin expression in those colonies that have already started differentiation toward neurons. We consider this study a step forward in the knowledge of zebrafish pluripotency markers and, therefore, an important tool for the monitoring of zebrafish embryonic cell cultures. PMID:21563922

  18. ASBESTOS AND GASTRO-INTESTINAL CANCER: CELL CULTURE STUDIES

    EPA Science Inventory

    Three forms of asbestos: amosite, crocidolite, and chrysotile, were assayed for their cytotoxicity and mutagenicity in cell clture. Using embjryonic human intestine derived and adult rat liver derived epitelial cells, the order of toxicity was chrysotile > amosite = crocidolite. ...

  19. Mechanically and Chemically Tunable Cell Culture System for Studying the Myofibroblast Phenotype

    PubMed Central

    2015-01-01

    Cell culture systems for studying the combined effects of matrix proteins and mechanical forces on the behavior of soft tissue cells have not been well developed. Here, we describe a new biomimetic cell culture system that allows for the study of mixtures of matrix proteins while controlling mechanical stiffness in a range that is physiological for soft tissues. This system consists of layer-by-layer (LbL)-assembled films of native matrix proteins atop mechanically tunable soft supports. We used hepatic stellate cells, which differentiate to myofibroblasts in liver fibrosis, for proof-of-concept studies. By culturing cells on collagen and lumican LbL-modified hydrogels, we demonstrate that this system is noncytotoxic and offers a valid control substrate, that the hydrogel determines the overall system mechanics, and that the addition of lumican to collagen influences the stellate cell phenotype. LbL-modified hydrogels offer the potential to study the influence of complex environmental factors on soft-tissue cells in culture. PMID:24787894

  20. Biocompatibility studies on fibrin glue cultured with bone marrow mesenchymal stem cells in vitro.

    PubMed

    Fang, Huang; Peng, Songlin; Chen, Anmin; Li, Fengfeng; Ren, Kai; Hu, Ning

    2004-01-01

    By culturing bone marrow mesenchymal stem cells of rabbits with fibrin glue in vitro, the biocompatibility of fibrin glue was investigated to study whether this material can be used as scaffolds in bone tissue engineering. After 2-months old New Zealand rabbits had been anesthetized, about 4-6 ml of bone marrow were aspirated from rabbit femoral trochanter. The monocytes suspension was aspirated after bone marrow was centrifuged with lymphocyte separating medium and cultured primarily. Then the cells were divided into two groups: one was cultured with complete medium and the other with induced medium. The cells of the two groups were collected and inoculated to the culture plate containing fibrin glue. In the control group, cells were inoculated without fibrin glue. The implanted cells and materials were observed at different stages under a phase-contrast microscope and scanning electron microscope. MTT and alkaline phosphatase (ALP) were measured. Bone marrow mesenchymal stem cells grew on the surface of fibrin glue and adhered to it gradually. Cells light absorption value (A value) and the ALP content showed no significant difference. Fibrin glue had no inhibitory effect on cell morphology, growth, proliferation and differentiation. It has good biocompatibility and can be used as scaffold materials for bone marrow mesenchymal stem cells in bone tissue engineering. PMID:15315346

  1. Functional Culture Models to Study Mechanisms Governing Apoptosis in Normal and Malignant Mammary Epithelial Cells

    PubMed Central

    Weaver, V. M.; Bissell, M. J.

    2010-01-01

    Mammary tissue homeostasis depends upon dynamic interactions between the epithelial cells, their microenvironment (including the basement membrane and the stroma), and the tissue architecture, which influence each other reciprocally to regulate growth, death and differentiation in the gland. To study how apoptosis is regulated in normal mammary cells, and to understand its role in breast tumor pathogenesis, we need model systems that recapitulate breast tissue architecture and microenvironment in culture. We have established culture models of primary and established nonmalignant mammary cell lines from both rodent and human, and defined procedures to study how cell and tissue architecture affect signaling by the basement membrane. We show that both a basement membrane and an organized tissue structure are required to achieve sustained mammary cell survival. These models could now be used to investigate how the basement membrane represses apoptosis in normal cells, and how breast cancers become death-resistant. PMID:10426398

  2. Organ Explant Culture of Neonatal Rat Ventricles: A New Model to Study Gene and Cell Therapy

    PubMed Central

    den Haan, A. Dénise; Veldkamp, Marieke W.; Bakker, Diane; Boink, Geert J. J.; Janssen, Rob B.; de Bakker, Jacques M. T.; Tan, Hanno L.

    2013-01-01

    Testing cardiac gene and cell therapies in vitro requires a tissue substrate that survives for several days in culture while maintaining its physiological properties. The purpose of this study was to test whether culture of intact cardiac tissue of neonatal rat ventricles (organ explant culture) may be used as a model to study gene and cell therapy. We compared (immuno) histology and electrophysiology of organ explant cultures to both freshly isolated neonatal rat ventricular tissue and monolayers. (Immuno) histologic studies showed that organ explant cultures retained their fiber orientation, and that expression patterns of ?-actinin, connexin-43, and ?-smooth muscle actin did not change during culture. Intracellular voltage recordings showed that spontaneous beating was rare in organ explant cultures (20%) and freshly isolated tissue (17%), but common (82%) in monolayers. Accordingly, resting membrane potential was -83.9±4.4 mV in organ explant cultures, ?80.5±3.5 mV in freshly isolated tissue, and ?60.9±4.3 mV in monolayers. Conduction velocity, measured by optical mapping, was 18.2±1.0 cm/s in organ explant cultures, 18.0±1.2 cm/s in freshly isolated tissue, and 24.3±0.7 cm/s in monolayers. We found no differences in action potential duration (APD) between organ explant cultures and freshly isolated tissue, while APD of monolayers was prolonged (APD at 70% repolarization 88.8±7.8, 79.1±2.9, and 134.0±4.5 ms, respectively). Organ explant cultures and freshly isolated tissue could be paced up to frequencies within the normal range for neonatal rat (CL 150 ms), while monolayers could not. Successful lentiviral (LV) transduction was shown via Egfp gene transfer. Co-culture of organ explant cultures with spontaneously beating cardiomyocytes increased the occurrence of spontaneous beating activity of organ explant cultures to 86%. We conclude that organ explant cultures of neonatal rat ventricle are structurally and electrophysiologically similar to freshly isolated tissue and a suitable new model to study the effects of gene and cell therapy. PMID:23516623

  3. A differentiated porcine bronchial epithelial cell culture model for studying human adenovirus tropism and virulence

    Microsoft Academic Search

    E. Lam; M. Ramke; S. Groos; G. Warnecke; A. Heim

    2011-01-01

    The species specificity of human adenoviruses (HAdV) almost precludes studying virulence and tropism in animal models, e.g. rodent models, or derived tissue and cell culture models. However, replication of HAdV type 5 (HAdV-C5) has been shown after intravenous injection in swine. In order to study adenovirus replication in airway tissue propagation of bronchial epithelial cells from porcine lungs was established.

  4. Primary culture of trigeminal satellite glial cells: a cell-based platform to study morphology and function of peripheral glia.

    PubMed

    Poulsen, Jeppe N; Larsen, Frederik; Duroux, Meg; Gazerani, Parisa

    2014-01-01

    Primary cell culture provides an experimental platform in which morphology, physiology, and cell-cell communication pathways can be studied under a well-controlled environment. Primary cell cultures of peripheral and central glia offer unique possibilities to clarify responses and pathways to different stimuli. Peripheral glia, satellite glial cells (SGCs), which surround neuronal cell bodies within sensory ganglia, have recently been known as key players in inflammation and neuronal sensitization. The objectives of this study were 1) to establish a cell-based platform of cultured trigeminal SGCs to study glial marker expression and functions under control conditions; 2) to validate the cell-based platform by prostaglandin E2 (PGE2) release response following administration of Cisplatin; and 3) to investigate inhibition of PGE2 release by glial modulators, Ibudilast and SKF86002. Primary cell cultures of SGCs from rat trigeminal ganglia were established following enzymatically and mechanically dissociation of the ganglia. Cultures were characterized in vitro for up to 21 days post isolation for morphological and immunocytochemical characteristics. PGE2 release, determined by ELISA, was used as a pro-inflammatory marker to characterize SGCs response to chemotherapeutic agent, Cisplatin, known to contribute in chemotherapy-induced peripheral neuropathy. Our results indicate that 1) isolated SGCs maintained their characteristics in vitro for up to 21 days; 2) Cisplatin enhanced PGE2 release from the SGCs, which was attenuated by Ibudilast and SKF86002. These findings confirm the utility and validity of the cultured trigeminal SGCs platform for glial activation and modulation; and suggest further investigation on Ibudilast and SKF86002 in prevention of chemotherapy-induced pain. PMID:24665354

  5. Ureaplasma infection of cell cultures.

    PubMed Central

    Kotani, H; McGarrity, G J

    1986-01-01

    Studies were performed to characterize the effects of ureaplasmas in HeLa, 3T6, and CV-1 cell cultures. The ureaplasmas studied were human Ureaplasma urealyticum T960 (serotype VIII), bovine U. diversum T95, simian strain T167-2, ovine strain 1202, canine strain D1M-C, and feline strains 382 and FT2-B. FT2-B was the only ureaplasma to grow in the cell free culture medium, Dulbecco modified Eagle-Earle medium containing 10% fetal bovine serum. The growth pattern of the ureaplasmas varied in the different cell cultures, but each strain grew in at least two of the cell cultures, suggesting a requirement for a product of the cell culture and for low concentrations of urea. When growth occurred, organisms grew to concentrations that approached, but did not equal, those observed in 10B broth. Most, but not all, ureaplasmas grew quickly, reaching peak titers 2 days after infection. Canine strain D1M-C did not grow in 3T6, but showed rapid growth in HeLa and CV-1 cells, killing both cultures, In some systems, e.g., U. urealyticum T960 and simian strain T167-2, the infection persisted, and ureaplasmas could be recovered from cell cultures four passages after infection, when studies were terminated. The cell culture ureaplasmas grew on T agar, but not on mycoplasma agar medium. Images PMID:3699891

  6. Studies on the differentiation pathway and growth characteristics of epithelial culture cells of the human prostate.

    PubMed

    Planz, B; Tabatabaei, S; Kirley, S D; Aretz, H T; Wang, QiFa; Lin, C-W; McDougal, W S; Marberger, M

    2004-01-01

    We established explant primary cultures in order to study the growth and hormone responsiveness, and the differentiation process of prostatic epithelial cells. Cell outgrowth was achieved from explant tissue by using a new DU145-cell-conditioned medium and special plastic coverslips. To define the present model, proliferation assays were tested by [3H]thymidine assay and planimetric analysis. Cells were analyzed using immunocytochemistry, light, phase contrast and electron microscopy, polymerase chain reaction, telomerase ELISA and immunoassay (PSA). Morphology and electron microscopy revealed typical epithelial differentiation. Immunocytochemistry showed the content of basal and secretory epithelial cells, endocrine paracrine cells and a high level of proliferation. With increasing culture time, mature epithelial differentiation (PSA) increases and the initial increase of alpha-smooth muscle actin (alpha-SMA) decreases again. After further passaging, alpha-SMA expression is no longer detected and PSA expression decreases. Furthermore, epithelial cells showed both androgen responsiveness and androgen receptor expression. These findings show the presence of epithelial cells in a process of differentiation with endocrine paracrine cells and a high level of proliferation. This model may maintain the cellular and functional properties more closely related to the human prostate and may provide a valuable tool for studying stem cells and differentiation characteristics. PMID:14999242

  7. A mechanistic study on the effect of dexamethasone in moderating cell death in Chinese Hamster Ovary cell cultures.

    PubMed

    Jing, Ying; Qian, Yueming; Ghandi, Mahmoud; He, Aiqing; Borys, Michael C; Pan, Shih-Hsie; Li, Zheng Jian

    2012-01-01

    Dexamethasone (DEX) was previously shown (Jing et al., Biotechnol Bioeng. 2010;107:488-496) to play a dual role in increasing sialylation of recombinant glycoproteins produced by Chinese Hamster Ovary (CHO) cells. DEX addition increased sialic acid levels of a recombinant fusion protein through increased expression of ?2,3-sialyltransferase and ?1,4-galactosyltransferase, but also decreased the sialidase-mediated, extracellular degradation of sialic acid through slowing cell death at the end of the culture period. This study examines the underlying mechanism for this cytoprotective action by studying the transcriptional response of the CHO cell genome upon DEX treatment using DNA microarrays and gene ontology term analysis. Many of those genes showing a significant transcriptional response were associated with the regulation of programmed cell death. The gene with the highest change in expression level, as validated by Quantitative PCR assays with TaqMan® probes and confirmed by Western Blot analysis, was the antiapoptotic gene Tsc22d3, also referred to as GILZ (glucocorticoid-induced leucine zipper). The pathway by which DEX suppressed cell death towards the end of the culture period was also confirmed by showing involvement of glucocorticoid receptors and GILZ through studies using the glucocorticoid antagonist mifepristone (RU-486). These findings advance the understanding of the mechanism by which DEX suppresses cell death in CHO cells and provide a rationale for the application of glucocorticoids in CHO cell culture processes. PMID:22140034

  8. [Mammalian cell culture as a model for studying the intracellular traffic of densovirus proteins].

    PubMed

    Kozlov, E N; Mukha, D V

    2015-02-01

    The intracellular localization of the fusion protein composed of green fluorescent protein (GFP) and one of the capsid proteins (namely VP1) of the German cockroach densovirus BgDV1 was investigated using the HeLa human cell culture. The intracellular localization of GFP was analyzed in a series of control experiments. Histochemical analysis with GFP antibodies showed that the fusion protein is localized exclusively inside the nucleus of cells because of the transitory expression of the corresponding vector constructions, whereas the GFP is located both in the nucleus and the cytoplasm. We can conclude that the signal of the nuclear localization of the capsid protein of the German cockroach densovirus is functionally active, not only within the cells of this insect but within the human cell culture as well. This observation extends the experimental possibilities for studying the genetic control of intracellular traffic of densovirus proteins. PMID:25966595

  9. Multigenerational Study of Chemically Induced Cytotoxicity and Proliferation in Cultures of Human Proximal Tubular Cells

    PubMed Central

    Lash, Lawrence H.; Putt, David A.; Benipal, Bavneet

    2014-01-01

    Primary cultures of human proximal tubular (hPT) cells are a useful experimental model to study transport, metabolism, cytotoxicity, and effects on gene expression of a diverse array of drugs and environmental chemicals because they are derived directly from the in vivo human kidney. To extend the model to investigate longer-term processes, primary cultures (P0) were passaged for up to four generations (P1–P4). hPT cells retained epithelial morphology and stained positively for cytokeratins through P4, although cell growth and proliferation successively slowed with each passage. Necrotic cell death due to the model oxidants tert-butyl hydroperoxide (tBH) and methyl vinyl ketone (MVK) increased with increasing passage number, whereas that due to the selective nephrotoxicant S-(1,2-dichlorovinyl)-l-cysteine (DCVC) was modest and did not change with passage number. Mitochondrial activity was lower in P2–P4 cells than in either P0 or P1 cells. P1 and P2 cells were most sensitive to DCVC-induced apoptosis. DCVC also increased cell proliferation most prominently in P1 and P2 cells. Modest differences with respect to passage number and response to DCVC exposure were observed in expression of three key proteins (Hsp27, GADD153, p53) involved in stress response. Hence, although there are some modest differences in function with passage, these results support the use of multiple generations of hPT cells as an experimental model. PMID:25411799

  10. May toxicity of amiodarone be prevented by antioxidants? A cell-culture study

    PubMed Central

    2012-01-01

    Background Atrial Fibrillation is the most common arrhythmia encountered following cardiac surgery. The most commonly administered drug used in treatment and prophylaxis is amiodarone which has several toxic effects on major organ functions. There are few clinical data concerning prevention of toxic effects and there is no routinely suggested agent. The aim of this study is to document the cytotoxic effects of amiodarone on cell culture media and compare the cytoprotective effects of commonly used antioxidant agents. Methods L929 mouse fibroblast cell line was cultured and 100,000 cells/well-plate were obtained. First group of cells were treated with increasing concentrations of amiodarone (20 to 180 ?M) alone. Second and third group of cells were incubated with one-fold equimolar dose of vitamin C and N-acetyl cysteine prior to amiodarone exposure. The viability of cells were measured by MTT assay and the cytoprotective effect of each agent was compared. Results The cytotoxicity of amiodarone was significant with concentrations of 100 ?M and more. The viabilities of both vitamin C and N-acetyl cysteine treated cells were higher compared to untreated cells. Conclusions Vitamin C and N-acetyl cysteine are commonly used in the clinical setting for different purposes in context of their known antioxidant actions. Their role in prevention of amiodarone induced cytotoxicity is not fully documented. The study fully demonstrates the cytoprotective role of both agents in amiodarone induced cytotoxicity on cell culture media; more pronounced with vitamin C in some concentrations. The findings may be projectile for further clinical studies. PMID:22741616

  11. Mammalian Cell Culture Simplified.

    ERIC Educational Resources Information Center

    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)

  12. A study of enzymatic activity in cell cultures via the analysis of volatile biomarkers.

    PubMed

    Chippendale, Thomas W E; Hu, Bin; El Haj, Alicia J; Smith, David

    2012-10-21

    Aldehyde dehydrogenase (ALDH) enzymes are responsible for the metabolism of aldehydes, including acetaldehyde (AA), and are linked to disease. We describe a method to study ALDH activity in cell cultures involving the measurement of AA concentrations in the gas/vapour phase. This has been achieved using selected ion flow tube mass spectrometry (SIFT-MS), developed for the rapid quantification of trace gases in humid media. Human cells of the hepG2 hepatocellular carcinoma cell line and primary bone marrow-derived mesenchymal stem cells (hMSCs) depleted AA from the culture media, but the application of ALDH inhibitors diethylaminobenzaldehyde (DEAB) and disulfiram (DSF), suppressed this depletion or in some cases resulted in elevated AA concentrations. Further, the cells were shown to reduce the dimethyl sulphoxide (DMSO) to dimethyl sulphide, which is mediated by methionine sulfoxide reductase A (MsrA) enzymes. Interestingly, this process was also inhibited by DEAB and DSF. The results of this study indicate that SIFT-MS gas phase analysis could be applied to the study of volatile metabolites of intracellular enzyme reactions, this having potential utility in disease research and drug discovery. PMID:22930361

  13. A microgroove patterned multiwell cell culture plate for high-throughput studies of cell alignment.

    PubMed

    Lücker, Petra B; Javaherian, Sahar; Soleas, John P; Halverson, Duncan; Zandstra, Peter W; McGuigan, Alison P

    2014-12-01

    Grooved substrates are commonly used to guide cell alignment and produce in vitro tissues that mimic certain aspects of in vivo cellular organization. These more sophisticated tissues provide valuable in vitro models for testing drugs and for dissecting out molecular mechanisms that direct tissue organization. To increase the accessibility of these tissue models we describe a simple and yet reproducible strategy to produce 1?µm-spaced grooved well plates suitable for conducting automated analysis of cellular responses. We characterize the alignment of four human cell types: retinal epithelial cells, umbilical vein endothelial cells, foreskin fibroblasts, and human pluripotent stem-cell-derived cardiac cells on grooves. We find all cells align along the grooves to differing extents at both sparse and confluent densities. To increase the sophistication of in vitro tissue organization possible, we also created hybrid substrates with controlled patterns of microgrooved and flat regions that can be identified in real-time using optical microscopy. Using our hybrid patterned surfaces we explore: (i) the ability of neighboring cells to provide a template to organize surrounding cells that are not directly exposed to grooved topographic cues, and (ii) the distance over which this template effect can operate in confluent cell sheets. We find that in fibroblast sheets, but not epithelial sheets, cells aligned on grooves can direct alignment of neighboring cells in flat regions over a limited distance of approximately 200??m. Our hybrid surface plate provides a novel tool for studying the collective response of groups of cells exposed to differential topographical cues. PMID:24889796

  14. Semliki Forest Virus Replication in Cultured Aedes albopictus Cells: studies on the Establishment of Persistence

    Microsoft Academic Search

    MARY W. DAVEY; L. DALGARNOt

    1974-01-01

    SUMMARY Semliki Forest virus (SFV) established a persistent, non-cytopathic infection in cultured Aedes albopictus cells with no effect on cumulative cell number as com- pared with control cultures. All cells were initially infected by SFV as judged by infective centre and immunofluorescence assay and released approx. 5o to 7o p.f.u.\\/cell in the initial a4 h after infection. At 12 h

  15. Development and characterization of a primary culture of chicken embryonic tracheal epithelial cells and their use in avian studies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A major route of infection of avian influenza is through cells of the airway epithelium. To study the molecular mechanism of infection and early host responses we created a primary chicken tracheal cell culture. Epithelial cells were isolated from the trachea of 18 day old chicken embryos and cult...

  16. A study on sonoporation of cells cultured on a soft collagen gel scaffold

    NASA Astrophysics Data System (ADS)

    Kudo, Nobuki; Kinoshita, Yuto

    2012-11-01

    Efficiencies of sonoporation were investigated using four types of monolayer-cell samples: cells cultured directly on a cover slip, cells cultured on a cover slip coated with collagen gel of several ?m in thickness, and cells cultured on collagen gel scaffolds of 0.4 and 1.0 mm in thicknesses. Cell samples attached with Levovist microbubbles were irradiated by one shot of a three- or 10,000-cycle ultrasound pulse, and cell detachment and membrane perforation were investigated. Experimental results showed that rates of cell detachment and membrane damage were markedly decreased in the presence of soft gel layer of 0.4 and 1.0 mm in thicknesses under the cells and that these rates were inversely proportional to the thickness of the gel layer. These results indicate that optimum conditions of sonoporation in vitro should be carefully translated into those in vivo.

  17. Cell Culturing of Cytoskeleton

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc., has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc., is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

  18. Cell Culturing of Cytoskeleton

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc. has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc. is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

  19. Studies on the renin-angiotensin system in primary monolayer cell cultures of the rat epididymis.

    PubMed

    Wong, P Y; Uchendu, C N

    1991-11-01

    Monolayer cell cultures formed from the rat cauda epididymidis exhibited renin-like and angiotensin-converting enzyme (ACE) activities and contained immunoreactive angiotensin I (AI) and angiotensin II (AII). Renin-like activity, determined indirectly by radioimmunoassay of generated AI at a near-neutral pH of 6.0, was demonstrated in the cell lysate but was almost undetectable in the serum-free cell culture medium, suggesting that renin expression in epididymal cells is an intracellular phenomenon. In contrast, both AI and AII were detected in the cell lysate and cell culture medium. The level of AI was enhanced by pretreating the cells with the ACE inhibitor captopril (100 nmol/l). Incubating the cell monolayers with thoroughly washed sperm cells obtained from the intact cauda epididymides of rats increase (P less than 0.01) the AII content of the cell culture medium, with a parallel decline (P less than 0.01) in the AI concentration. However, adrenaline (0.23 mumol/l), which was found to stimulate electrogenic anion secretion by cell monolayers grown on previous supports, was without effect on the renin-like activity or concentration of angiotensins. The ACE activity in cells was confirmed by its strong dependence on chloride ion and its susceptibility to inhibition by captopril (100 nmol/l). Enzyme activity was significantly (P less than 0.005) higher in the culture medium than in the cell lysate and cell membrane fragments. Angiotensinogen, which is obligatory for an intrinsic renin-angiotensin system, is present in epididymal cells. Presumably, it is synthesized and processed in the cell cytosol by intracellular renin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1660520

  20. Radiation oncogenesis in cell culture

    SciTech Connect

    Borek, C.

    1982-01-01

    This review article examines the oncogenic effects of radiation with emphasis on ionizing radiations. Cell transformation in vitro is examined with respect to culture systems currently used in these studies, initiation and phenotypic expression of transformation and criteria for transformation. The section of radiation oncogenesis in vitro includes ionizing and nonionizing radiation studies and cocarcinogens and modulators of radiogenic transformations.

  1. Comparative studies on sorting cells from Artemia sinica at different developmental stages for in vitro cell culture.

    PubMed

    Jiang, Guojian; Xu, Xiaohui; Jing, Yi; Wang, Ruixin; Fan, Tingjun

    2011-06-01

    Cell growth in primary cell culture of the brine shrimp (Artemia sinica) embryo at 12 and 20 h after rehydration at 25°C was examined comparatively in modified Leibovitz-15 medium. The cells from A. sinica embryo at 12 h after rehydration were dispersed, and the cells disseminated but did not attach to the surface of wells and multiply at 2 d of culture, and 12 d later, the cells were degenerated and dead. The best growth of the brine shrimp cells was obtained from the prenauplii of A. sinica at 20 h after dormant embryo rehydration. The fibroblast-like cells attached to the well surface and multiplied at 15 d after the primary culture was set up. Confluent monolayer was formed at 50 d. The prenauplii cells have been subcultured up to passage 3 and maintained for approximately 200 d. The reasons for cell growth potential at the different developmental stages of Artemia embryo were discussed. PMID:21512890

  2. Studies on Culture and Osteogenic Induction of Human Mesenchymal Stem Cells under CO2-Independent Conditions

    PubMed Central

    Chen, Jian; Zhang, Cui; Feng, Yiding; Zong, Chen; Chen, Jiarong; Tang, Zihua; Jia, Bingbing; Tong, Xiangming; Zheng, Qiang

    2013-01-01

    Abstract Human mesenchymal stem cells (hMSCs) are one of the important factors that regulate bone anabolism. Osteoporosis resulting from microgravity during spaceflight may possibly be due to a decrease in osteogenesis mediated by hMSCs. This speculation should be verified through culture and osteogenic induction of hMSCs in a microgravity environment during spaceflight. Control of CO2 is a key component in current experimental protocols for growth, survival, and proliferation of in vitro cultured cells. However, carrying CO2 tanks on a spaceflight and devoting space/mass allowances for classical CO2 control protocols make experimentation on culture and osteogenesis difficult during most missions. Therefore, an experimental culture and osteogenic medium was developed through modifying the components of buffer salts in conventional culture medium. This experimental medium was used to culture and induce hMSCs under CO2-independent conditions. The results showed that culture and induction of hMSCs with conventional culture medium and conventional osteogenic medium under CO2-independent conditions resulted in an increase of pH in medium. The proliferation of hMSCs was also inhibited. hMSCs cultured with experimental culture medium under CO2-independent conditions showed a proliferation potential that was the same as those cultured with conventional culture medium under CO2-dependent conditions. The experimental osteogenic medium could promote hMSCs to differentiate into osteoblast-like cells under CO2-independent conditions. Cells induced by this induction system showed high alkaline phosphatase activity. The expression levels of osteogenic genes in cells induced with experimental osteogenic medium under CO2-independent conditions were not significantly different from those cells induced with conventional osteogenic medium under CO2-dependent conditions. These results suggest that the experimental culture and induction system could be used to culture hMSCs and induce the osteogenesis of hMSCs in the atmospheric conditions common to spaceflights without additional CO2. Key Words: hMSCs—CO2-independent culture—Osteogenic differentiation—Proliferation. Astrobiology 13, 370–379. PMID:23577816

  3. A microfluidic system for automatic cell culture

    NASA Astrophysics Data System (ADS)

    Huang, Chun-Wei; Lee, Gwo-Bin

    2007-07-01

    This study presents a new chip capable of automating the cell culture process by using microfluidic technology. This microfluidic cell culture system comprising microheaters, a micro temperature sensor, micropumps, microvalves, microchannels, a cell culture area and several reservoirs was fabricated by using micro-electro-mechanical-systems' fabrication processes. Traditional manual cell culture processes can be performed on this chip. A uni-directional pneumatic micropump was developed to transport the culture reagents and constraint the solutions to flow only in one direction, safeguarding the entire culture process from contamination. A new micro check valve was also used to prevent the culture solutions from flowing back into the microchannels. The microheaters and the micro temperature sensor were used to maintain a constant temperature during the cell culturing process. The pH value suitable for cell growth was also regulated during the cell culture process. A typical cell culturing process for human lung cancer cells (A549) was successfully performed to demonstrate the capability of the developed microfluidic system. This automatic cell culturing system can be eventually integrated with subsequent microfluidic modules for cell purification, collection, counting and lysis to form a cell-based micro-total-analysis system. Preliminary results have been presented in The Asia-Pacific Conference of Transducers and Micro-Nano Technology (APCOT), 25-28 June 2006

  4. Microfluidic-driven viral infection on cell cultures: Theoretical and experimental study.

    PubMed

    Cimetta, Elisa; Franzoso, Mauro; Trevisan, Marta; Serena, Elena; Zambon, Alessandro; Giulitti, Stefano; Barzon, Luisa; Elvassore, Nicola

    2012-06-01

    Advanced cell culture systems creating a controlled and predictable microenvironment together with computational modeling may be useful tools to optimize the efficiency of cell infections. In this paper, we will present a phenomenological study of a virus-host infection system, and the development of a multilayered microfluidic platform used to accurately tune the virus delivery from a diffusive-limited regime to a convective-dominated regime. Mathematical models predicted the convective-diffusive regimes developed within the system itself and determined the dominating mass transport phenomena. Adenoviral vectors carrying the enhanced green fluorescent protein (EGFP) transgene were used at different multiplicities of infection (MOI) to infect multiple cell types, both in standard static and in perfused conditions. Our results validate the mathematical models and demonstrate how the infection processes through perfusion via microfluidic platform led to an enhancement of adenoviral infection efficiency even at low MOIs. This was particularly evident at the longer time points, since the establishment of steady-state condition guaranteed a constant viral concentration close to cells, thus strengthening the efficiency of infection. Finally, we introduced the concept of effective MOI, a more appropriate variable for microfluidic infections that considers the number of adenoviruses in solution per cell at a certain time. PMID:23734169

  5. Electrical Impedance Study of Cultured Endothelial Cells Under Fluid Shear Stress

    NASA Astrophysics Data System (ADS)

    Dong, Chunzhi; Depaola, Natacha; Keese, Charles R.; Giaever, Ivar

    2004-03-01

    The lumen of blood vessels is lined with a monolayer of endothelial cells (EC). In this work, electric cell-substrate impedance sensing (ECIS) was used to monitor the effect of fluid shear stress (FSS) on the morphology and function of cultured EC layers. Confluent layers of bovine aortic endothelial cells (BAEC) were grown on small gold electrodes and exposed to different flow conditions, while the impedance of the system was monitored. When the cells are subjected to FSS, the impedance rapidly increases 5-10%, and if the FSS is removed after a few minutes duration, the impedance returns back to the initial level in about 10 minutes. If the FSS remains for a long duration, the impedance will decrease 20-30% over a 10-15 hour period but ultimately returns to the original value, as the cells apparently accommodate to the FSS condition. These results suggest that ECIS may provide a sensitive means to study the response of EC to shear stress in vitro.

  6. Perfusion Based Cell Culture Chips

    Microsoft Academic Search

    A. Heiskanen; J. Emnéus; M. Dufva

    2010-01-01

    \\u000a 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\\u000a composition, long term unattended cultures and tissue like structuring of the cultures. However, as this chapter illustrates,\\u000a many issues remain to be identified regarding perfusion cell culture

  7. Studies on the effects of microgravity on the ultrastructure and functions of cultured mammalian cells (L-6)

    NASA Technical Reports Server (NTRS)

    Sato, Atsushige

    1993-01-01

    The human body consists of 10(exp 13) cells. Understanding the mechanisms by which the cells sense and respond to microgravity is very important as the basis for space biology. The cells were originally isolated aseptically from mammalian bodies and cultured in vitro. A set of cell culture vessels was developed to be applied to three kinds of space flight experiments. Experiment 1 is to practice the cell culture technique in a space laboratory and obtain favorable growth of the cells. Aseptic handling in tryspin treatment and medium renewal will be tested. The cells, following space flight, will be returned to the ground and cultured continuously to investigate the effects of space flight on the cellular characteristics. Experiment 2 is to examine the cytoskeletal structure of the cells under microgravity conditions. The cytoskeletal structure plays essential roles in the morphological construction, movements, axonal transport, and differentiation of the cells. The cells fixed during space flight will be returned and the cytoskeleton and ultrastructure observed using electron microscopy and fluorescence microscopy. Experiment 3 is to study the cellular productivity of valuable substances. The waste medium harvested during space flight are returned and quantitated for the cellular products. The effects of microgravity on mammalian cells will be clarified from the various aspects.

  8. Use of plant cell cultures to study graminicide effects on lipid metabolism.

    PubMed

    Price, Lindsey J; Herbert, Derek; Cole, David J; Harwood, John L

    2003-07-01

    Graminicides belonging to the cyclohexanedione and aryloxyphenoxypropionate classes are well established to act by disrupting acyl lipid biosynthesis via specific inhibition of acetyl-CoA carboxylase. Species of grass inherently resistant to such herbicides, or biotypes of grassy weed species which display acquired resistance to recommended rates of graminicide application, are known to possess an altered plastidic multifunctional acetyl-CoA carboxylase showing reduced sensitivity to these herbicides in vitro. Studies reported here demonstrate that cell suspension cultures of maize, a graminicide-sensitive species and Poa annua, a graminicide-insensitive species, display a similar differential sensitivity of acyl lipid biosynthesis as tissue from corresponding intact plants. Acyl lipid biosynthesis in P. annua can be inhibited if sufficiently high concentrations of graminicide are used. The major plastidic form and the minor cytosolic forms of acetyl-CoA carboxylase were successfully purified from maize cell suspensions, were compared to those from leaf tissue and were shown to be differentially inhibited by graminicides in a similar manner to their counterparts from leaf tissue. These studies demonstrate that cell suspensions are useful for studying the mode of action of graminicides, especially in view of the limited amount of material obtainable from many grassy species which are very fine-growing. PMID:12809713

  9. 'From Cultural Studies to Cultural Science

    Microsoft Academic Search

    John Hartley

    2009-01-01

    This paper, first presented at a symposium on the 'past, present and future of cultural studies,' traces disciplinary changes in the study of culture from the perspective of 'cultural science,' a term that was used by some of the earliest practitioners of cultural studies, including Raymond Williams. The paper goes on to describe some problems with cultural studies as it

  10. Phenol Red in Tissue Culture Media is a Weak Estrogen: Implications concerning the Study of Estrogen-Responsive Cells in Culture

    Microsoft Academic Search

    Yolande Berthois; John A. Katzenellenbogen; Benita S. Katzenellenbogen

    1986-01-01

    Although much attention has been paid to the removal of hormones from sera and to the development of serum-free media for studies on hormone-responsive cells in culture, little consideration has been given to the possibility that the media components themselves may have hormonal activity. We have found that phenol red, which bears a structural resemblance to some nonsteroidal estrogens and

  11. Cell culture model for antiulcerogenic agents.

    PubMed

    Terano, A; Hiraishi, H; Shimada, T; Takahashi, M; Yoshiura, K; Horie-Sakata, K

    2001-06-15

    To elucidate the mechanisms of antiulcerogenic agents, we established the cell culture model derived from rat gastric epithelium. The cultured cells were identified as mucus-producing cells by using histological analysis. This culture model is useful for investigating the untiulcer effect of various agents and to reveal the mechanisms of the drug action. In particular, the ulcer-healing model using the cultured monolayer is promising and convenient for the study of several growth factors such as HGF as well as antiulcerogenic agents. The effect of polaporezinc in the cultured model is introduced. PMID:11525256

  12. Three-dimensional culture of HeLa-FUCCI cells for study of bystander cell-cycle effect of high LET particles

    PubMed Central

    Sakamoto, Yuka; Kaminaga, Kiichi; Kanari, Yukiko; Noguchi, Miho; Yokoya, Akinari

    2014-01-01

    It has been recognized that bystander effect is one of the key factors for radiobiological effects, particularly in low-dose region. Although <1% of cell nuclei were actually traversed by an alpha particle, 30% of the cells showed an increased frequency of sister chromatid exchanges at very low dose (0.31 mGy) of alpha particles exposed to a CHO culture dish [ 1]. Since then, a number of studies including high LET microbeam experiments have revealed that the bystander effect is possibly mediated through both gap-junction signal transfer and releasing bystander-transmitter molecules from the irradiated cell into medium. Although it has particularly been given considerable attention to the latter process, however, the signal transfer through medium seems very specific to the artificially system of monolayer culture dishes, which are substantially different from in vivo system in which cells contact each other to form a functionally three-dimensional (3D) structure. Bystander signals must mainly be transferred through gap junctions. In order to examine bystander effects in the 3D cell system, we have developed a HeLa-FUCCI spheroid system. FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator) cells show specific colors of cell nuclei depending on cell cycle. Thus, we can easily trace cell-cycle modifications by irradiation. We observed bystander cell-cycle delay as preliminary tests using monolayer culture of the HeLa-FUCCI cells. It will be very interesting to examine whether the cell-cycle effect also appear in the 3D cell system exposed to single high LET particles. We have studied suitable conditions for the spheroid culture, such as size of spheroids and methods of stable fixing a spheroid in a dish to perform the microbeam irradiation, and observation of the cell cycles of each cell in a spheroid after irradiation using time-lapse micro-imaging technique. The first day of the culture, single cells were seeded in a commercial 96-well multi-plate. A typical spheroid image observed for 3 days after seeding was shown in Fig. 1. The cells substantially formed a 3D structure of spheroid, in which the cells showed different cell cycle as shown by green (S/G2) and red (G1). This result indicates that the cells in a spheroid keep cell division to grow. We further investigate the effect of high LET particle irradiation on cell cycle in a spheroid in the future. Fig. 1.Spheroid after 3-day culture. Red and green cells are in G1 and S/G2 phase, respectively. The cells form a globular three-dimensional structure with about a few hundred micrometer.

  13. Cell culture's spider silk road.

    PubMed

    Perkel, Jeffrey

    2014-06-01

    A number of synthetic and natural materials have been tried in cell culture and tissue engineering applications in recent years. Now Jeffrey Perkel takes a look at one new culture component that might surprise you-spider silk. PMID:24924388

  14. Fabrication of pseudo three-dimensional PADC cell culture substrates for dosimetric studies

    E-print Network

    Yu, K.N.

    ) as a marker of DNA damages (Tartier et al. 2007; Han et al. 2010a,b) to quantify the radiation effects (w10 mm) fabricated by UV polymerization. PADC substrates record alpha-particle hit positionsLa cells cultured on these substrates, including the radiation- induced bystander effect (RIBE). RIBE

  15. Transplantation of Cultured Bovine Corneal Endothelial Cells to Rabbit Cornea: Clinical Implications for Human Studies

    Microsoft Academic Search

    Denis Gospodarowicz; Gary Greenburg; Jorge Alvarado

    1979-01-01

    Rabbit corneas denuded of their endothelium were coated with bovine corneal endothelial cells (from steers) previously maintained in tissue culture for short (20 generations) or prolonged (200 generations) periods. When grafted back into female rabbits, the corneal buttons remained clear and showed no edema. In contrast, denuded corneas coated with bovine keratocytes and grafted into rabbits became opaque and edematous

  16. Psyllid cell culture: A system to study Candidatus Liberibacter species replication

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Primary cell cultures were made from the Potato Psyllid, Bactericera cockerelli (Hemiptera: Psyllidae). The potato psyllid is an important agricultural pest insect due to its ability to transmit the bacterial pathogen Candidatus Liberibacter psyllaurous, CLp. The pathogen is a phloem limited bacteri...

  17. Cell culture experiments planned for the space bioreactor

    NASA Technical Reports Server (NTRS)

    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.

  18. Hydrogels as extracellular matrix mimics for 3D cell culture

    Microsoft Academic Search

    Mark W. Tibbitt; Kristi S. Anseth

    2009-01-01

    Methods for culturing mammalian cells ex vivo are increasingly needed to study cell and tissue physiology and to grow replacement tissue for regenerative medicine. Two-dimensional culture has been the paradigm for typical in vitro cell culture; however, it has been demonstrated that cells behave more natively when cultured in three- dimensional environments. Permissive, synthetic hydrogels and promoting, natural hydrogels have

  19. Recent advances in crayfish hematopoietic stem cell culture: a model for studies of hemocyte differentiation and immunity.

    PubMed

    Söderhäll, Irene

    2013-10-01

    Hematopoiesis is the process by which blood cells (hemocytes) mature and subsequently enter the circulation and we have developed a new technique to culture the hematopoietic progenitor cells in vitro. The reason for the successful culture was the isolation of a plasma protein that turned out to be a novel cytokine, astakine 1 (Ast1) containing a domain present in several vertebrates, so-called prokineticins. Now we have detected several astakines from other invertebrate species. Depending on our discovery of the cytokine Ast1 we have an opportunity to study in detail the differentiation of cells in the hematopoietic tissue of a crustacean, a tissue of evolutionary interest for studies of the connection between the vascular system and the nervous system. We have been able to isolate the entire hematopoietic tissue and for the first time detected a link between this tissue and the brain. We have further localized a proliferation center in the tissue and characterized its different parts. We have also used this system to isolate a new hematopoietic factor CHF that is important in the crossroad between apoptosis and hemocyte differentiation. Our technique for culture of crayfish hematopoietic stem cells provides a simple tool for studying the mechanism of hematopoiesis, but also enables detailed studies of immune defense reactions. Further, the culture system has been used for studies of viral defense and the system is suitable for gene silencing which allows functional characterization of different molecules involved in host defense as well as in hemocyte differentiation. PMID:23686548

  20. Study of the cells proliferating in parent versus F hybrid mixed lymphocyte culture.

    PubMed

    Piguet, P F; Dewey, H K; Vassalli, P

    1975-04-01

    Caryotypic analysis of the cells dividing in mouse parent-hybrid MLC showed an F1 hybrid cell proliferation, which varied depending upon the source of lymphoid cells used: strong in spleen MLC (sometimes equal to that of the parental cells), less marked in lymph node cell MLC, and most often absent in MLC between cortisone-resistant (CR) thymocytes. MLC between parental spleen cells and F1 CR thymocytes showed, however, that in certain conditions of culture F thymocytes can also proliferate. Using parental or F1 spleen cells lacking T lymphocytes, it was found that F1 cell proliferation is entirely dependent upon the presence of parental T cells, but does not require the presence of T lymphocytes among the F1 cells. Immunofluorescence analysis of the blasts observed in one-way MLC showed that about 70% of the parental blasts were T blasts, and 25%B blasts (containing a high proportion of plasmablasts); among the F1 blasts, there was also the same percentage of B blasts and plasmablasts, but many of the T blasts bore only small amounts of T-cell antigen (MTLA), and there was also about 20%of unstained blasts, possibly T blasts bearing MTLA in amounts undetectable by immunofluorescence. The possibility is discussed that the F1 responding T cells belong to a subpopulation performing a suppressive function; MLC lacking F1 T cells showed increased [3H] thymidine incorporation. The proliferation and differentiation of parental and F1 B cells may result mainly from an unspecific, "polyclonal" triggering. PMID:1092790

  1. Some properties of Bomirski Ab amelanotic melanoma cells, which underwent spontaneous melanization in primary cell culture

    Microsoft Academic Search

    Andrzej Stomifiski

    1985-01-01

    Summary Four types of the Bomirski Ab amelanotic melanoma primary cell culture, differing in the presence of calf serum in the medium and in the cell number used for starting the culture, were employed in the study. In all types of cell culture, rapid melanization occurred in the cytoplasm of the cultured cells. Calf serum in the culture medium stimulated

  2. Stromal-epithelial interaction study: The effect of corneal epithelial cells on growth factor expression in stromal cells using organotypic culture model.

    PubMed

    Kobayashi, Takeshi; Shiraishi, Atsushi; Hara, Yuko; Kadota, Yuko; Yang, Lujun; Inoue, Tomoyuki; Shirakata, Yuji; Ohashi, Yuichi

    2015-06-01

    Interactions between stromal and epithelial cells play important roles in the development, homeostasis, and pathological conditions of the cornea. Soluble cytokines are critical factors in stromal-epithelial interactions, and growth factors secreted from corneal stromal cells contribute to the regulation of proliferation and differentiation of corneal epithelial cells (CECs). However, the manner in which the expression of growth factors is regulated in stromal cells has not been completely determined. To study stromal-epithelial cell interactions, we used an organotypic culture model. Human or rabbit CECs (HCECs or RCECs) were cultured on amniotic membranes placed on human corneal fibroblasts (HCFs) embedded in a collagen gel. The properties of the organotypic culture were examined by hematoxylin-eosin staining and immunofluorescence. In the organotypic culture, HCECs or RCECs were stratified into two-three layers after five days and five-seven layers after nine days. However, stratification was not observed when the HCECs were seeded on a collagen gel without fibroblasts. K3/K12 were expressed on day 9. The HCF-embedded collagen gels were collected on days 3, 5, or 9 after seeding the RCECs, and mRNA expression of growth factors FGF7, HGF, NGF, EGF, TGF-?, SCF, TGF-?1, TGF-?2, and TGF-?3 were quantified by real-time PCR. mRNA expression of the growth factors in HCFs cultured with RCECs were compared with those cultured without RCECs, as well as in monolayer cultures. mRNA expression of TGF-? was markedly increased in HCFs cultured with RCECs. However, mRNA expression of the TGF-? family was suppressed in HCFs cultured with RCECs. Principal component analysis revealed that mRNA expression of the growth factors in HCFs were generally similar when they were cultured with RCECs. In organotypic cultures, the morphological changes in the CECs and the expression patterns of the growth factors in the stromal cells clearly demonstrated stromal-epithelial cell interactions, and the results suggest that stromal cells and epithelial cells may act in concert in the cornea. PMID:25682729

  3. A novel 3-dimensional culture system as an in vitro model for studying oral cancer cell invasion

    PubMed Central

    Duong, Hai S; Le, Anh D; Zhang, Qunzhou; Messadi, Diana V

    2005-01-01

    Tissue microenvironment plays a critical role in tumour growth and invasion. This study established a novel 3-dimensional (3-D) cell invasion model for direct microscopic observation of oral cancer cell invasion into the underlying basement membrane and connective tissue stroma. A multilayer cell construct was developed using the OptiCell chamber, consisting of a lower layer of oral mucosa fibroblasts embedded in collagen gel and an overlaying upper layer of oral cancer cells. The two layers are separated by a basement membrane composed of reconstituted extracellular matrix. To verify the applicability of the cell invasion model, multilayer cell constructs of oral squamous cell carcinoma and oral mucosal fibroblasts were exposed to extrinsic urokinase-type plasminogen activator (uPA) or plasminogen activator inhibitor (PAI-1), which are known effectors of cell migration. In addition, the constructs were exposed to both normoxic and hypoxic culture conditions. Microscopic study showed that the presence of uPA enhanced cell invasion, while PAI-1 inhibited cell migration. Western blot and zymographic analysis demonstrated that hypoxia up-regulated uPA and matrix metalloproteinases (MMPs) expression and activity; conversely, PAI-1 level was down-regulated in response to hypoxic challenge as compared to normoxic condition. Our results indicated that the novel 3-D invasion model could serve as an excellent in vitro model to study cancer cell invasion and to test conditions or mediators of cellular migration. PMID:16309542

  4. A COMPREHENSIVE STUDY ON APOPTOSIS INDUCTION BY AZADIRACHTIN IN Spodoptera frugiperda CULTURED CELL LINE Sf9.

    PubMed

    Shu, Benshui; Wang, Wenxiang; Hu, Qingbo; Huang, Jingfei; Hu, Meiying; Zhong, Guohua

    2015-07-01

    The induction of apoptosis by azadirachtin, a well-known botanical tetranortriterpenoid isolated from the neem tree (Azadirachta indica A. Juss) and other members of the Meliaceae, was investigated in Spodoptera frugiperda cultured cell line (Sf9). Morphological changes in Sf9 cells treated by various concentrations of azadirachtin were observed at different times under light microscopy. Morphological and biochemical analysis indicated that Sf9 cells treated by 1.5 ?g/mL azadirachtin showed typical morphological changes, which were indicative of apoptosis and a clear DNA ladder. The flow cytometry analysis showed the apoptosis rate reached a maximum value of 32.66% at 24 h with 1.5 ?g/mL azadirachtin in Sf9 cells. The inhibition of Sf9 cell proliferation suggested that the effect of azadirachtin was dose dependent and the EC50 at 48 and 72 h was 2.727 × 10(-6) and 6.348 × 10(-9) ?g/mL, respectively. The treatment of azadirachtin in Sf9 cells could significantly increase the activity of Sf caspase-1, but showed no effect on the activity of Topo I, suggesting that the apoptosis induced by azadirachtinin Sf9 cells is through caspase-dependent pathway. These results provided not only a series of morphological, biochemical, and toxicological comprehensive evidences for induction of apoptosis by azadirachtin, but also a reference model for screening insect cell apoptosis inducers from natural compounds. PMID:25828604

  5. A study on proliferation and gene expression in normal human urothelial cells in culture.

    PubMed

    Chamorro, Clara Ibel; Zeiai, Said; Engberg, Gisela Reinfeldt; Brodin, David; Nordenskjöld, Agneta; Fossum, Magdalena

    2015-02-01

    Cultured human urothelial cells can be used in tissue engineering for reconstruction of urothelial defects. For safety reasons, a fine characterization of the cells is required before use in reconstructive surgery. For these reasons, we aimed to characterize the effect of in vitro propagation of urothelial cells on gene expression and proliferative capacity. Gene expression of urothelial cells in passage two and eight was captured by using a microarray chip covering the whole human genome. To find relationships in biological functions and pathways, differentially regulated genes were subjected to pathway analysis using the WEB-based Gene Set Analysis Toolkit (WebGestalt). Proliferative capacity was tested with population doubling time, efficiency in colony formation assays, and immunocytochemistry. In addition, senescence markers were evaluated. Bioinformatics analysis revealed gene expression profile differences. Downregulated genes at passage eight clustered in biological pathways of cell cycle and DNA repair processes; upregulated genes had no obvious association to any specific biological function or pathway according to WebGestalt analysis, but individual genes with extracellular matrix, apoptosis, and cell morphology. Data were supported by reverse transcription-polymerase chain reaction (RT-PCR) and in vitro growth experiments. Passage two urothelial cells had higher efficiency in colony formation and lower population doubling time. An increase in senescence markers was detected at passage eight. We conclude that pretransplantation quality controls are important and, for reconstructive purposes, cells should be transplanted back to the patient as soon as possible to procure good proliferative capacity also after transplantation. PMID:25159583

  6. A novel human cell culture model for the study of familial prostate cancer.

    PubMed

    Yasunaga, Y; Nakamura, K; Ewing, C M; Isaacs, W B; Hukku, B; Rhim, J S

    2001-08-15

    Research into molecular and genetic mechanisms underlying familial prostate cancer would be greatly advanced by in vitro models of prostate tumor cells representing primary tumors. We have successfully established an immortalized human prostate epithelial cell culture derived from primary tumors of familial prostate cancer patients with telomerase. The actively proliferating early-passaged 957E cells were transduced through infection with a retrovirus expressing the human telomerase catalytic subunit, human telomerase reverse transcriptase (hTERT). A high level of telomerase activity was detected in 957E/hTERT cells, but not in 957E cells. 957E/hTERT cells are currently growing well at passage 40, whereas 957E cells senesced at passage 5. 957E/hTERT cells exhibit epithelial morphology. Expression of an androgen-regulated prostate specific homeobox gene NKX3.1 and an epithelial cell-specific cytokeratin 8, but not prostate specific antigen or androgen receptor, was detected in 957E/hTERT cells. Prostatic stem cell antigen and p16 were also expressed in this line. 957E/hTERT cells showed growth inhibition when exposed to retinoic acid and transforming growth factor beta1, potent inhibitors of prostate epithelial cell growth. Chromosome analysis showed that the 957E/hTERT cell line (passage 10) was near diploid human male (XY), with most chromosome counts in the 44-46 range. However, there was random loss of chromosomes 8, 13, X, Y, and alteration in chromosome 4q. The late passage 957E/hTERT cell line (passage 32) was karyologically similar to the early passage 957E/hTERT cell line (passage 10) and also had the same alteration of 4q observed in the early passage 957E/hTERT cell line (passage 10) as well as a trisomy of chromosome 20. The well-characterized human cancer lines derived from such patients will be useful for the identification and characterization of prostate cancer susceptibility genes. This is the first documented case of an established human prostate cancer cell line from primary tumor of a familial prostate cancer patient. PMID:11507036

  7. Knockdown of Drosha in human alveolar type II cells alters expression of SP-A in culture: a pilot study

    PubMed Central

    Silveyra, Patricia; Chroneos, Zissis C; DiAngelo, Susan L; Thomas, Neal J; Noutsios, Georgios T; Tsotakos, Nikolaos; Howrlylak, Judie A; Umstead, Todd M; Floros, Joanna

    2014-01-01

    Human surfactant protein A (SP-A) plays an important role in surfactant metabolism and lung innate immunity. SP-A is synthesized and secreted by alveolar type II cells (ATII), one of the two cell types of the distal lung epithelium (ATII and ATI). We have shown that miRNA interactions with sequence polymorphisms on the SP-A mRNA 3?UTRs mediate differential expression of SP-A1 and SP-A2 gene variants in vitro. In the present study, we describe a physiologically relevant model to study miRNA regulation of SP-A in human ATII. For these studies, we purified and cultured human ATII on an air-liquid interface matrix (A/L) or plastic wells without matrix (P). Gene expression analyses confirmed that cells cultured in A/L maintained the ATII phenotype for over 5 days, whereas P-cultured cells differentiated to ATI. When we transfected ATII with siRNAs to inhibit the expression of Drosha, a critical effector of miRNA maturation, the levels of SP-A mRNA and protein increased in a time dependent manner. We next characterized cultured ATII and ATI by studying expression of 1,066 human miRNAs using miRNA PCR arrays. We detected expression of >300 miRNAs with 24 miRNAs differentially expressed in ATII vs. ATI, 12 of which predicted to bind SP-A 3?UTRs, indicating that these may be implicated in SP-A downregulation in ATI. Thus, miRNAs not only affect SPA expression, but also may contribute to the maintenance of the ATII cell phenotype and/or the trans-differentiation of ATII to ATI cells, and may represent new molecular markers that distinguish ATII and ATI. PMID:25058539

  8. Studies of formation and efflux of methotrexate polyglutamates with cultured hepatic cells

    SciTech Connect

    Galivan, J.; Balinska, M.

    1983-01-01

    Methotrexate polyglutamates are extensively synthesized when cultured hepatocytes and H35 hepatoma cells are exposed to micromolar concentrations of methotrexate. The predominant species found within the cell have from two to four additional gamma-linked glutamate residues. When either cell type containing a mixture of methotrexate and its polyglutamate derivatives is exposed to medium lacking methotrexate, there is a rapid release of methotrexate. This release has a T/sub 1/2/ of 2 to 4 min and is apparently complete within 30 to 60 min. Methotrexate polyglutamates leave the cells much more slowly and appear to do so by two mechanisms. Although cleavage to methotrexate and subsequent efflux appears to be quantitatively the more important pathway, there is also a slow, finite loss of intact methotrexate polyglutamates from cells which exclude trypan blue. The T/sub 1/2/ for the loss of methotrexate polyglutamates by both cell types, when placed in medium lacking methotrexate, is approximately 6 to 8 hr. These results suggest that the polyglutamate derivatives are forms of methotrexate which are as cytotoxic as methotrexate but which offer a potentially greater capacity for cellular destruction because they are retained longer in the tissue.

  9. THE COMPARISON OF TWO VITRO PALATAL ORGAN CULTURE MODELS TO STUDY CELL SIGNALING PATHWAYS DURING PALATOGENESIS

    EPA Science Inventory

    This study was performed to determine the best palatal organ culture model to use in evaluating the role of epidermal growth factor (EGF) signaling in the response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Previous work has shown that TCDD and EGF can induce teratogenic effe...

  10. High density cell culture system

    NASA Technical Reports Server (NTRS)

    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.

  11. Dynamized Preparations in Cell Culture

    PubMed Central

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

    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 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. PMID:18955237

  12. Development of an in vitro cell culture model to study milk to plasma ratios of therapeutic drugs

    PubMed Central

    Athavale, Maithili A.; Maitra, Anurupa; Patel, Shahnaz; Bhate, Vijay R.; Toddywalla, Villi S.

    2013-01-01

    Objective: To create an in vitro cell culture model to predict the M/P (concentration of drug in milk/concentration in maternal plasma) ratios of therapeutic drugs viz. rifampicin, theophylline, paracetamol, and aspirin. Materials and Methods: An in vitro cell culture model using CIT3 cells (mouse mammary epithelial cells) was created by culturing the cells on transwells. The cells formed an integral monolayer, allowing only transcellular transport as it happens in vivo. Functionality of the cells was confirmed through scanning electron microscopy. Time wise transfer of the study drugs from plasma to milk was studied and compared with actual (in vivo) M/P ratios obtained at reported tmax for the respective drugs. Results: The developed model mimicked two important intrinsic factors of mammary epithelial cells viz. secretory and tight-junction properties and also the passive route of drug transport. The in vitro M/P ratios at reported tmax were 0.23, 0.61, 0.87, and 0.03 respectively, for rifampicin, theophylline, paracetamol, and salicylic acid as compared to 0.29, 0.65, 0.65, and 0.22, respectively, in vitro. Conclusion: Our preliminary effort to develop an in vitro physiological model showed promising results. Transfer rate of the drugs using the developed model compared well with the transfer potential seen in vivo except for salicylic acid, which was transferred in far lower concentration in vitro. The model has a potential to be developed as a non-invasive alternative to the in vitro technique for determining the transfer of therapeutic drugs into breast milk. PMID:24014904

  13. Comparative Evaluation of Different Cell Lysis and Extraction Methods for Studying Benzo(a)pyrene Metabolism in HT-29 Colon Cancer Cell Cultures

    PubMed Central

    Myers, Jeremy N.; Rekhadevi, Perumalla V.; Ramesh, Aramandla

    2011-01-01

    Lysis and extraction of cells are essential sample processing steps for investigations pertaining to metabolism of xenobiotics in cell culture studies. Of particular importance to these procedures are maintaining high lysis efficiency and analyte integrity as they influence the qualitative and quantitative distribution of drug and toxicant metabolites in the intra- and extracellular milieus. In this study we have compared the efficiency of different procedures viz. homogenization, sonication, bead beating, and molecular grinding resin treatment for disruption of HT-29 colon cells exposed to benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH) compound and a suspected colon carcinogen. Also, we have evaluated the efficiency of various procedures for extracting BaP parent compound/metabolites from colon cells and culture media prior to High Performance Liquid Chromatography (HPLC) analyses. The extraction procedures include solid phase extraction, solid-supported liquid- liquid extraction, liquid-liquid extraction, and homogeneous liquid- liquid extraction. Our findings showed that bead-beating in combination with detergent treatment of cell pellet coupled with liquid-liquid extraction yielded greater concentrations of BaP metabolites compared to the other methods employed. Our method optimization strategy revealed that disruption of HT-29 colon cells by a combination of mechanical and chemical lysis followed by liquid-liquid extraction is efficient and robust enough for analyzing BaP metabolites from cell culture studies. PMID:21865728

  14. Cultured hepatoma cells for the study of enzyme regulation: Induction of ornithine decarboxylase by insulin and asparagine

    Microsoft Academic Search

    R. Van Potter; Theresa Ruh Evanson; Debra P. Gayda; James A. Gurr

    1984-01-01

    Summary  The induction and decay of ornithine decarboxylase (ODC) by insulin and asparagine in cultures of H4-II-EC3 (H35) hepatoma\\u000a cells was studied in a modified Waymouth medium in the presence of fetal bovine serum (FBS) and in serum-free media. The insulin\\u000a response was enhanced by the presence of asparagine although the effect of asparagine was not so much on the initial

  15. CELL CULTURE STUDIES WITH THE IMC-HZ-1 NONOCCLUDED VIRUS

    EPA Science Inventory

    Studies were conducted on an adventitious agent (Hz-lv) isolated from the IMC-Hz-1 cell line. It appeared identical to the virus first obtained by Granados et al. from a persistent infection of this cell line. Restriction endonuclease digestion of Hz-lv DNA indicated the agent wa...

  16. Fibroin hydrogels for biomedical applications: preparation, characterization and in vitro cell culture studies.

    PubMed

    Motta, A; Migliaresi, C; Faccioni, F; Torricelli, P; Fini, M; Giardino, R

    2004-01-01

    Silk fibroin hydrogels prepared either by treating a 2% (w/v) silk fibroin aqeuous solution at 4 degrees C (thermgel) or by adding 30% (v/v) of glycerol (glygel), were characterized by using Environmental Scanning Electron Microscopy (ESEM), Fourier Transform Infrared Spectroscopy (FT-IR), Differential Scanning Calorimetry (DSC), Thermogravimetrical Analysis (TGA) and molecular weight determination. The preparation procedure affected morphology and molecular weight of hydrogels, with no or negligible differences being displayed by FT-IR and DSC analyses. While thermgel presented a well uniform porous structure, the morphology of glygel appeared to be non-porous and heterogeneous. Glygel presented lower water content and lower degradation temperatures, associated with the presence of glycerol but likely also to less-organized protein structures. Cytoxicity tests with human osteoblast-like cells indicated that both gels were not cytoxic, while cell cultures pointed out a faster cell proliferation on glygel and a higher cell activation and differentiation on thermgel. These gels could be used as scaffolds able to promote in situ bone regeneration. PMID:15318796

  17. PCR amplification of 16S rDNA from lyophilized cell cultures facilitates studies in molecular systematics

    NASA Technical Reports Server (NTRS)

    Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.

    1990-01-01

    The sequence of the major portion of a Bacillus cycloheptanicus strain SCH(T) 16S rRNA gene is reported. This sequence suggests that B. cycloheptanicus is genetically quite distinct from traditional Bacillus strains (e.g., B. subtilis) and may be properly regarded as belonging to a different genus. The sequence was determined from DNA that was produced by direct amplification of ribosomal DNA from a lyophilized cell pellet with straightforward polymerase chain reaction (PCR) procedures. By obviating the need to revive cell cultures from the lyophile pellet, this approach facilitates rapid 16S rDNA sequencing and thereby advances studies in molecular systematics.

  18. The demonstration of acid phosphatase in in vitro cultured tissue cells. Studies on the significance of fixation, tonicity and permeability

    Microsoft Academic Search

    Ulf T. Brunk; Jan L. E. Ericsson

    1972-01-01

    Synopsis  \\u000a1. \\u000aMethods for the fine structural demonstration of acid phosphatase were studied in monolayers ofin vitro cultured cells after fixation with glutaraldehyde.\\u000a2. \\u000aInactivation of enzyme activity occurred rapidly during the initial phase of glutaraldehyde fixation.\\u000a3. \\u000aFixation for more than 5 min did not cause further marked inactivation of enzyme activity.\\u000a4. \\u000aStabilization of the cells for cytochemical

  19. Chemical Synthesis, Characterisation, and Biocompatibility of Nanometre Scale Porous Anodic Aluminium Oxide Membranes for Use as a Cell Culture Substrate for the Vero Cell Line: A Preliminary Study

    PubMed Central

    Poinern, Gérrard Eddy Jai; Le, Xuan Thi; Becker, Thomas; Fawcett, Derek

    2014-01-01

    In this preliminary study we investigate for the first time the biomedical potential of using porous anodic aluminium oxide (AAO) membranes as a cell substrate for culturing the Cercopithecus aethiops (African green monkey) Kidney (Vero) epithelial cell line. One advantage of using the inorganic AAO membrane is the presence of nanometre scale pore channels that allow the exchange of molecules and nutrients across the membrane. The size of the pore channels can be preselected by adjusting the controlling parameters of a temperature controlled two-step anodization process. The cellular interaction and response of the Vero cell line with an in-house synthesised AAO membrane, a commercially available membrane, and a glass control were assessed by investigating cell adhesion, morphology, and proliferation over a 72?h period. The number of viable cells proliferating over the respective membrane surfaces revealed that the locally produced in-house AAO membrane had cells numbers similar to the glass control. The study revealed evidence of focal adhesion sites over the surface of the nanoporous membranes and the penetration of cellular extensions into the pore structure as well. The outcome of the study has revealed that nanometre scale porous AAO membranes have the potential to become practical cell culture scaffold substrates with the capability to enhance adhesion and proliferation of Vero cells. PMID:24579077

  20. Chemical synthesis, characterisation, and biocompatibility of nanometre scale porous anodic aluminium oxide membranes for use as a cell culture substrate for the vero cell line: a preliminary study.

    PubMed

    Poinern, Gérrard Eddy Jai; Le, Xuan Thi; O'Dea, Mark; Becker, Thomas; Fawcett, Derek

    2014-01-01

    In this preliminary study we investigate for the first time the biomedical potential of using porous anodic aluminium oxide (AAO) membranes as a cell substrate for culturing the Cercopithecus aethiops (African green monkey) Kidney (Vero) epithelial cell line. One advantage of using the inorganic AAO membrane is the presence of nanometre scale pore channels that allow the exchange of molecules and nutrients across the membrane. The size of the pore channels can be preselected by adjusting the controlling parameters of a temperature controlled two-step anodization process. The cellular interaction and response of the Vero cell line with an in-house synthesised AAO membrane, a commercially available membrane, and a glass control were assessed by investigating cell adhesion, morphology, and proliferation over a 72?h period. The number of viable cells proliferating over the respective membrane surfaces revealed that the locally produced in-house AAO membrane had cells numbers similar to the glass control. The study revealed evidence of focal adhesion sites over the surface of the nanoporous membranes and the penetration of cellular extensions into the pore structure as well. The outcome of the study has revealed that nanometre scale porous AAO membranes have the potential to become practical cell culture scaffold substrates with the capability to enhance adhesion and proliferation of Vero cells. PMID:24579077

  1. Studies of the ultrastructure of embryonic Boophilus microplus cells in culture and interaction of Babesia bovis with these cells 

    E-print Network

    Droleskey, Robert Edward

    1981-01-01

    of 100% ethanol. After dehydration, cultures were embedded in Epon-Araldite plastic (29) in the following order: I) 50/ plastic/ethanol for 30 min, Z) 75% plastic/ethanol for 30 min, and 3) two changes of 100K plastic over the course of the next Z hr... of mylar plastic containing a few drops of epoxy resin. The mylar was then covered with a petri dish and placed in a 60 C oven to polymerize. After polymerization, the coverslips containing embedded cells were removed from the mylar plastic sheet. Small...

  2. Cold response of dedifferentiated barley cells at the gene expression, hormone composition, and freezing tolerance levels: studies on callus cultures.

    PubMed

    Vashegyi, Ildikó; Marozsán-Tóth, Zsuzsa; Galiba, Gábor; Dobrev, Petre I; Vankova, Radomira; Tóth, Balázs

    2013-06-01

    In this study, data is presented how dark-grown, embryogenic barley callus cells respond to cold without any light-dependent, chloroplast-related mechanism, independently of the systemic signals. The expression of HvCBF9, HvCBF14, and HvCOR14b genes, members of one of the most important cold-inducible regulatory system, was measured by real-time PCR. Characteristic of the cold response was similar in the crowns of seedlings and in dark-grown callus cultures, however, gene expression levels were lower in calli. Endogenous concentration of auxins, abscisic acid, and salicylic acid did not change, but phaseic acid and neophaseic acid showed robust accumulation after cold acclimation. Freezing tolerance of the cultures was also higher after 7 days of cold-hardening. The results suggest the presence of a basal, light-independent, cold-responsive activation of the CBF-COR14b pathway in barley cultures. The effects of Dicamba, the exogenous auxin analog used for maintaining tissue cultures were also studied. Dicamba seems to be a general enhancer of the gene expression and physiological responses to cold stress, but has no specific effect on the activation. Our data along with previous findings show that this system might be a suitable model for studying certain basic cellular mechanisms involved in the cold acclimation process in cereals. PMID:22669585

  3. Culture and transfection of axolotl cells.

    PubMed

    Denis, Jean-François; Sader, Fadi; Ferretti, Patrizia; Roy, Stéphane

    2015-01-01

    The use of cells grown in vitro has been instrumental for multiple aspects of biomedical research and especially molecular and cellular biology. The ability to grow cells from multicellular organisms like humans, squids, or salamanders is important to simplify the analyses and experimental designs to help understand the biology of these organisms. The advent of the first cell culture has allowed scientists to tease apart the cellular functions, and in many situations these experiments help understand what is happening in the whole organism. In this chapter, we describe techniques for the culture and genetic manipulation of an established cell line from axolotl, a species widely used for studying epimorphic regeneration. PMID:25740487

  4. Culturing primary mouse pancreatic ductal cells.

    PubMed

    Reichert, Maximilian; Rhim, Andrew D; Rustgi, Anil K

    2015-01-01

    The most common subtype of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). PDAC resembles ductal cells morphologically. To study pancreatic ductal cell (PDC) and pancreatic intraepithelial neoplasia (PanIN)/PDAC biology, it is essential to have reliable in vitro culture conditions. Here we describe a methodology to isolate, culture, and passage PDCs and duct-like cells from the mouse pancreas. It can be used to isolate cells from genetically engineered mouse models (GEMMs), providing a valuable tool to study disease models in vitro to complement in vivo findings. The culture conditions allow epithelial cells to outgrow fibroblast and other "contaminating" cell types within a few passages. However, the resulting cultures, although mostly epithelial, are not completely devoid of fibroblasts. Regardless, this protocol provides guidelines for a robust in vitro culture system to isolate, maintain, and expand primary pancreatic ductal epithelial cells. It can be applied to virtually all GEMMs of pancreatic disease and other diseases and cancers that arise from ductal structures. Because most carcinomas resemble ductal structures, this protocol has utility in the study of other cancers in addition to PDAC, such as breast and prostate cancers. PMID:26034301

  5. Cell Culture in 3Dimensional Microfluidic Structure of PDMS (polydimethylsiloxane)

    Microsoft Academic Search

    Eric Leclerc; Yasuyuki Sakai; Teruo Fujii

    2003-01-01

    In this paper, a device with 3-dimensional microfluidic structure composed of two stacked layers of PDMS (polydimethylsiloxane) is fabricated for mammalian cell culture. This microdevice is tested with Hepatocarcinoma liver cells (Hep G2 cells). The purpose of this study is to understand to what extent cell culture in a PDMS microdevice is available. The experimental protocols for Hep G2 cell

  6. TRANSFORMATION OF MONOCYTES IN TISSUE CULTURE INTO MACROPHAGES, EPITHELIOID CELLS, AND MULTINUCLEATED GIANT CELLS: An Electron Microscope Study

    Microsoft Academic Search

    JERRY S. SUTTON; LEON WEISS

    1966-01-01

    The scqucntial transformation of chickcn monocytcs into macrophages, cpithelioid cells, and multinucleatcd giant cells in vitro was studied by electron microscopy after fixation and cmbcdment in situ. The following changes occur. In the nucleus, margination of chro- matin, cvidcnt in monocytes, decreases in later forms. Nucleoli become more complcx and nuclear pores increase in number. In cytoplasm, a progressive increase

  7. Metabolomics in cell culture--a strategy to study crucial metabolic pathways in cancer development and the response to treatment.

    PubMed

    Halama, Anna

    2014-12-15

    Metabolomics is a comprehensive tool for monitoring processes within biological systems. Thus, metabolomics may be widely applied to the determination of diagnostic biomarkers for certain diseases or treatment outcomes. There is significant potential for metabolomics to be implemented in cancer research because cancer may modify metabolic pathways in the whole organism. However, not all biological questions can be answered solely by the examination of small molecule composition in biofluids; in particular, the study of cellular processes or preclinical drug testing requires ex vivo models. The major objective of this review was to summarise the current achievement in the field of metabolomics in cancer cell culture-focusing on the metabolic pathways regulated in different cancer cell lines-and progress that has been made in the area of drug screening and development by the implementation of metabolomics in cell lines. PMID:25218088

  8. [Study on the Rheum palmatum volatile oil against HBV in cell culture in vitro].

    PubMed

    Zhang, B; Chen, J; Li, H; Xu, X

    1998-10-01

    The antiHBV effect of Rheum palmatum Volatile oil was studied by using 2215 cell line transfected with HBV DNA. At the same time MTT method was applied for the detection of cytoxicity of drugs, selecting acyclovir(ACV) as control medicine. It turns out that the toxic concentration of Rheum palmatum Volatile oil for 50% cells was (CD50) > 1.25 x 10(-1) g/L. When concentration was below 0.625 x 10(-1) g/L, the survival rate of cells was over 90%. The maximum inhibitory rates for HBsAg and HBeAg were 70.71 +/- 5.4% and 30.99 +/- 5.3% respectively. This shows Rheum palmatum Volatile oil possesses the effect of antiHBV in vitro. PMID:12569832

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

    PubMed Central

    Fox, Derek B.; Stoker, Aaron M.; Beatty, Mark; Cockrell, Mary; Janicek, John C.; Cook, James L.

    2014-01-01

    Introduction. Tissue engineering is a new methodology for addressing meniscal injury or loss. Synovium may be an ideal source of cells for in vitro meniscal fibrocartilage formation, however, favorable in vitro culture conditions for synovium must be established in order to achieve this goal. The objective of this study was to determine cellularity, cell distribution, and extracellular matrix (ECM) formation of equine fibroblast-like synoviocytes (FLS) cultured on synthetic scaffolds, for potential application in synovium-based meniscal tissue engineering. Scaffolds included open-cell poly-L-lactic acid (OPLA) sponges and polyglycolic acid (PGA) scaffolds cultured in static and dynamic culture conditions, and PGA scaffolds coated in poly-L-lactic (PLLA) in dynamic culture conditions. Materials and Methods. Equine FLS were seeded on OPLA and PGA scaffolds, and cultured in a static environment or in a rotating bioreactor for 12 days. Equine FLS were also seeded on PGA scaffolds coated in 2% or 4% PLLA and cultured in a rotating bioreactor for 14 and 21 days. Three scaffolds from each group were fixed, sectioned and stained with Masson’s Trichrome, Safranin-O, and Hematoxylin and Eosin, and cell numbers and distribution were analyzed using computer image analysis. Three PGA and OPLA scaffolds from each culture condition were also analyzed for extracellular matrix (ECM) production via dimethylmethylene blue (sulfated glycosaminoglycan) assay and hydroxyproline (collagen) assay. PLLA coated PGA scaffolds were analyzed using double stranded DNA quantification as areflection of cellularity and confocal laser microscopy in a fluorescent cell viability assay. Results. The highest cellularity occurred in PGA constructs cultured in a rotating bioreactor, which also had a mean sulfated glycosaminoglycan content of 22.3 µg per scaffold. PGA constructs cultured in static conditions had the lowest cellularity. Cells had difficulty adhering to OPLA and the PLLA coating of PGA scaffolds; cellularity was inversely proportional to the concentration of PLLA used. PLLA coating did not prevent dissolution of the PGA scaffolds. All cell scaffold types and culture conditions produced non-uniform cellular distribution. Discussion/Conclusion. FLS-seeding of PGA scaffolds cultured in a rotating bioreactor resulted in the most optimal cell and matrix characteristics seen in this study. Cells grew only in the pores of the OPLA sponge, and could not adhere to the PLLA coating of PGA scaffold, due to the hydrophobic property of PLA. While PGA culture in a bioreactor produced measureable GAG, no culture technique produced visible collagen. For this reason, and due to the dissolution of PGA scaffolds, the culture conditions and scaffolds described here are not recommended for inducing fibrochondrogenesis in equine FLS for meniscal tissue engineering. PMID:24765587

  10. Microfluidic Cell-Culture Devices

    Microsoft Academic Search

    Yasuyuki Sakai; Eric Leclerc; Teruo Fujii

    Microfluidics is the emerging technologies that could bring favorable features to tissue engineering applications. Fundamental\\u000a techniques to fabricated microfluidic cell-culture devices and experimental attempts towards in vitro liver tissue reconstitution\\u000a are presented for further discussion on the possible developments in the field of lab-on-a-chip for cellomics.

  11. Protein Inhibition by Microinjection and RNA-Mediated Interference in Tissue Culture Cells: Complementary Approaches to Study Protein Function

    NASA Astrophysics Data System (ADS)

    Stout, Jane R.; Rizk, Rania S.; Walczak, Claire E.

    A major goal in cell biology is to understand the molecular mechanisms of the biological process under study, which requires functional information about the roles of individual proteins in the cell. For many non-genetic model organisms researchers have relied on the use of inhibitory reagents, such as antibodies that can be microinjected into cells. More recently, the advent of RNA-mediated interference (RNAi) has allowed scientists to knockdown individual proteins and to examine the consequences of the knockdown. In this chapter we present a comparison between microinjection of inhibitory reagents and RNAi for the analysis of protein function in mammalian tissue culture cells, providing both a description of the techniques as well as a discussion of the benefits and drawbacks of each approach. In addition, we present a strategy to employ RNAi for organisms without a sequenced genome. While the focus of our research is on the organization of the mitotic spindle during cell division and thus the examples utilized are from that system, the approaches described here should be readily applicable to multiple experimental models.

  12. A novel in vitro co-culture system for the study of maternal decidual endothelial cell–trophoblast interactions in human pregnancy

    Microsoft Academic Search

    Eileen D. M. Gallery; Suzanne Campbell; Biljana Ilkovski; Michael J. Sinosich; Christopher Jackson

    2001-01-01

    Investigation of the pathophysiology of pre-eclampsia (characterised by insufficient invasion of the intrauterine vasculature by cytotrophoblasts) has been hampered by the absence of a suitable animal model, and ethical constraints in clinical studies. We have developed a novel in vitro human cell co-culture system allowing direct assessment of cytotrophoblast invasion of a decidual endothelial cell monolayer from the abluminal side,

  13. A study of peritoneal cells from healthy and Schistosoma mansoni-infected mice with special reference to myofibroblasts arising in culture.

    PubMed

    Godoy, M; Geuskens, M; el Ouagmiri, M; Semal, P; Van Gansen, P

    1992-10-01

    Adherent, trypsin-resistant, peritoneal cells from mice with chronic schistosomiasis mansoni, and from control mice, were cultivated in vitro up to 20 days. Fibroblasts regularly appeared, about 6 days after seeding, in cultures of the manyfold more numerous cells from infected mice, concomitantly with a dramatic increase, detected by autoradiography, in the percentage of DNA-replicating cells of the monocyte-macrophage lineage. Peritoneal cells from healthy and from infected mice were fractionated on discontinuous Percoll gradients. Eight cell subsets were harvested in both cases, quantitated, and studied by electron microscopy. Two fractions (2 and 3: 1.041 < densities < 1.060 g/ml) from infected mice were greatly enriched in monoblasts and promonocytes. The cells of the different subsets were seeded separately, trypsin-treated and cultivated in vitro. Cultures of cell fractions 2 and 3 from infected mice contained the majority of the DNA-synthesizing cells and gave regularly rise to fibroblasts. Cultures of the different fractions were used for sequential morphological observations (2-11 days) at the electron microscope level. Early cultures were also used for the ultrastructural detection of the Mac-1 (CD 18/CD 11b) surface antigen by gold immunocytochemistry. A few fibroblasts were rarely observed in cultures of fractions 2 and 3 from control mice, while cells with ultrastructural features of myofibroblasts were regularly observed in cultures of the same fractions harvested from mice with chronic schistosomiasis. Fractions 2 and 3 from infected mice contained a large number of Mac-1 positive monoblasts. The correlations between the presence of monoblasts, DNA replication in cells of the monocyte-macrophage lineage and the appearance of myofibroblasts in cultures of the same fractions derived from infected mice are discussed. PMID:1458434

  14. Cerebellar Granule Cells in Culture: Monosynaptic Connections with Purkinje Cells and Ionic Currents

    Microsoft Academic Search

    Tomoo Hirano; Yoshihiro Kubo; Michael M. Wu

    1986-01-01

    Electrophysiological properties of cerebellar granule cells and synapses between granule and Purkinje cells were studied in dissociated cultures. Electrophysiological properties of neurons and synapses in the mammalian central nervous system are best studied in dissociated cell cultures because of good target cell visibility, control over the contents of the extracellular solution, and the feasibility of whole-cell patch electrode recording, which

  15. QUANTITATIVE STUDIES OF THE GROWTH OF MOUSE EMBRYO CELLS IN CULTURE AND THEIR DEVELOPMENT INTO ESTABLISHED LINES

    Microsoft Academic Search

    GEORGE J. TODARO; HOWARD GREEN

    1963-01-01

    Disaggrcgatcd mouse embryo cells, grown in monolaycrs, undcrwcnt a progrcssivc dcclinc in growth ratc upon succcssivc transfer, the rapidity of the decline dcpcnding, among othcr things, on the inoculation density. Ncvcrthclcss, ncarly all culturcs dcvclopcd into cstablishcd lincs within 3 months of culture. Thc first sign of thc emcrgcncc of an established line was the ability of thc cells to

  16. A novel cell culture model for studying differentiation and apoptosis in the mouse mammary gland

    E-print Network

    Gordon, Katrina E; Binas, Bert; Chapman, Rachel S; Kurian, Kathreena M; Clarkson, Richard W E; Clark, A John; Birgitte Lane, E; Watson, Christine J

    2000-03-07

    Abstract Background This paper describes the derivation and characterization of a novel, conditionally immortal mammary epithelial cell line named KIM-2. These cells were derived from mid-pregnant mammary glands of a mouse harbouring one to two...

  17. Establishment of primary cell cultures: Experiences with 155 cell strains

    Microsoft Academic Search

    M. Dietel; H. Arps; D. Gerding; M. Trapp; A. Niendorf

    1987-01-01

    Summary Cell culture systems allow the examination of cell populations in a functional state. To simulate in vivo conditions as closely as possible freshly established cell strains are superior to permanent cell lines. Different aspects for the establishment of primary cell cultures obtained from various tissues are compared: (1) Disintegration, (2) culture media supplemented with basal additions, (3) special supplements

  18. Interaction of atherogenic lipoproteins with cultured cells: a confocal laser scanning microscopy study

    NASA Astrophysics Data System (ADS)

    Hofer, Gerald; Gorges, Roland; Paltauf, Fritz; Kostner, Gerhard M.; Hermetter, Albin

    1994-08-01

    Low density lipoprotein (LDL) and lipoprotein (a) [Lp(a)] were covalently labeled with the fluorescent dyes BODIPY succinimidyl ester (green) or Rhodamine iodoacetamide (red). The interaction of the fluorescent lipoproteins with HepG2 cells was visualized by means of a confocal laser scanning fluorescence microscope operating in the dual wavelength mode. If LDL or Lp(a) were incubated with the cells both lipoproteins bound to the cell surface at 4 degree(s)C or were internalized by the cells at 37 degree(s)C. In all cases larger amounts of LDL interacted with the cells compared with Lp(a). When mixtures of LDL and Lp(a), each labeled with a different dye, were incubated with cells again both lipoproteins bound to the cell surface (4 degree(s)C) or were internalized by the cells (37 degree(s)C). In addition, the major part of the lipoproteins colocalized either on the cell surface or inside the cells. thus, we conclude that interactions of Lp(a) with cells is mediated by LDL, probably via the LDL receptor, to a large extent.

  19. Carrier-free Cultured Autologous Oral Mucosa Epithelial Cell Sheet (CAOMECS) for Corneal Epithelium Reconstruction: A Histological Study.

    PubMed

    Bardag-Gorce, Fawzia; Oliva, Joan; Wood, Andrew; Hoft, Richard; Pan, Derek; Thropay, Jacquelyn; Makalinao, Andrew; French, Samuel W; Niihara, Yutaka

    2015-04-01

    This study investigates the therapeutic effects of carrier-free cultured autologous oral mucosa epithelial cell sheet (CAOMECS) transplantation for experimentally induced severe rabbit limbal stem cell deficiency (LSCD). Buccal biopsies were performed and CAOMECS were cultured and transplanted onto diseased corneas. Six-month follow-up examinations indicated that three out of four corneas with CAOMECS grafts showed a decrease in superficial vascularization, while almost all the sham corneas did not show a similar decrease. H&E staining of corneas showed that CAOMECS transplantation reduced blood vessel invasion of central cornea, reduced lymphocyte infiltration and fibrotic tissue formation. DeltaNp63 stained markedly in the grafted cornea and to a lesser extent in the sham corneas. PCNA and Ki-67 staining were much greater in the sham corneas than in the grafted and normal corneas. K3 and K13 staining demonstrated that CAOMECS transplanted corneas had much more K3- and less K13- positive cells compared to the sham corneas. Muc5AC was decreased in the central region of grafted corneas. Very little alpha-smooth muscle actin (aSMA) staining was detected in grafted corneas, while there was a greater amount of aSMA staining in sham corneas. Staining for anti-angiogenic factor TIMP -3 was also increased, and pro-angiogenic factor MMP-3 was decreased in grafted corneas compared to sham corneas. Our results indicate that CAOMECS grafts resulted in improved epithelialization of the corneal surface and decreased vascularization and fibrosis of the diseased corneas. PMID:25881998

  20. A simple method to quickly and simultaneously purify and enrich intact rat brain microcapillaries and endothelial and glial cells for ex vivo studies and cell culture.

    PubMed

    Lenhard, Thorsten; Hülsermann, Uta; Martinez-Torres, Francisco; Fricker, Gert; Meyding-Lamadé, Uta

    2013-06-26

    The blood-brain barrier is morphologically composed of cerebral microcapillary endothelium through its tight junctions. It serves as a mechanical, metabolic and cellular barrier and can also protect the brain from pathogen invasion. Many brain diseases involve a disturbance of blood-brain barrier function either as a consequence of a noxa or as primary failure. In vitro models of the blood-brain barrier are suitable tools to study drug transport, pathogen transmigration and leukocyte diapedesis across the cerebral endothelium. Such models have previously been derived mainly from porcine or bovine brain tissues. We describe here a simple method by which rat cerebral microcapillaries and cells of glial origin can be quickly and simultaneously purified. By using a capillary fragment size restriction method based on glass bead columns different fractions can be separated: vital, long capillary fragments for ex vivo uptake studies and smaller capillary fragments for endothelial culture. Furthermore, fractions can be obtained for astroglial and oligodendroglial cell cultures. With this method both microcapillary enrichment and glial cell purification are quickly achieved, which reduces expenditure, number of required animals and laboratory working time. PMID:23665392

  1. Hydrogels as Extracellular Matrix Mimics for 3D Cell Culture

    PubMed Central

    Tibbitt, Mark W.; Anseth, Kristi S.

    2010-01-01

    Methods for culturing mammalian cells ex vivo are increasingly needed to study cell and tissue physiology and to grow replacement tissue for regenerative medicine. Two-dimensional culture has been the paradigm for typical in vitro cell culture; however, it has been demonstrated that cells behave more natively when cultured in three-dimensional environments. Permissive, synthetic hydrogels and promoting, natural hydrogels have become popular as three-dimensional cell culture platforms; yet, both of these systems possess limitations. In this perspective, we discuss the use of both synthetic and natural hydrogels as scaffolds for three-dimensional cell culture as well as synthetic hydrogels that incorporate sophisticated biochemical and mechanical cues as mimics of the native extracellular matrix. Ultimately, advances in synthetic–biologic hydrogel hybrids are needed to provide robust platforms for investigating cell physiology and fabricating tissue outside of the organism. PMID:19472329

  2. Progress Towards Drosophila Epithelial Cell Culture

    PubMed Central

    Simcox, Amanda

    2015-01-01

    Drosophila epithelial research is at the forefront of the field; however, there are no well-characterized epithelial cell lines that could provide a complementary in vitro model for studies conducted in vivo. Here, a protocol is described that produces epithelial cell lines. The method uses genetic manipulation of oncogenes or tumor suppressors to induce embryonic primary culture cells to rapidly progress to permanent cell lines. It is, however, a general method and the type of cells that comprise a given line is not controlled experimentally. Indeed, only a small fraction of the lines produced are epithelial in character. For this reason, additional work needs to be done to develop a more robust epithelial cell-specific protocol. It is expected that Drosophila epithelial cell lines will have great utility for in vitro analysis of epithelial biology, particularly high-throughput analyses such as RNAi screens. PMID:23097097

  3. CHARACTERIZATION OF ALVEOLAR EPITHELIAL CELLS CULTURED IN SEMIPERMEABLE HOLLOW FIBERS

    PubMed Central

    Grek, Christina L.; Newton, Danforth A.; Qiu, Yonhzhi; Wen, Xuejun; Spyropoulos, Demetri D.; Baatz, John E.

    2012-01-01

    Cell culture methods commonly used to represent alveolar epithelial cells in vivo have lacked airflow, a 3-dimensional air-liquid interface, and dynamic stretching characteristics of native lung tissue—physiological parameters critical for normal phenotypic gene expression and cellular function. Here the authors report the development of a selectively semipermeable hollow fiber culture system that more accurately mimics the in vivo microenvironment experienced by mammalian distal airway cells than in conventional or standard air-liquid interface culture. Murine lung epithelial cells (MLE-15) were cultured within semipermeable polyurethane hollow fibers and introduced to controlled airflow through the microfiber interior. Under these conditions, MLE-15 cells formed confluent monolayers, demonstrated a cuboidal morphology, formed tight junctions, and produced and secreted surfactant proteins. Numerous lamellar bodies and microvilli were present in MLE-15 cells grown in hollow fiber culture. Conversely, these alveolar type II cell characteristics were reduced in MLE-15 cells cultured in conventional 2D static culture systems. These data support the hypothesis that MLE-15 cells grown within our microfiber culture system in the presence of airflow maintain the phenotypic characteristics of type II cells to a higher degree than those grown in standard in vitro cell culture models. Application of our novel model system may prove advantageous for future studies of specific gene and protein expression involving alveolar epithelial or bronchiolar epithelial cells. PMID:19263283

  4. Plastid transformation of tobacco suspension cell cultures.

    PubMed

    Staub, Jeffrey M

    2014-01-01

    Chloroplast transformation has been extremely valuable for the study of plastid biology and gene expression, but the tissue culture methodology involved can be laborious, and it can take several months to obtain homoplasmic regenerated plants useful for molecular or physiological studies. In contrast, transformation of tobacco suspension cell plastids provides an easy and efficient system to rapidly evaluate the efficacy of multiple constructs prior to plant regeneration. Suspension cell cultures can be initiated from many cell types, and once established, can be maintained by subculture for more than a year with no loss of transformation efficiency. Using antibiotic selection, homoplasmy is readily achieved in uniform cell colonies useful for comparative gene expression analyses, with the added flexibility to subsequently regenerate plants for in planta studies. Plastids from suspension cells grown in the dark are similar in size and cellular morphology to those in embryogenic culture systems of monocot species, thus providing a useful model for understanding the steps leading to plastid transformation in those recalcitrant species. PMID:24599853

  5. Cultural Studies and Curricular Change.

    ERIC Educational Resources Information Center

    Carr, Jean Ferguson

    1990-01-01

    The renaming of literature appreciation as cultural studies marks a rethinking of what is experienced as cultural materials, going beyond reading and writing to media, popular culture, newspapers, advertising, textbooks, and advice manuals. It also marks the movement away from the study of an object to the study of criticism. (MSE)

  6. Cultured Human Epidermal Cells Do Not Synthesize HLA-DR

    Microsoft Academic Search

    Vera B. Morhenn; Claudia J. Benike; Alvin J. Cox; Dominique J. Charron; Edgar G. Engleman

    1982-01-01

    All nucleated cells express HLA-A, B, and C antigens. However, only a few cells, including epidermal cells, demonstrate HLA-DR antigens which are potent transplantation immunogens in man. The current study was undertaken to determine if epidermal cells continue to synthesize and\\/or express HLA-DR antigens after prolonged in vitro culture. Epidermal cells cultured for 7 days or more no longer stimulated

  7. Gonococcal and meningococcal pathogenesis as defined by human cell, cell culture, and organ culture assays.

    PubMed Central

    Stephens, D S

    1989-01-01

    Human cells, cell cultures, and organ cultures have been extremely useful for studying the events that occur when gonococci and meningococci encounter human mucosal surfaces. The specificity and selectivity of these events for human cells are striking and correlate with the adaptation of these pathogens for survival on human mucous membranes. To colonize these sites, meningococci and gonococci have developed mechanisms to damage local host defenses such as the mucociliary blanket, to attach to epithelial cells, and to invade these cells. Attachment to epithelial cells mediated by pili, and to some types of cells mediated by PIIs, serves to anchor the organism close to sources of nutrition and allows multiplication. Intracellular invasion, possibly initiated by the major porin protein, may provide additional nutritional support and protection from host defenses. Mucosal invasion may also result in access of gonococci and meningococci to the bloodstream, leading to dissemination. Images PMID:2497953

  8. Culture surface influence on T-cell phenotype and function.

    PubMed

    Hashimdeen, Shaikh Shimaz; Römhild, Andy; Schmueck, Michael; Kratz, Karl; Lendlein, Andreas; Kurtz, Andreas; Reinke, Petra

    2013-01-01

    When dealing with T lymphocyte culture there is currently very less information available about the interaction between T-cells and the culture system. In this study we look at the influence of the culture chamber on T-cell proliferation in two main aspects of the culture system, namely: culture chamber material and geometry. The study was carried out using unique polymeric closed cell culture inserts, which were processed via injection moulding from polystyrene (PS), polycarbonate (PC), polyetherurethane (PEU), polystyrene-co-acrylonitrile (PSAN) and polyetherimide (PEI). Furthermore culture chamber geometry was studied using commercially available 24, 12 and 6-well plates prepared from tissue culture plastic (TCP). For T lymphocyte stimulation two methods were used involving either EBV peptide pools or MACS iBead particles depending on the experiment performed. Culture was done with 1645 RPMI medium supplemented with foetal calf serum, penicillin, streptomycin and rhIL-2. We found four materials out of five we tested (PS, PC, PSAN and PEI) exhibited similar fold expansions with minimal influence on proportions of CD4 and CD8, while PEU had a negative influence on T cell growth along with adversely affected CD4/CD8 proportions. Changes in the geometry of TCP had no effect on T cell growth or maturation rather the size of geometry seems to have more influence on proliferation. T-cells appear to prefer smaller geometries during initial stages of culture while towards the end of the culture size becomes less significant to cell proliferation. The parameters tested in this study have significant influences on T-cell growth and are necessary to consider when designing and constructing expansion systems for antigen specific T lymphocytes. This is important when culturing T-cells for immunotherapeutic applications where antigen specificity, T-cell maturation and function should remain unaffected during culture. PMID:24099989

  9. Catechin production in cultured Polygonum hydropiper cells

    Microsoft Academic Search

    Kanji Ono; Mayumi Nakao; Masao Toyota; Yoshimi Terashi; Masashi Yamada; Tetsuya Kohno; Yoshinori Asakawa

    1998-01-01

    Callus and suspension-cultured cells were induced from hypocotyls of Polygonum hydropiper seedlings. Both the callus and suspension-cultured cells produced mainly (+)-catechin accompanied by (?)-epicatechin and (?)-epicatechin-3-O-gallate. The (+)-catechin production of suspension-cultured cells increased with cell growth and reached the maximal value (29.0mgg?1 dry wt) after 6days from the start of subculture. This is the highest value of (+)-catechin content among

  10. Somaclonal Variation Is Induced De Novo via the Tissue Culture Process: A Study Quantifying Mutated Cells in Saintpaulia

    PubMed Central

    Sato, Mitsuru; Hosokawa, Munetaka; Doi, Motoaki

    2011-01-01

    Background The origin of somaclonal variation has not been questioned previously, i.e., “pre-existing mutations” in explants and “newly induced mutations” arising from the tissue culture process have not been distinguished. This is primarily because there has been no reliable molecular method for estimating or quantifying variation. Methodology/Principal Findings We adopted a petal-variegated cultivar of Saintpaulia ‘Thamires’ (Saintpaulia sp.) as the model plant. Based on the difference between the pre- and post-transposon excision sequence of the promoter region of flavonoid 3?, 5?-hydoroxylase (F3?5?H), we estimated mutated (transposon-excised) cell percentages using a quantitative real-time PCR. Mutated cell percentages in leaf laminae used as explants was 4.6 and 2.4% in highly or low variegation flower plants, respectively, although the occurrences of blue color mutants in their regenerants were more than 40%. Preexisting mutated cell percentages in cultured explants were considerably lower than the mutated plant percentage among total regenerants via tissue culture. Conclusions/Significance The estimation of mutated cell percentages became possible using the quantitative real-time PCR. The origins of mutations were successfully distinguished; it was confirmed that somaclonal variations are mainly caused by newly generated mutations arising from tissue culture process. PMID:21853148

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

    PubMed Central

    Antoni, Delphine; Burckel, Hélène; Josset, Elodie; Noel, Georges

    2015-01-01

    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. PMID:25768338

  12. Chromosome-wide aneuploidy study of cultured circulating myeloid progenitor cells from workers occupationally exposed to formaldehyde.

    PubMed

    Lan, Qing; Smith, Martyn T; Tang, Xiaojiang; Guo, Weihong; Vermeulen, Roel; Ji, Zhiying; Hu, Wei; Hubbard, Alan E; Shen, Min; McHale, Cliona M; Qiu, Chuangyi; Liu, Songwang; Reiss, Boris; Beane-Freeman, Laura; Blair, Aaron; Ge, Yichen; Xiong, Jun; Li, Laiyu; Rappaport, Stephen M; Huang, Hanlin; Rothman, Nathaniel; Zhang, Luoping

    2015-01-01

    Formaldehyde (FA) is an economically important industrial chemical to which millions of people worldwide are exposed environmentally and occupationally. Recently, the International Agency for Cancer Research concluded that there is sufficient evidence that FA causes leukemia, particularly myeloid leukemia. To evaluate the biological plausibility of this association, we employed a chromosome-wide aneuploidy study approach, which allows the evaluation of aneuploidy and structural chromosome aberrations (SCAs) of all 24 chromosomes simultaneously, to analyze cultured myeloid progenitor cells from 29 workers exposed to relatively high levels of FA and 23 unexposed controls. We found statistically significant increases in the frequencies of monosomy, trisomy, tetrasomy and SCAs of multiple chromosomes in exposed workers compared with controls, with particularly notable effects for monosomy 1 [P = 6.02E-06, incidence rate ratio (IRR) = 2.31], monosomy 5 (P = 9.01E-06; IRR = 2.24), monosomy 7 (P = 1.57E-05; IRR = 2.17), trisomy 5 (P = 1.98E-05; IRR = 3.40) and SCAs of chromosome 5 (P = 0.024; IRR = 4.15). The detection of increased levels of monosomy 7 and SCAs of chromosome 5 is particularly relevant as they are frequently observed in acute myeloid leukemia. Our findings provide further evidence that leukemia-related cytogenetic changes can occur in the circulating myeloid progenitor cells of healthy workers exposed to FA, which may be a potential mechanism underlying FA-induced leukemogenesis. PMID:25391402

  13. Maintenance of primary cell cultures of immunocytes from Cacopsylla spp. psyllids: a new in vitro tool for the study of crop pest insects.

    PubMed

    Monti, M; Mandrioli, M; Bextine, B; Hunter, W B; Alma, A; Tedeschi, R

    2014-10-01

    Primary cell cultures of immunocytes have been developed from the three psyllid species Cacopsylla melanoneura, Cacopsylla pyri (vectors of 'Candidatus Phytoplasma mali' and 'Candidatus Phytoplasma pyri', respectively) and Cacopsylla crataegi. The medium most suitable of those evaluated was Hert-Hunter 70 (HH70) psyllid medium. In fact, good survival and proliferation of the Cacopsylla immunocytes for over 60 d were observed, with mitosis activities starting at 15-d post culture. Moreover, adhesion and phagocytosis activities were confirmed for all the psyllid cell cultures by functionality tests. Morphological examination of cultured immunocytes revealed the presence of different cell types in all the three psyllid species in accordance to published data about insect immunocytes. The in vitro maintenance of psyllid immunocytes represents a powerful tool for a wide range of applications, especially for psyllid cell biology. In particular, in-depth studies on the biology of psyllids as vector insects as well as analyses to understand the mechanisms behind the interactions with pathogens and symbionts are now possible. These cultures can be used as an in vitro model to study psyllid humoral immune responses, which also will allow in-depth investigations on the abilities of psyllids as vectors of phytoplasmas. All these applications provide new opportunities to develop more focused and specific pest control strategies. PMID:24934235

  14. Centrifugation of Cultured Osteoblasts And Macrophages as a Model To Study How Gravity Regulates The Function of Skeletal Cells

    NASA Technical Reports Server (NTRS)

    Globus, Ruth K.; Searby, Nancy D.; Almeida, Eduardo A. C.; Sutijono, Darrell; Yu, Joon-Ho; Malouvier, Alexander; Doty, Steven B.; Morey-Holton, Emily; Weinstein, Steven L.; Dalton, Bonnie P. (Technical Monitor)

    2000-01-01

    Mechanical loading helps define the architecture of weight-bearing bone via the tightly regulated process of skeletal turnover. Turnover occurs by the concerted activity of osteoblasts, responsible for bone formation. and osteoclasts, responsible for bone resorption. Osteoclasts are specialized megakaryon macrophages, which differentiate from monocytes in response to resorption stimuli, such as reduced weight-bearing. Habitation in space dramatically alters musculoskeletal loading, which modulates both cell function and bone structure. Our long-term objective is to define the molecular and cellular mechanisms that mediate skeletal adaptations to altered gravity environments. Our experimental approach is to apply hypergravity loads by centrifugation to rodents and cultured cells. As a first step, we examined the influence of centrifugation on the structure of cancellous bone in rats to test the ability of hypergravity to change skeletal architecture. Since cancellous bone undergoes rapid turnover we expected the most dramatic structural changes to occur in the shape of trabeculae of weight-bearing, cancellous bone. To define the cellular responses to hypergravity loads, we exposed cultured osteoblasts and macrophages to centrifugation. The intraosseous and intramedullary pressures within long bones in vivo reportedly range from 12-40 mm Hg, which would correspond to 18-59 gravity (g) in our cultures. We assumed that hydrostatic pressure from the medium above the cell layer is at least one major component of the mechanical load generated by centrifuging cultured cells. and therefore we exposed the cells to 10-50g. In osteoblasts, we examined the structure of their actin and microtubule networks, production of prostaglandin E2 (PGE2), and cell survival. Analysis of the shape of the cytoskeletal networks provides evidence for the ability of centrifugation to affect cell structure, while the production of PGE2 serves as a convenient marker for mechanical stimulation. We examined cell survival, reasoning that osteoblasts might mold skeletal structure in a hypergravity environment in part by regulating apoptosis and thus the duration of osteoblast productivity. Finally, we tested the influence of centrifugation on microbial activation of a macrophage cell line (RAW264.7). In response to the appropriate hormonal stimulation, this cell line is reportedly capable of undergoing differentiation to express osteoclast markers. In addition, a component of the cell wall of gram-negative bacteria, lipopolysaccaride (LPS), stimulates the formation of osteoclasts in vivo. Thus we tested the influence on centrifugation on RAW264.7 cells stimulated with LPS to provide an index of the function of osteoclast precursors.

  15. Three-dimensional tissue culture based on magnetic cell levitation

    NASA Astrophysics Data System (ADS)

    Souza, Glauco R.; Molina, Jennifer R.; Raphael, Robert M.; Ozawa, Michael G.; Stark, Daniel J.; Levin, Carly S.; Bronk, Lawrence F.; Ananta, Jeyarama S.; Mandelin, Jami; Georgescu, Maria-Magdalena; Bankson, James A.; Gelovani, Juri G.; Killian, T. C.; Arap, Wadih; Pasqualini, Renata

    2010-04-01

    Cell culture is an essential tool in drug discovery, tissue engineering and stem cell research. Conventional tissue culture produces two-dimensional cell growth with gene expression, signalling and morphology that can be different from those found in vivo, and this compromises its clinical relevance. Here, we report a three-dimensional tissue culture based on magnetic levitation of cells in the presence of a hydrogel consisting of gold, magnetic iron oxide nanoparticles and filamentous bacteriophage. By spatially controlling the magnetic field, the geometry of the cell mass can be manipulated, and multicellular clustering of different cell types in co-culture can be achieved. Magnetically levitated human glioblastoma cells showed similar protein expression profiles to those observed in human tumour xenografts. Taken together, these results indicate that levitated three-dimensional culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and may be more feasible for long-term multicellular studies.

  16. Three-dimensional Tissue Culture Based on Magnetic Cell Levitation

    PubMed Central

    Souza, Glauco R.; Molina, Jennifer R.; Raphael, Robert M.; Ozawa, Michael G.; Stark, Daniel J.; Levin, Carly S.; Bronk, Lawrence F.; Ananta, Jeyarama S.; Mandelin, Jami; Georgescu, Maria-Magdalena; Bankson, James A.; Gelovani, Juri G.

    2015-01-01

    Cell culture is an essential tool for drug discovery, tissue engineering, and stem cell research. Conventional tissue culture produces two-dimensional (2D) cell growth with gene expression, signaling, and morphology that can differ from those in vivo and thus compromise clinical relevancy1–5. Here we report a three-dimensional (3D) culture of cells based on magnetic levitation in the presence of hydrogels containing gold and magnetic iron oxide (MIO) nanoparticles plus filamentous bacteriophage. This methodology allows for control of cell mass geometry and guided, multicellular clustering of different cell types in co-culture through spatial variance of the magnetic field. Moreover, magnetic levitation of human glioblastoma cells demonstrates similar protein expression profiles to those observed in human tumor xenografts. Taken together, these results suggest levitated 3D culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and allows for long-term multi-cellular studies. PMID:20228788

  17. Glial activation modulates glutamate neurotoxicity in cerebellar granule cell cultures.

    PubMed

    Pérez-Capote, Kamil; Serratosa, Joan; Solà, Carme

    2004-02-01

    We studied the influence of glial cells on the neuronal response to glutamate toxicity in cerebellar granule cell cultures. We compared the effect of glutamate on neuronal viability in neuronal vs. neuronal-glial cultures and determined this effect after pretreating the cultures with the lipopolysaccharide (LPS) of Escherichia coli, agent widely used to induce glial activation. Morphological changes in glial cells and nitric oxide (NO) production were evaluated as indicators of glial activation. We observed that glutamate neurotoxicity in neuronal-glial cultures was attenuated in a certain range of glutamate concentration when compared to neuronal cultures, but it was enhanced at higher glutamate concentrations. This enhanced neurotoxicity was associated with morphological changes in astrocytes and microglial cells in the absence of NO production. LPS treatment induced morphological changes in glial cells in neuronal-glial cultures as well as NO production. These effects occurred in the absence of significant neuronal death. However, when LPS-pretreated cultures were treated with glutamate, the sensitivity of neuronal-glial cultures to glutamate neurotoxicity was increased. This was accompanied by additional morphological changes in glial cells in the absence of a further increase in NO production. These results suggest that quiescent glial cells protect neuronal cells from glutamate neurotoxicity, but reactive glial cells increase glutamate neurotoxicity. Therefore, glial cells play a key role in the neuronal response to a negative stimulus, suggesting that this response can be modified through an action on glial cells. PMID:14730699

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

    NASA Technical Reports Server (NTRS)

    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.

  19. Cell culture techniques in honey bee research

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cell culture techniques are indispensable in most if not all life science disciplines to date. Wherever cell culture models are lacking scientific development is hampered. Unfortunately this has been and still is the case in honey bee research because permanent honey bee cell lines have not yet been...

  20. Seed coat removal improves Fe bioavailability in cooked lentils: studies using an in vitro digestion/Caco-2 cell culture model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study examined the range of Fe concentration and relative Fe bioavailability of 24 varieties of cooked lentils, as well as the impact of seed coat removal on lentil Fe nutritional quality. Relative Fe bioavailability was assessed by the in vitro/Caco-2 cell culture method. While Fe concentrat...

  1. Applicability of integrated cell culture quantitative PCR (ICC-qPCR) for the detection of infectious adenovirus type 2 in UV disinfection studies

    EPA Science Inventory

    Human adenovirus is relatively resistant to UV radiation and has been used as a conservative testing microbe for evaluations of UV disinfection systems as components of water treatment processes. In this study, we attempted to validate the applicability of integrated cell culture...

  2. Serial culturing of human bronchial epithelial cells derived from biopsies

    Microsoft Academic Search

    Petra M. de Jong; Marianne A. J. A. van Sterkenburg; Johanna A. Kempenaar; Joop H. Dijkman; Maria Ponec

    1993-01-01

    Summary  In the present study we describe the establishment of serial cultures of human bronchial epithelial cells derived from biopsies\\u000a obtained by fiberoptic bronchoscopy. The cell cultures were initiated from small amounts of material (2 mm forceps biopsies)\\u000a using either explants or epithelial cell suspensions in combination with a feeder-layer technique. The rate of cell proliferation\\u000a and the number of passages

  3. Culture medium study of human mesenchymal stem cells for practical use of tissue engineering and regenerative medicine.

    PubMed

    Nakamura, Sayaka; Yamada, Yoichi; Baba, Shunsuke; Kato, Harumi; Kogami, Hiroyuki; Takao, Makoto; Matsumoto, Naoyuki; Ueda, Minoru

    2008-01-01

    Mesenchymal stem cells (MSCs) are multipotent cells that have potential to differentiate into various phenotypes and appear useful for therapeutic applications in regenerative medicine. Mesenchymal Stem Cell Basal Medium (MSCBM; Cambrex) is a widespread and suitable medium used in MSC cultivation, but it is extremely difficult to use generally for clinical treatment because of its unclear traceability and cost. Assessment of cost-effectiveness is a critical issue for successful practical application; therefore, we have evaluated the effects of a generally used medium, Dulbecco's Modified Eagle's Medium (D-MEM) on the expansion of MSCs in comparison with MSCBM. To isolate human MSCs, bone marrow aspirates were taken and cultured in MSCBM or D-MEM. Proliferation assay indicated that MSCs isolated in both media showed a similar growth rate. When supplemented with osteo-inductive reagents, alkaline phosphatase activity was not significantly different between cells in D-MEM and MSCBM. Moreover, the cells expressed identical mesenchymal lineage markers, but not endothelial and hematopoietic lineage markers. Our findings suggest that cells obtained from bone marrow and cultured in D-MEM might possess proliferative capacity and the potential to differentiate into an osteogenic lineage. In conclusion, D-MEM might be a suitable basal medium for the cultivation of MSCs for clinical applications. PMID:18725693

  4. [Polysaccharides of cell cultures of Silene vulgaris].

    PubMed

    Giunter, E A; Ovodov, Iu S

    2007-01-01

    Callus and suspension cultures of campion (Silene vulgaris) produced pectin polysaccharides, similar in structure to the polysaccharides of intact plants. The major components of the pectins were D-galacturonic acid, galactose, arabinose, and rhamnose residues. The maximum content of pectins was found in callus. The monosaccharide composition of arabinogalactans isolated from cells and a culture medium of callus cultures were similar, with the ratio between arabinose and galactose of 1: (2.3-6.5) being retained. The arabinogalactans from the cells and culture medium of the suspension cultures also had a similar structure, and the arabinose to galactose ratio was 1: (1.5-1.8). In contrast to the callus cultures, the suspension cultures produced arabinogalactans with an increased content of arabinose residues and a decreased content of galactose residues. The greatest content of arabinogalactan was detected in the culture medium of the suspension cultures. PMID:17345866

  5. Establishment of callus and cell suspension cultures from Gypsophila paniculata leaf segments and study of the attachment of host cells by Erwinia herbicola pv. gypsophilae

    Microsoft Academic Search

    Mazen Nayef Salman

    2002-01-01

    Callus and cell suspension cultures were initiated from leaf segments of G. paniculata. Fresh and dry weights measurements of callus showed that callus growth was optimal on MS medium supplemented with 1.0 mg l-1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 mg l-1 benzyladenin (BA). Calli cultured on this medium, showed a two-fold increase in fresh weight by the fourth week of

  6. Study on reproductive endocrinology of human placenta--culture of highly purified cytotrophoblast cell in serum-free hormone supplemented medium.

    PubMed

    Li, R H; Zhuang, L Z

    1991-08-01

    A new method of long-term culture of cytotrophoblast cells in serum-free medium has been developed. Cytotrophoblast cells were isolated with cold trypsin and purified by unit gravity sedimentation through BSA density gradients. The cells were cultured in the FD medium with supplement of EGF, insulin, transferrin and sodium selenite. They could survive over three weeks. The results showed that both EGF and insulin stimulated hCG and progesterone secretion and that sodium selenite elevated hCG output but not progesterone secretion. Transferrin produced synergistic effect with EGF and insulin on hCG and progesterone secretion but it was ineffective when used alone. This study demonstrates that the four growth factors mentioned above are essential for the survival of cytotrophoblast cells in vitro. It is therefore suggested that EGF, insulin and selenium may possibly be involved in the regulation of hCG and progesterone secretion in the human placenta. PMID:1801845

  7. Effect of Latanoprost on the Extracellular Matrix of the Ciliary Muscle. A Study on Cultured Cells and Tissue Sections

    Microsoft Academic Search

    ANETTE OCKLIND

    1998-01-01

    Prostaglandin F2?and its analogue latanoprost, both prostanoid FP receptor agonists, reduce the intraocular pressure mainly by enhancing uveoscleral outflow. Changes in the extracellular matrix of the ciliary muscle may be involved in the increased outflow. The effect of latanoprost and prostaglandin F2?on the extracellular matrix of the ciliary muscle was investigated.Cell cultures of human ciliary muscle were treated with latanoprost

  8. Immunohistochemical analyses of cell–cell interactions during hepatic organoid formation from fetal mouse liver cells cultured in vitro

    Microsoft Academic Search

    Yoshinori Sugiyama; Toru Koike; Nobuyoshi Shiojiri

    2007-01-01

    Cell–cell interactions among cell types constituting the fetal liver such as hepatoblasts, stellate cells and endothelial\\u000a cells lead to functional lobule development. The present study was undertaken to investigate hepatic histogenesis in the primary\\u000a culture of E12.5 mouse livers, including cell–cell and cell–matrix interactions. Fetal livers were dispersed with protease\\u000a treatment and cultured for 5 days. Cellular adhesion of each hepatic

  9. Acetaldehyde and hexanaldehyde from cultured white cells

    PubMed Central

    Shin, Hye-Won; Umber, Brandon J; Meinardi, Simone; Leu, Szu-Yun; Zaldivar, Frank; Blake, Donald R; Cooper, Dan M

    2009-01-01

    Background Noninvasive detection of innate immune function such as the accumulation of neutrophils remains a challenge in many areas of clinical medicine. We hypothesized that granulocytes could generate volatile organic compounds. Methods To begin to test this, we developed a bioreactor and analytical GC-MS system to accurately identify and quantify gases in trace concentrations (parts per billion) emitted solely from cell/media culture. A human promyelocytic leukemia cell line, HL60, frequently used to assess neutrophil function, was grown in serum-free medium. Results HL60 cells released acetaldehyde and hexanaldehyde in a time-dependent manner. The mean ± SD concentration of acetaldehyde in the headspace above the cultured cells following 4-, 24- and 48-h incubation was 157 ± 13 ppbv, 490 ± 99 ppbv, 698 ± 87 ppbv. For hexanaldehyde these values were 1 ± 0.3 ppbv, 8 ± 2 ppbv, and 11 ± 2 ppbv. In addition, our experimental system permitted us to identify confounding trace gas contaminants such as styrene. Conclusion This study demonstrates that human immune cells known to mimic the function of innate immune cells, like neutrophils, produce volatile gases that can be measured in vitro in trace amounts. PMID:19402909

  10. Culture of Cells from Amphibian Embryos.

    ERIC Educational Resources Information Center

    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)

  11. Effects of pulsing electromagnetic fields on cultured cartilage cells

    Microsoft Academic Search

    A. Sakai; K. Suzuki; T. Nakamura; T. Norimura; T. Tsuchiya

    1991-01-01

    In order to evaluate the effects of pulsing electromagnetic fields (PEMFs) on cell proliferation and glycosaminoglycan (GAG) synthesis and to study the action site of PEMF stimulation in the cells, we performed a series of experiments on rabbit costal growth cartilage cells and human articular cartilage cells in culture. A PEMF stimulator was made using a Helmholz coil. Repetitive pulse

  12. Skeletal muscle satellite cells cultured in simulated microgravity

    Microsoft Academic Search

    Greg Molnar; Nancy A. Schroedl; Steve R. Gonda; Charles R. Hartzell

    1997-01-01

    Summary  Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells.\\u000a Satellite cells retain the capacity to proliferate and differentiate in vitro and, therefore, provide a useful model to study postnatal muscle development. Most culture systems used to study postnatal\\u000a muscle development are limited by the two-dimensional (2-D) confines of the culture dish. Limiting proliferation

  13. Lymphokine-activated killer cells lyse human renal cancer cell lines and cultured normal kidney cells.

    PubMed Central

    Miltenburg, A M; Meijer-Paape, M E; Daha, M R; Paul, L C

    1988-01-01

    In this study, we investigated whether or not lymphokine-activated killer (LAK) cells can damage renal tissue and therefore whether they may contribute to graft destruction during kidney allograft rejection. Human peripheral blood mononuclear cells were activated with a lymphokine preparation and the resulting LAK cells were tested against kidney cells from various sources. Renal cancer cells as well as cultured normal kidney cells were efficiently lysed by LAK cells, as assessed with Cr-labelled target cells, showing that both cell types are sensitive to LAK cell-mediated cytolysis. PMID:3259208

  14. Nanotechnology in drug delivery: the need for more cell culture based studies in screening

    PubMed Central

    2014-01-01

    Advances in biomedical science are leading to upsurge synthesis of nanodelivery systems for drug delivery. The systems were characterized by controlled, targeted and sustained drug delivery ability. Humans are the target of these systems, hence, animals whose systems resembles humans were used to predict outcome. Thus, increasing costs in money and time, plus ethical concerns over animal usage. However, with consideration and planning in experimental conditions, in vitro pharmacological studies of the nanodelivery can mimic the in vivo system. This can function as a simple method to investigate the effect of such materials without endangering animals especially at screening phase. PMID:25057288

  15. Regulating apoptosis in mammalian cell cultures

    Microsoft Academic Search

    Nilou Arden; M. J. Betenbaugh

    2006-01-01

    Cell culture technology has become a widely accepted method used to derive therapeutic and diagnostic protein products. Mammalian\\u000a cells adapted to grow in bioreactors now play an integral role in the development of these biologicals. A major limiting factor\\u000a determining the output efficiency of mammalian cell cultures however, is apoptosis or programmed cell death. Methods to delay\\u000a apoptosis and increase

  16. Contact inhibition of movement in the cultures of transformed cells.

    PubMed

    Guelstein, V I; Ivanova, O Y; Margolis, L B; Vasiliev, J M; Gelfand, I M

    1973-07-01

    Results of cell-cell collisions were studied with the aid of time-lapse microcinematography in primary cultures of normal mouse-embryo fibroblast-like cells and in cultures of transformed mouse cells of two types: (a) primary fibroblast-like cells transformed by Moloney mouse sarcoma virus; (b) neoplastic fibroblasts of the CIM strain. Collisions of normal fibroblast-like cells and CIM cells in mixed cultures were also analyzed. Classification of the results of collisions was based on observation of the movements of the active cell edge during the first hour after the moment when this edge had contacted another cell. Three types of collision results were detected: halt of the active edge, overlapping, and underlapping. The relative number of overlappings was not higher and that of halts not lower in the cultures of transformed cells as compared with those of normal cells. Analysis of the collisions of normal fibroblasts with transformed cells gave similar results. Thus, the altered morphology of the cultures of these transformed cells cannot be explained by loss of contact inhibition of movement leading to increased ability of cells to move over the surfaces of other cells after collision. PMID:4516201

  17. Silk Protein Sericin Improves Mammalian Cell Culture

    Microsoft Academic Search

    Satoshi Terada; Naoki Takada; Kazuaki Itoh; Takuya Saitoh; Masahiro Sasaki; Hideyuki Yamada

    Mammalian cell cultures generally require supplementation with fetal bovine serum (FBS), or its replacement, into the culture\\u000a media. Sera contain various unidentified and unknown factors and the risk of infections, including bovine spongiform encephalopathy\\u000a (BSE), is of serious concern. Therefore, the supplementation of sera into culture media is a major obstacle for purification\\u000a to recover cell products and this limits

  18. In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

    PubMed Central

    Ahmad, Shama; Ahmad, Aftab; Neeves, Keith B.; Hendry-Hofer, Tara; Loader, Joan E.; White, Carl W.; Veress, Livia

    2014-01-01

    Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types. PMID:24837339

  19. Mutagenesis of haploid cultured frog cells

    SciTech Connect

    Mezger-Freed, L.

    1973-01-01

    From 13th international congress of genetics; Berkeley, California (20 Aug 1973). Haploid cells afford an opportunity to test some of the assumptions from bacterial genetics which have been adopted by somatic cell geneticists. Haploid cultured cell lines derived from the grass frog Rana pipiens were compared to diploid cell lines in order to test a model which predicts that recessive mutations will be expressed in diploid cells with a frequency equal to the square of that in haploid cells. Haploid and diploid monolayer cultures were compared for (1) survival after exposure to compounds known to be mutagenic for bacteria (a measure of the frequency with which lethal mutations are expressed), and (2) the induction of drug-resistant variants (putative mutants) by such compounds. The proportion of cells that survived from diploid cultures was no more than ten times that from haploid cultures, a much smaller difference than predicted. Furthermore, the frequency of drug-resistant variants was independent of ploidy. (auth)

  20. Use of an insect cell culture growth medium to isolate bacteria from horses with effusive, fibrinous pericarditis: a preliminary study.

    PubMed

    Jones, Samuel L; Valenzisi, Amy; Sontakke, Sushama; Sprayberry, Kimberly A; Maggi, Ricardo; Hegarty, Barbara; Breitschwerdt, Edward

    2007-03-31

    Effusive, fibrinous pericarditis is an uncommon disease entity in horses. In 2001, pericarditis occurred in conjunction with an epizootic in central Kentucky that was associated with exposure to eastern tent caterpillars (ETCs). Bacterial isolation from equine pericardial fluid samples was attempted using an insect cell culture growth medium (ICCGM). Using previously cultured, stored frozen samples from four horses with fibrinous pericarditis, inoculation of 10% blood agar plates yielded no growth, whereas simultaneous inoculation of ICCGM resulted in the isolation of Proprionibacterium acnes, Staphylococcus equorum, a Streptococcus sp. and Pseudomonas rhodesiae from pericardial fluid samples. A similar or novel caterpillar-associated bacteria was not identified; however, use of an ICCGM might enhance isolation of bacteria from equine pericardial fluid. PMID:17204376

  1. Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)-based Quantitative Proteomics Study of a Thyroid Hormone-regulated Secretome in Human Hepatoma Cells*

    PubMed Central

    Chen, Cheng-Yi; Chi, Lang-Ming; Chi, Hsiang-Cheng; Tsai, Ming-Ming; Tsai, Chung-Ying; Tseng, Yi-Hsin; Lin, Yang-Hsiang; Chen, Wei-Jan; Huang, Ya-Hui; Lin, Kwang-Huei

    2012-01-01

    The thyroid hormone, 3, 3?,5-triiodo-l-thyronine (T3), regulates cell growth, development, differentiation, and metabolism via interactions with thyroid hormone receptors (TRs). However, the secreted proteins that are regulated by T3 are yet to be characterized. In this study, we used the quantitative proteomic approach of stable isotope labeling with amino acids in cell culture coupled with nano-liquid chromatography-tandem MS performed on a LTQ-Orbitrap instrument to identify and characterize the T3-regulated proteins secreted in human hepatocellular carcinoma cell lines overexpressing TR?1 (HepG2-TR?1). In total, 1742 and 1714 proteins were identified and quantified, respectively, in three independent experiments. Among these, 61 up-regulated twofold and 11 down-regulated twofold proteins were identified. Eight proteins displaying increased expression and one with decreased expression in conditioned media were validated using Western blotting. Real-time quantitative RT-PCR further disclosed induction of plasminogen activator inhibitor-1 (PAI-1), a T3 target, in a time-course and dose-dependent manner. Serial deletions of the PAI-1 promoter region and subsequent chromatin immunoprecipitation assays revealed that the thyroid hormone response element on the promoter is localized at positions –327/–312. PAI-1 overexpression enhanced tumor growth and migration in a manner similar to what was seen when T3 induced PAI-1 expression in J7-TR?1 cells, both in vitro and in vivo. An in vitro neutralizing assay further supported a crucial role of secreted PAI-1 in T3/TR-regulated cell migration. To our knowledge, these results demonstrate for the first time that proteins involved in the urokinase plasminogen activator system, including PAI-1, uPAR, and BSSP4, are augmented in the extra- and intracellular space of T3-treated HepG2-TR?1 cells. The T3-regulated secretome generated in the current study may provide an opportunity to establish the mechanisms underlying T3-associated tumor progression and prognosis. PMID:22171322

  2. Biochemical Assays of Cultured Cells

    NASA Technical Reports Server (NTRS)

    Barlow, G. H.

    1985-01-01

    Subpopulations of human embryonic kidney cells isolated from continuous flow electrophoresis experiments performed at McDonnell Douglas and on STS-8 have been analyzed. These analyses have included plasminogen activator assays involving indirect methodology on fibrin plated and direct methodology using chromogenic substrates. Immunological studies were performed and the conditioned media for erythropoietin activity and human granulocyte colony stimulating (HGCSF) activity was analyzed.

  3. Sodium 22+ washout from cultured rat cells

    SciTech Connect

    Kino, M.; Nakamura, A.; Hopp, L.; Kuriyama, S.; Aviv, A.

    1986-10-01

    The washout of Na/sup +/ isotopes from tissues and cells is quite complex and not well defined. To further gain insight into this process, we have studied /sup 22/Na/sup +/ washout from cultured Wistar rat skin fibroblasts and vascular smooth muscle cells (VSMCs). In these preparations, /sup 22/Na/sup +/ 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), /sup 22/Na/sup +/ 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 /sup 22/Na/sup +/ is apparently monoexponential. Calculations of the cellular Na/sup +/ concentrations, based on the /sup 22/Na/sup +/ 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 /sup 22/Na/sup +/ 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/sup +/ 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.

  4. [Studies on growth of brown adipose tissue by means of cell culture and angiogenesis in vitro techniques].

    PubMed

    Ueno, N; Nakamura, K; Yamashita, H; Kizaki, T; Ookawara, T; Ohno, H

    1994-02-01

    To elucidate the mechanism of brown adipose tissue (BAT) enlargement, we investigated the effects of norepinephrine (NE) and insulin on the in vitro growth of rat brown adipose cells (RBAC) and the capillary growth in angiogenesis in vitro using co-culture of bovine capillary endothelial cells (BCEC) and rat smooth muscle cells. In the primary cell culture, NE significantly enhanced the growth of RBAC in the range of 10(-9)-10(-5)M, whereas it did not stimulate the growth of BCEC. Insulin showed the same trend. Moreover, both NE and insulin appeared to increase the expression of mRNA for basic fibroblast growth factor (bFGF), which is a potent angiogenic factor, in RBAC. At 4 h after NE stimulation, the bFGF mRNA expression was considerably increased but it decreased markedly after 16 h. These results suggest that the bFGF mRNA expression in RBAC is quickly simulated by NE, wih resulting bFGF production. Actually, bFGF stimulated the RBAC growth up to about 170% of the control level. However, neither NE nor insulin stimulated the expression of the bFGF gene in BCEC. On the other hand, NE notably increased the capillary growth in vitro compared with insulin. It is thus possible that NE and insulin contribute to the growth of RBAC and endothelial cells partly through bFGF production by an autocrine mechanism, suggesting that both agonists play an important role not only in the thermogenic function of BAT but also in BAT enlargement. PMID:7510348

  5. Emulsions Containing Perfluorocarbon Support Cell Cultures

    NASA Technical Reports Server (NTRS)

    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.

  6. Multinucleated giant cells from fibroblast cultures

    PubMed Central

    Holt, DJ; Grainger, DW

    2011-01-01

    Many multinucleated giant cells are well-known to form from macrophage origin. Those formed from other cell types are less described, but may be as prevalent in pathological tissue. Giant multinucleated cells derived from secondary and primary fibroblast sources in various cultures with similar characteristics to foreign body giant cells are reported. Secondary-transformed NIH 3T3 fibroblasts rapidly fuse within 24 hours in contact co-cultures with RAW 264.7 immortalized macrophages, while 3T3 mono-cultures, non-contact (transwell) co-cultures, and macrophage-conditioned media-treated 3T3 mono-cultures all do not fuse. Primary-derived murine fibroblasts also form multinucleated cells, both in the presence or absence of co-cultured macrophages that increase during long-term culture (5–30 days). In contrast to 3T3 fusion, this primary cell phenomenon is not due to fibroblast fusion, but rather to nuclear division without cytokinesis. That these multinucleated fibroblasts can originate via different mechanisms may influence and distinguish their behaviors in conditions under which they may arise, including various in vitro culture assays, and in certain fibroblastic pathologies such as the foreign body response, fibrosis, cancer and aged tissue. PMID:21397323

  7. Constructing a High Density Cell Culture System

    NASA Technical Reports Server (NTRS)

    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.

  8. Primary cell cultures from fetal bovine hypothalamus and cerebral cortex: A reliable model to study P450 Arom and ? and ? estrogen receptors in vitro

    Microsoft Academic Search

    Antonella Peruffo; Genny Buson; Bruno Cozzi; Cristina Ballarin

    2008-01-01

    Estrogens synthesized by neural P450 aromatase (P450Arom) are implicated in many aspects of mammalian brain development and particularly in sexual differentiation of the central nervous system (CNS). This study analyzes the usefulness of an in vitro model based on bovine primary cell cultures from the hypothalamus and frontal cortex to investigate the role of P450Arom and estrogen receptors (ERs) in

  9. FINE STRUCTURE OF SMOOTH MUSCLE CELLS GROWN IN TISSUE CULTURE

    Microsoft Academic Search

    GORDON R. CAMPBELL; YASUO UEHARA; GERDA MARK; GEOFFREY BURNSTOCK

    1971-01-01

    The fine structure of smooth muscle cells of the embryo chicken gizzard cultured in mono- layer was studied by phase-contrast optics and electron microscopy . The smooth muscle cells were irregular in shape, but tended to be elongate . The nucleus usually contained prominent nucleoli and was large in relation to the cell body . When fixed with glutaralde- hyde,

  10. Algal culture studies for CELSS

    NASA Technical Reports Server (NTRS)

    Radmer, R.; Behrens, P.; Arnett, K.; Gladue, R.; Cox, J.; Lieberman, D.

    1987-01-01

    Microalgae are well-suited as a component of a Closed Environmental Life Support System (CELSS), since they can couple the closely related functions of food production and atmospheric regeneration. The objective was to provide a basis for predicting the response of CELSS algal cultures, and thus the food supply and air regeneration system, to changes in the culture parameters. Scenedesmus growth was measured as a function of light intensity, and the spectral dependence of light absorption by the algae as well as algal respiration in the light were determined as a function of cell concentration. These results were used to test and confirm a mathematical model that describes the productivity of an algal culture in terms of the competing processes of photosynthesis and respiration. The relationship of algal productivity to cell concentration was determined at different carbon dioxide concentrations, temperatures, and light intensities. The maximum productivity achieved by an air-grown culture was found to be within 10% of the computed maximum productivity, indicating that CO2 was very efficiently removed from the gas stream by the algal culture. Measurements of biomass productivity as a function of cell concentration at different light intensities indicated that both the productivity and efficiency of light utilization were greater at higher light intensities.

  11. Three dimensional spheroid cell culture for nanoparticle safety testing.

    PubMed

    Sambale, Franziska; Lavrentieva, Antonina; Stahl, Frank; Blume, Cornelia; Stiesch, Meike; Kasper, Cornelia; Bahnemann, Detlef; Scheper, Thomas

    2015-07-10

    Nanoparticles are widely employed for many applications and the number of consumer products, incorporating nanotechnology, is constantly increasing. A novel area of nanotechnology is the application in medical implants. The widespread use of nanoparticles leads to their higher prevalence in our environment. This, in turn, raises concerns regarding potential risks to humans. Previous studies have shown possible hazardous effects of some nanoparticles on mammalian cells grown in two-dimensional (2D) cultures. However, 2D in vitro cell cultures display several disadvantages such as changes in cell shape, cell function, cell responses and lack of cell-cell contacts. For this reason, the development of better models for mimicking in vivo conditions is essential. In the present work, we cultivated A549 cells and NIH-3T3 cells in three-dimensional (3D) spheroids and investigated the effects of zinc oxide (ZnO-NP) and titanium dioxide nanoparticles (TiO2-NP). The results were compared to cultivation in 2D monolayer culture. A549 cells in 3D cell culture formed loose aggregates which were more sensitive to the toxicity of ZnO-NP in comparison to cells grown in 2D monolayers. In contrast, NIH-3T3 cells showed a compact 3D spheroid structure and no differences in the sensitivity of the NIH-3T3 cells to ZnO-NP were observed between 2D and 3D cultures. TiO2-NP were non-toxic in 2D cultures but affected cell-cell interaction during 3D spheroid formation of A549 and NIH-3T3 cells. When TiO2-NP were directly added during spheroid formation in the cultures of the two cell lines tested, several smaller spheroids were formed instead of a single spheroid. This effect was not observed if the nanoparticles were added after spheroid formation. In this case, a slight decrease in cell viability was determined only for A549 3D spheroids. The obtained results demonstrate the importance of 3D cell culture studies for nanoparticle safety testing, since some effects cannot be revealed in 2D cell culture. PMID:25595712

  12. Studies on the synthesis of sterol carrier protein-2 in rat adrenocortical cells in monolayer culture. Regulation by ACTH and dibutyryl cyclic 3',5'-AMP

    SciTech Connect

    Trzeciak, W.H.; Simpson, E.R.; Scallen, T.J.; Vahouny, G.V.; Waterman, M.R.

    1987-03-15

    The effects of ACTH or dibutyryl cyclic AMP (Bt2cAMP) on the synthesis of sterol carrier protein-2 (SCP2) have been studied in rat adrenocortical cells in monolayer culture. Radiolabeling of total cellular proteins with (/sup 35/S)methionine and immunoprecipitation with antibodies directed against rat liver SCP2, followed by polyacrylamide gel electrophoresis and fluorography, showed a 3-4-fold increase in the rate of synthesis of SCP2 in cells treated for 48 h with ACTH (1 microM) or Bt2cAMP (0.1 mM). The induction of SCP2 synthesis depended upon the concentrations of ACTH or Bt2cAMP with an ED50 of 8 and 100 nM, respectively, and increased linearly with time between 12 and 48 h of treatment. Immunoprecipitation of SCP2 synthesized in a rabbit reticulocyte in vitro translation system programmed with RNA isolated from cells treated with ACTH or Bt2cAMP revealed increased synthesis of SCP2 compared to RNA from control cells. The immunoprecipitable rat adrenal SCP2, synthesized in a cell-free translation system, showed mobility corresponding to Mr of 14,400 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was clearly larger than immunodetectable SCP2 synthesized in cultured adrenal cells (Mr = 11,300). The electrophoretic mobilities of rat liver SCP2 synthesized in cultured cells and in a cell-free translation system were the same as the respective forms from rat adrenal.

  13. The effect of TRAIL molecule on cell viability in in vitro beta cell culture.

    PubMed

    Tekmen, I; Ozyurt, D; Pekçetin, C; Buldan, Z

    2007-06-01

    Insulin-dependent diabetes mellitus (IDDM) is an organ-specific autoimmune disorder triggered by autoreactive T cells directed to pancreas beta-cell antigens. In this disorder, more than 90% of beta cells are destroyed. Cell death may be mediated via soluble or membrane-bound cell death ligands. One of these ligands may be tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF-alpha superfamily. In the present study, we examined whether TRAIL had cytotoxic effects on adult rat pancreas beta cell cultures and INS1-E rat insulinoma cell line cultures or not. In this study, cell destruction models were built with TRAIL concentrations of 10, 100 and 1000 ng. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used for evaluating cell viability. It was detected that cell cultures with TRAIL added showed no differences statistically when compared with control cultures containing no toxic additions. These results showed that TRAIL did not have significant cytotoxic effects on pancreas beta cell culture and INS-1E rat insulinoma cell line cultures. Detection of the expression of TRAIL receptors and natural apoptosis inhibitor proteins will be favourable to investigate the resistance mechanisms to TRAIL-induced cell death in this cell culture system. PMID:17530468

  14. The Corticostriatal System in Dissociated Cell Culture

    PubMed Central

    Randall, Fiona E.; Garcia-Munoz, Marianela; Vickers, Catherine; Schock, Sarah C.; Staines, William A.; Arbuthnott, Gordon W.

    2011-01-01

    The sparse connectivity within the striatum in vivo makes the investigation of individual corticostriatal synapses very difficult. Most studies of the corticostriatal input have been done using electrical stimulation under conditions where it is hard to identify the precise origin of the cortical input. We have employed an in vitro dissociated cell culture system that allows the identification of individual corticostriatal pairs and have been developing methods to study individual neuron inputs to striatal neurons. In mixed corticostriatal cultures, neurons had resting activity similar to the system in vivo. Up/down states were obvious and seemed to encompass the entire culture. Mixed cultures of cortical neurons from transgenic mice expressing green fluorescent protein with striatal neurons from wild-type mice of the same developmental stage allowed visual identification of individual candidate corticostriatal pairs. Recordings were performed between 12 and 37 days in vitro (DIV). To investigate synaptic connections we recorded from 69 corticostriatal pairs of which 44 were connected in one direction and 25 reciprocally. Of these connections 41 were corticostriatal (nine inhibitory) and 53 striatocortical (all inhibitory). The observed excitatory responses were of variable amplitude (?10 to ?370?pA, n?=?32). We found the connections very secure – with negligible failures on repeated stimulation (approximately 1?Hz) of the cortical neuron. Inhibitory corticostriatal responses were also observed (?13 to ?314?pA, n?=?9). Possibly due to the mixed type of culture we found an inhibitory striatocortical response (?14 to ?598?pA, n?=?53). We are now recording from neurons in separate compartments to more closely emulate neuroanatomical conditions but still with the possibility of the easier identification of the connectivity. PMID:21743806

  15. Primary human epithelial cell culture system for studying interactions between female upper genital tract and sexually transmitted viruses, HSV-2 and HIV-1.

    PubMed

    Kaushic, Charu; Nazli, Aisha; Ferreira, Victor H; Kafka, Jessica K

    2011-10-01

    Evidence from clinical and epidemiological studies indicates that women are disproportionately susceptible to sexually transmitted viral infections. To understand the underlying biological basis for this increased susceptibility, more studies are needed to examine the acute events in the female reproductive tract following exposure to viruses during sexual transmission. The epithelial lining of the female reproductive tract is the primary barrier that sexually transmitted viruses, such as HIV-1 and HSV-2 need to infect or traverse, in order to initiate and establish productive infection. We have established an ex-vivo primary culture system to grow genital epithelial cells from upper reproductive tract tissues of women. Using these cultures, we have extensively examined the interactions between epithelial cells of the female genital tract and HSV-2 and HIV-1. In this review, we describe in detail the experimental protocol to grow these cultures, monitor their differentiation and inoculate with HSV-2 and HIV-1. Prospective use of these cultures to re-create the microenvironment in the reproductive tract is discussed. PMID:21996033

  16. Multiwell cell culture plate format with integrated microfluidic perfusion system

    NASA Astrophysics Data System (ADS)

    Domansky, Karel; Inman, Walker; Serdy, Jim; Griffith, Linda G.

    2006-01-01

    A new cell culture analog has been developed. It is based on the standard multiwell cell culture plate format but it provides perfused three-dimensional cell culture capability. The new capability is achieved by integrating microfluidic valves and pumps into the plate. The system provides a means to conduct high throughput assays for target validation and predictive toxicology in the drug discovery and development process. It can be also used for evaluation of long-term exposure to drugs or environmental agents or as a model to study viral hepatitis, cancer metastasis, and other diseases and pathological conditions.

  17. Microglial cells in astroglial cultures: a cautionary note

    PubMed Central

    Saura, Josep

    2007-01-01

    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. PMID:17937799

  18. Biotransformation of valencene by cultured cells of Gynostemma pentaphyllum

    Microsoft Academic Search

    Hiroshi Sakamaki; Ken-ichi Itoh; Tetsuyuki Taniai; Susumu Kitanaka; Yoshikazu Takagi; Wen Chai; C. Akira Horiuchi

    2005-01-01

    It has been found that the suspension cultures of Gynostemma pentaphyllum convert valencene (1) into nootkatone (2) as the major product and nootkatol (3) as the minor product. Based on this finding, a further study was conducted to investigate the biotransformation of 1 by other cultured plant cells (Caragana chamlagu, Hibiscus cannabinus).

  19. Aminoacid transport into cultured tobacco cells

    Microsoft Academic Search

    S. L. Berry; H. M. Harrington; R. L. Bernsteint; R. R. Henkel

    1981-01-01

    Arginine transport in suspension-cultured cells of Nicotiana tabacum L. cv. Wisconsin-38 was investigated. Cells that were preincubated in the presence of Ca2+ for 6 h prior to transport exhibited stimulated transport rates. After the preincubation treatment, initial rates of uptake were constant for at least 45 min. Arginine accumulated in the cells against a concentration gradient; this accumulation was not

  20. [In vitro suspension and bioreactor culture of hematopoietic cells].

    PubMed

    Chi, Zhan-You; Xia, Quan-Ming; Kang, Zi-Zhen; Tan, Wen-Song; Dai, Gan-Ce

    2003-09-01

    Stirred culture offers a number of advantages over static systems as it maintains a stable, homogeneous culture environment and is easy to scale-up. This paper focused on the development and application of stirred tank bioreactor to culture hematopoietic cells. Preliminary study of stirred culture of hematopoietic cells was carried out in cord blood mononuclear cells culture in spinner flask. The results showed that the amplification rates of total cell, CFU-GM and BFU-E, with the exception of CFU-Mk, were greater in spinner flask than T-flask. The number of total cells increased 20 fold after 14 days incubation in spinner flask. The amplification rates of CFU-GM, CFU-Mk and BFU-E reached maximum at 10th day, 10th day and 7th day respectively, and the maximal amplification rates were 9.2-fold, 5.5-fold and 2.4-fold respectively, whereas the rate of CD34+ cells in spinner flask was (6.7 +/- 4.0)-fold at day 10. These results indicated that the stirred culture system is better than the static culture systems for hematopoietic cell proliferation. The biocompatibility of cord blood MNC to different types of materials used in bioreactors was also tested. The results showed that glass, stainless steel 316L and polytetraflouroethylene (PTFE) supported the growth of hematopoietic cells well. A higher cell density was reached in stirred bioreactors with controlled pH and DO than static culture. These findings suggested that the controlled large-scale culture could be used to overcome the clinical shortage of hematopoietic cells. PMID:15969089

  1. Isolation, Culture and Identification of Porcine Skeletal Muscle Satellite Cells.

    PubMed

    Li, Bo-Jiang; Li, Ping-Hua; Huang, Rui-Hua; Sun, Wen-Xing; Wang, Han; Li, Qi-Fa; Chen, Jie; Wu, Wang-Jun; Liu, Hong-Lin

    2015-08-01

    The objective of this study was to establish the optimum protocol for the isolation and culture of porcine muscle satellite cells. Mononuclear muscle satellite cells are a kind of adult stem cell, which is located between the basal lamina and sarcolemma of muscle fibers and is the primary source of myogenic precursor cells in postnatal muscle. Muscle satellite cells are a useful model to investigate the mechanisms of muscle growth and development. Although the isolation and culture protocols of muscle satellite cells in some species (e.g. mouse) have been established successfully, the culture system for porcine muscle satellite cells is very limited. In this study, we optimized the isolation procedure of porcine muscle satellite cells and elaborated the isolation and culture process in detail. Furthermore, we characterized the porcine muscle satellite cells using the immunofluorecence. Our study provides a reference for the isolation of porcine muscle satellite cells and will be useful for studying the molecular mechanisms in these cells. PMID:26104526

  2. Bioprocessing technology for plant cell suspension cultures

    Microsoft Academic Search

    Wei wen Su

    1995-01-01

    Considering various forms of in vitro plant tissue cultures, cell suspension culture is most amenable to large-scale production\\u000a of natural compounds, owing primarily to its superior culture homogeneity. This fact has already been demonstrated in several\\u000a largescale applications, including the commercial shikonin process. The scope of this work is to review the state of the art\\u000a in bioprocessing technologies pertinent

  3. Endogenous amyloidogenesis in long-term rat hippocampal cell cultures

    PubMed Central

    2011-01-01

    Background Long-term primary neuronal cultures are a useful tool for the investigation of biochemical processes associated with neuronal senescence. Improvements in available technology make it possible to observe maturation of neural cells isolated from different regions of the rodent brain over a prolonged period in vitro. Existing experimental evidence suggests that cellular aging occurs in mature, long-term, primary neuronal cell cultures. However, detailed studies of neuronal development in vitro are needed to demonstrate the validity of long-term cell culture-based models for investigation of the biochemical mechanisms of in vitro neuronal development and senescence. Results In the current study, neuron-enriched hippocampal cell cultures were used to analyze the differentiation and degeneration of hippocampal neurons over a two month time period. The expression of different neuronal and astroglial biomarkers was used to determine the cytochemical characteristics of hippocampal cells in long-term cultures of varying ages. It was observed that the expression of the intermediate filament nestin was absent from cultures older than 21 days in vitro (DIV), and the expression of neuronal or astrocytic markers appeared to replace nestin. Additionally, morphological evaluations of neuronal integrity and Hoescht staining were used to assess the cellular conditions in the process of hippocampal culture development and aging. It was found that there was an increase in endogenous production of A?1-42 and an increase in the accumulation of Congo Red-binding amyloidal aggregates associated with the aging of neurons in primary culture. In vitro changes in the morphology of co-existing astrocytes and cell culture age-dependent degeneration of neurodendritic network resemble features of in vivo brain aging at the cellular level. Conclusion In conclusion, this study suggests that long-term primary CNS culture is a viable model for the study of basic mechanisms and effective methods to decelerate the process of neuronal senescence. PMID:21569253

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

    NASA Astrophysics Data System (ADS)

    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.

  5. Cryopreservation of Taxus chinensis suspension cell cultures.

    PubMed

    Kim, S I; Choi, H K; Son, J S; Yun, J H; Jang, M S; Kim, H R; Song, J Y; Kim, J H; Choi, H J; Hong, S S

    2001-01-01

    A simple cryopreservation method for suspension cells of Taxus chinensis was established. In this procedure 7 days old suspension cells were used without any pre-culture treatment. At first, cells were incubated in cryoprotectant solution (0.5M DMSO and 0.5M glycerol) on ice for 30 min and then frozen at a cooling rate of 1 degree C/min to -40 degrees C prior to immersion in liquid nitrogen. The average viability of frozen-thawed cells was between 30 to 40%. The recovery of cryopreserved cells in liquid nitrogen for 1 month was accomplished. After rapid thawing, cells were transferred to solid medium and cultivated for 4-6 weeks. The treatment of trehalose as a cryoprotectant enhanced re-growth of frozen-thawed cells. The stable maintenance of paclitaxel biosynthetic ability in cryopreserved cells was confirmed by comparing with that of regularly sub-cultured suspension cells. PMID:11788843

  6. Oscillatory behavior of cells in tissue culture.

    NASA Astrophysics Data System (ADS)

    Giaever, Ivar; Linton, Michael F. A.; Keese, Charles R.

    1998-03-01

    Fibroblasts and epithelial cells organize themselves in distinct patterns in tissue culture which indicates that neighboring cells communicate. A striking example of such communication is the oscillatory behavior of Madin-Darby canine kidney (MDCK) cells reported here. These oscillations were discovered using a biosensor referred to as ECIS (Electric Cell-substrate Impedance Sensing). In this measurement cells are seeded out on a small electrode deposited at the bottom of a tissue culture well and immersed in ordinary culture medium. By measuring the changes in the impedance of the electrode as a function of time, many important properties of the cells on the electrode can be inferred, such as motion, morphology changes and membrane capacitance. The impedance oscillations of MDCK cells were observed with highly confluent cell layers, where the approximately 100 cells on the electrode acted in unison. The communication between cells can be demonstrated directly by a variation of the ECIS concept, where cells are cultured on two closely spaced electrodes. The impedance fluctuations are measured independently on each electrode and compared by using a cross-correlation function.

  7. Cell Culture on MEMS Platforms: A Review

    E-print Network

    Ni, Ming

    Microfabricated systems provide an excellent platform for the culture of cells, and are an extremely useful tool for the investigation of cellular responses to various stimuli. Advantages offered over traditional methods ...

  8. Decellularized Feeders: An Optimized Method for Culturing Pluripotent Cells

    PubMed Central

    Lim, Mei Ling; Jungebluth, Philipp; Sjöqvist, Sebastian; Nikdin, Hero; Kjartansdóttir, Kristín Rós; Unger, Christian; Vassliev, Ivan

    2013-01-01

    Pluripotent cells such as human embryonic stem cells and human induced pluripotent stem cells are useful in the field of regenerative medicine because they can proliferate indefinitely and differentiate into all cell types. However, a limiting factor for maintaining and propagating stem cells is the need for inactivated fibroblasts as a growth matrix, since these may potentially cause cross-contamination. In this study, we aimed to maintain stem cells on the extracellular matrix (ECM) of either nonirradiated or ?-irradiated fibroblasts. It has been demonstrated that the ECM contains factors and proteins vital for the adhesion, proliferation, and differentiation of pluripotent cells. In order to preserve the ECM, the cell layers of the fibroblasts were decellularized by treatment with 0.05% sodium dodecyl sulfate (SDS), which resulted in an absence of DNA as compared with conventional feeder culture. However, SDS treatment did not cause a detectable change in the ECM architecture and integrity. Furthermore, immunohistochemistry demonstrated that expressions of major ECM proteins, such as fibronectin, collagen, and laminin, remained unaltered. The human pluripotent cells cultured on this decellularized matrix maintained gene expression of the pluripotency markers NANOG and OCT4 and had the potency to differentiate to three germ layers. The in vitro culture system shown here has an excellent potential since the main allogeneic components (i.e., DNA of the feeder cells) are removed. It is also a technically easy, fast, safe, and cheap method for maintaining a refined feeder-free stem cell culture for further cell differentiation studies. PMID:24167316

  9. Microfabricated polymeric vessel mimetics for 3-D cancer cell culture

    PubMed Central

    Jaeger, Ashley A.; Das, Chandan K.; Morgan, Nicole Y.; Pursley, Randall H.; McQueen, Philip G.; Hall, Matthew D.; Pohida, Thomas J.; Gottesman, Michael M.

    2013-01-01

    Modeling tumor growth in vitro is essential for cost-effective testing of hypotheses in preclinical cancer research. 3-D cell culture offers an improvement over monolayer culture for studying cellular processes in cancer biology because of the preservation of cell-cell and cell-ECM interactions. Oxygen transport poses a major barrier to mimicking in vivo environments and is not replicated in conventional cell culture systems. We hypothesized that we can better mimic the tumor microenvironment using a bioreactor system for controlling gas exchange in cancer cell cultures with silicone hydrogel synthetic vessels. Soft-lithography techniques were used to fabricate oxygen-permeable silicone hydrogel membranes containing arrays of micropillars. These membranes were inserted into a bioreactor and surrounded by basement membrane extract (BME) within which fluorescent ovarian cancer (OVCAR8) cells were cultured. Cell clusters oxygenated by synthetic vessels showed a ?100um drop-off to anoxia, consistent with in vivo studies of tumor nodules fed by the microvasculature. We showed oxygen tension gradients inside the clusters oxygenated by synthetic vessels had a ?100 µm drop-off to anoxia, which is consistent with in vivo studies. Oxygen transport in the bioreactor system was characterized by experimental testing with a dissolved oxygen probe and finite element modeling of convective flow. Our study demonstrates differing growth patterns associated with controlling gas distributions to better mimic in vivo conditions. PMID:23911071

  10. [Studies on IFN-gamma production by peripheral blood mononuclear cells cultured with Candida antigen in asthmatics].

    PubMed

    Kimura, G; Ogurusu, K; Soda, R; Tada, S; Takahashi, K; Kimura, I

    1992-12-01

    T-cell-derived lymphokine, IFN-gamma, has potent effects on B-cell differentiation and leukotriene C4 production in leukocytes, and inhibits the effect of IL-4 on IgE production. To investigate the role of IFN-gamma in the pathogenesis of bronchial asthma, we examined IFN-gamma production by peripheral blood mononuclear cells cultured with Candida antigen and serum Candida specific IgG1 antibody from 20 asthmatics. The results were as follows: 1) IFN-gamma production in non-atopic severe asthmatics was significantly higher than in healthy subjects, atopic mild and moderate asthmatics, and atopic severe asthmatics (p < 0.05). 2) There was a significant correlation between IFN-gamma production induced by Candida antigen and serum Candida specific IgG1 antibody (r = 0.72, p < 0.01). These results suggest that IFN-gamma may play an important role in the pathogenesis of non-atopic severe bronchial asthma. PMID:1290416

  11. Optimization of Buffalo (Bubalus bubalis) Embryonic Stem Cell Culture System

    PubMed Central

    Zandi, Mohammad; Muzaffar, Musharifa; Shah, Syed Mohmad; Kumar Singh, Manoj; Palta, Prabhat; Kumar Singla, Suresh; Manik, Radheysham; Chauhan, Manmohan Singh

    2015-01-01

    Objective In order to retain an undifferentiated pluripotent state, embryonic stem (ES) cells have to be cultured on feeder cell layers. However, use of feeder layers limits stem cell research, since experimental data may result from a combined ES cell and feeder cell response to various stimuli. Materials and Methods In this experimental study, a buffalo ES cell line was established from in vitro derived blastocysts and characterized by the Alkaline phosphatase (AP) and immunoflourescence staining of various pluripotency markers. We examined the effect of various factors like fibroblast growth factor 2 (FGF-2), leukemia inhibitory factor (LIF) and Y-27632 to support the growth and maintenance of bubaline ES cells on gelatin coated dishes, in order to establish feeder free culture systems. We also analyzed the effect of feeder-conditioned media on stem cell growth in gelatin based cultures both in the presence as well as in the absence of the growth factors. Results The results showed that Y-27632, in the presence of FGF-2 and LIF, resulted in higher colony growth and increased expression of Nanog gene. Feeder-Conditioned Medium resulted in a significant increase in growth of buffalo ES cells on gelatin coated plates, however, feeder layer based cultures produced better results than gelatin based cultures. Feeder layers from buffalo fetal fibroblast cells can support buffalo ES cells for more than two years. Conclusion We developed a feeder free culture system that can maintain buffalo ES cells in the short term, as well as feeder layer based culture that can support the long term maintenance of buffalo ES cells. PMID:26199905

  12. Improvement of photoaged skin wrinkles with cultured human fibroblasts and adipose-derived stem cells: a comparative study.

    PubMed

    Jeong, Jae Hoon; Fan, Yingfang; You, Ga Young; Choi, Tae Hyun; Kim, Sukwha

    2015-03-01

    We investigated the antiwrinkle effects of cultured human fibroblasts and adipose-derived stem cells (ADSCs) and the mechanisms underlying the reduction of wrinkles in photoaged skin. The fibroblasts and ADSCs were isolated from human tissue and cultured. A total of 28 6-week-old female BALB/c nude mice were classified into four groups, including the normal control group and three groups that were irradiated six times a week for 6-weeks using ultraviolet B radiation to induce photoaged wrinkles. ADSCs were injected into the wrinkles in the skin of the second group and fibroblasts were injected into the wrinkles in the skin of the third group. The fourth group was the irradiated negative control group (no therapy). After 4 weeks of injections, the wrinkles were compared by replica analysis, biopsies were performed, and the dermal thickness and collagen densities were measured. We determined the amounts of type 1 collagen and matrix metalloproteinases (MMPs) 1, 2, 3, 9, and 13 using real-time polymerase chain reaction and Western blot analysis, and we assessed tropoelastin and fibrillin-1 expression in the dermis by immunohistochemistry. Replica analysis showed significant wrinkle reduction in the fibroblast group and the ADSC group. ADSCs stimulated collagen expression and decreased MMP expression. Although fibroblasts stimulated more collagen expression than ADSCs, they also increased MMP expression. Overall, the ADSC group showed higher collagen density and had better outcomes in the tropoelastin and fibrillin-1 assessments. Both cultured fibroblasts and ADSCs could play an important role in wrinkle reduction despite differences in their mechanisms of action. PMID:25484240

  13. Cytotoxicity of TSP in 3D Agarose Gel Cultured Cell

    PubMed Central

    Chun, Song-I; Mun, Chi-Woong

    2015-01-01

    Purpose A reference reagent, 3-(trimethylsilyl) propionic-2, 2, 3, 3-d4 acid sodium (TSP), has been used frequently in nuclear magnetic resonance (NMR) and magnetic resonance spectroscopy (MRS) as an internal reference to identify cell and tissue metabolites, and determine chemical and protein structures. This reference material has been exploited for the quantitative and dynamic analyses of metabolite spectra acquired from cells. The aim of this study was to evaluate the cytotoxicity of TSP on three-dimensionally, agarose gel, cultured cells. Materials and Methods A human osteosarcoma cell line (MG-63) was selected, and cells were three dimensionally cultured for two weeks in an agarose gel. The culture system contained a mixture of conventional culture medium and various concentrations (0, 1, 3, 5, 7, 10, 20 30 mM) of TSP. A DNA quantification assay was conducted to assess cell proliferation using Quant-iT PicoGreen dsDNA reagent and kit, and cell viability was determined using a LIVE/DEAD Viability/Cytotoxicity kit. Both examinations were performed simultaneously at 1, 3, 7 and 14 days from cell seeding. Results In this study, the cytotoxicity of TSP in the 3D culture of MG-63 cells was evaluated by quantifying DNA (cell proliferation) and cell viability. High concentrations of TSP (from 10 to 30 mM) reduced both cell proliferation and viability (to 30% of the control after one week of exposure), but no such effects were found using low concentrations of TSP (0–10mM). Conclusions This study shows that low concentrations of TSP in 3D cell culture medium can be used for quantitative NMR or MRS examinations for up to two weeks post exposure. PMID:26058017

  14. Establishment, Culture, and Characterization of Guinea Pig Fetal Fibroblast Cell

    PubMed Central

    Mahboobi, Reza; Dianatpour, Mehdi; Zare, Shahrokh; Hosseini, Seyed Ebrahim

    2014-01-01

    Establishment of Guinea pig fetal fibroblast cells and their biological evaluation before and after cryopreservation were the main purposes of this study. After determination of the proper age of pregnancy by ultrasonography, 30 days old fetuses of Guinea pigs were recovered. Their skins were cut into small pieces (1?mm2) and were cultured. When reaching 80–90% confluence, the cells were passaged. Cells of the second and eighth passages were cultured in 24-well plates (4 × 104 cells/well) for 6 days and three wells per day were counted. The average cell counts at each time point were then plotted against time and the population doubling time (PDT) was determined. Then, vials of cells (2 × 106 cells/mL) were cryopreserved for 1 month and after thawing, the cell viability was evaluated. The PDT of the second passage was about 23?h and for the eighth passage was about 30?h. The viability of the cultures was 95% in the second passage and 74.5% in the eighth passage. It was shown that the Guinea pig fetal fibroblast cell culture can be established using the adherent culture method while, after freezing, the viability indices of these cells were favorable. PMID:24790770

  15. Characterizing the mechanics of cultured cell monolayers

    PubMed Central

    Peter, Loic; Bellis, Julien; Baum, Buzz; Kabla, Alexandre J.; Charras, Guillaume T.

    2012-01-01

    One-cell-thick monolayers are the simplest tissues in multicellular organisms, yet they fulfill critical roles in development and normal physiology. In early development, embryonic morphogenesis results largely from monolayer rearrangement and deformation due to internally generated forces. Later, monolayers act as physical barriers separating the internal environment from the exterior and must withstand externally applied forces. Though resisting and generating mechanical forces is an essential part of monolayer function, simple experimental methods to characterize monolayer mechanical properties are lacking. Here, we describe a system for tensile testing of freely suspended cultured monolayers that enables the examination of their mechanical behavior at multi-, uni-, and subcellular scales. Using this system, we provide measurements of monolayer elasticity and show that this is two orders of magnitude larger than the elasticity of their isolated cellular components. Monolayers could withstand more than a doubling in length before failing through rupture of intercellular junctions. Measurement of stress at fracture enabled a first estimation of the average force needed to separate cells within truly mature monolayers, approximately ninefold larger than measured in pairs of isolated cells. As in single cells, monolayer mechanical properties were strongly dependent on the integrity of the actin cytoskeleton, myosin, and intercellular adhesions interfacing adjacent cells. High magnification imaging revealed that keratin filaments became progressively stretched during extension, suggesting they participate in monolayer mechanics. This multiscale study of monolayer response to deformation enabled by our device provides the first quantitative investigation of the link between monolayer biology and mechanics. PMID:22991459

  16. Applicability of integrated cell culture quantitative PCR (ICC-qPCR) for the detection of infectious adenovirus type 2 in UV disinfection studies.

    PubMed

    Ryu, Hodon; Cashdollar, Jennifer L; Fout, G Shay; Schrantz, Karen A; Hayes, Samuel

    2015-07-01

    Practical difficulties of the traditional adenovirus infectivity assay such as intensive labor requirements and longer turnaround period limit the direct use of adenovirus as a testing microorganism for systematic, comprehensive disinfection studies. In this study, we attempted to validate the applicability of integrated cell culture quantitative PCR (ICC-qPCR) as an alternative to the traditional cell culture method with human adenovirus type 2 (HAdV2) in a low-pressure UV disinfection study and to further optimize the procedures of ICC-qPCR for 24-well plate format. The relatively high stability of the hexon gene of HAdV2 was observed after exposure to UV radiation, resulting in a maximum gene copy reduction of 0.5 log10 at 280 mJ cm(-2). Two-day post-inoculation incubation period and a maximum spiking level of 10(5) MPN mL(-1) were selected as optimum conditions of ICC-qPCR with the tested HAdV2. An approximate 1:1 correlation of virus quantities by the traditional and ICC-qPCR cell culture based methods suggested that ICC-qPCR is a satisfactory alternative for practical application in HAdV2 disinfection studies. ICC-qPCR results, coupled with a first-order kinetic model (i.e., the inactivation rate constant of 0.0232 cm(2) mJ(-1)), showed that an UV dose of 172 mJ cm(-2) achieved a 4-log inactivation credit for HAdV2. This estimate is comparable to other studies with HAdV2 and other adenovirus respiratory types. The newly optimized ICC-qPCR shows much promise for further study on its applicability of other slow replicating viruses in disinfection studies. PMID:26030683

  17. Comparison between Culture Conditions Improving Growth and Differentiation of Blood and Bone Marrow Cells Committed to the Endothelial Cell Lineage

    Microsoft Academic Search

    Claudio Muscari; Chiara Gamberini; Ilaria Basile; Francesca Bonafé; Simond Valgimigli; Ombretta Capitani; Carlo Guarnieri; Claudio Marcello Caldarera

    2010-01-01

    The aim of this study was to compare different cell sources and culture conditions to obtain endothelial progenitor cells (EPCs) with predictable antigen pattern, proliferation potential and in vitro vasculogenesis. Pig mononuclear cells were isolated from blood (PBMCs) and bone marrow (BMMCs). Mesenchymal stem cells (MSCs) were also derived from pig bone marrow. Cells were cultured on fibronectin in the

  18. Identifying viable regulatory and innovation pathways for regenerative medicine: a case study of cultured red blood cells.

    PubMed

    Mittra, J; Tait, J; Mastroeni, M; Turner, M L; Mountford, J C; Bruce, K

    2015-01-25

    The creation of red blood cells for the blood transfusion markets represents a highly innovative application of regenerative medicine with a medium term (5-10 year) prospect for first clinical studies. This article describes a case study analysis of a project to derive red blood cells from human embryonic stem cells, including the systemic challenges arising from (i) the selection of appropriate and viable regulatory protocols and (ii) technological constraints related to stem cell manufacture and scale up to clinical Good Manufacturing Practice (GMP) standard. The method used for case study analysis (Analysis of Life Science Innovation Systems (ALSIS)) is also innovative, demonstrating a new approach to social and natural science collaboration to foresight product development pathways. Issues arising along the development pathway include cell manufacture and scale-up challenges, affected by regulatory demands emerging from the innovation ecosystem (preclinical testing and clinical trials). Our discussion reflects on the efforts being made by regulators to adapt the current pharmaceuticals-based regulatory model to an allogeneic regenerative medicine product and the broader lessons from this case study for successful innovation and translation of regenerative medicine therapies, including the role of methodological and regulatory innovation in future development in the field. PMID:25094050

  19. Microtechnology for Stem Cell Culture

    Microsoft Academic Search

    Elena Serena; Elisa Cimetta; Camilla Luni; Nicola Elvassore

    \\u000a Advances in stem cell research in recent decades have been aided by progress in the development of novel technologies aimed\\u000a at biological systems. At the same time mimicking stem cell niches in vitro has become crucial for both basic stem cell research\\u000a and the development of innovative therapies based on stem cells. Innovative microscale technologies can contribute to our\\u000a quantitative

  20. Challenges in Using Cultured Primary Rodent Hepatocytes or Cell Lines to Study Hepatic HDL Receptor SR-BI Regulation by Its Cytoplasmic Adaptor PDZK1

    PubMed Central

    Tsukamoto, Kosuke; Buck, Lorenna; Inman, Walker; Griffith, Linda; Kocher, Olivier; Krieger, Monty

    2013-01-01

    Background PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1 - <5% of normal hepatic SR-BI is seen in the livers of PDZK1 knockout mice. Progress has been made in identifying features of PDZK1 required to control hepatic SR-BI in vivo using hepatic expression of wild-type and mutant forms of PDZK1 in wild-type and PDZK1 KO transgenic mice. Such in vivo studies are time consuming and expensive, and cannot readily be used to explore many features of the underlying molecular and cellular mechanisms. Methodology/Principal Findings Here we have explored the potential to use either primary rodent hepatocytes in culture using 2D collagen gels with newly developed optimized conditions or PDZK1/SR-BI co-transfected cultured cell lines (COS, HEK293) for such studies. SR-BI and PDZK1 protein and mRNA expression levels fell rapidly in primary hepatocyte cultures, indicating this system does not adequately mimic hepatocytes in vivo for analysis of the PDZK1 dependence of SR-BI. Although PDZK1 did alter SR-BI protein expression in the cell lines, its influence was independent of SR-BI’s C-terminus, and thus is not likely to occur via the same mechanism as that which occurs in hepatocytes in vivo. Conclusions/Significance Caution must be exercised in using primary hepatocytes or cultured cell lines when studying the mechanism underlying the regulation of hepatic SR-BI by PDZK1. It may be possible to use SR-BI and PDZK1 expression as sensitive markers for the in vivo-like state of hepatocytes to further improve primary hepatocyte cell culture conditions. PMID:23936087

  1. Characteristics of cardiac cell cultures derived from human myocardial explants.

    PubMed

    Pavlova, S V; Perovskii, P P; Chepeleva, E V; Malakhova, A A; Dement'eva, E V; Pokushalov, E A; Sukhikh, G T; Zakiyan, S M

    2013-11-01

    Primary cell cultures derived from human myocardial explants were obtained and characterized. The explant cultures contained cardiac stem cells (c-kit(+); ? 4%), microvascular cells (endothelial cells and pericytes), fibroblasts, and myofibroblasts. It was demonstrated that culturing of cardiac cells in cardiospheres did not promote enrichment of the cell culture with stem cells. MACS-sorted c-kit(+) cells from the explant culture were characterized by limited proliferative capacity and were capable of cardiomyogenic differentiation. The presence of microvascular cells determined general angiogenic potential of the culture. PMID:24319709

  2. Silencing of exogenous DNA in cultured cells.

    PubMed

    Ochiai, Hiroshi; Harashima, Hideyoshi; Kamiya, Hiroyuki

    2006-06-01

    The intranuclear disposition of exogenous DNA is highly important for the therapeutic effects of the administrated DNA. Naked luciferase-plasmid DNA was transfected into cultured cells including HeLa by electroporation, and the amounts of intranuclear plasmid DNA and luciferase were quantitated at various time points. Decrease in expression efficiency from one copy of the exogenous DNA over time occurred as the case of mouse liver, and its degrees varied between cell lines. These results suggest that exogenous DNA is 'silenced' in the cultured cells as well as in mouse hepatocytes. PMID:16755038

  3. Phenotypic and Functional Characterization of Human Mammary Stem\\/Progenitor Cells in Long Term Culture

    Microsoft Academic Search

    Devaveena Dey; Meera Saxena; Anurag N. Paranjape; Visalakshi Krishnan; Rajashekhar Giraddi; M. Vijaya Kumar; Geetashree Mukherjee; Annapoorni Rangarajan; Gianni Parise

    2009-01-01

    BackgroundCancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures. Since

  4. Cell, tissue and organ culture as in vitro models to study the biology of squamous cell carcinomas of the head and neck

    Microsoft Academic Search

    Peter G. Sacks

    1996-01-01

    In vitro models are currently being used to study head and neck squamous cell carcinoma (HNSCC). Several hundred HNSCC cell lines have been established by various investigators and used to study a broad spectrum of questions related to head and neck cancer. The head and neck model with respect to multistage carcinogenesis is now complete. Several techniques exist for the

  5. Large-scale culture of plant cells.

    PubMed

    Scragg, A H; Fowler, M W

    1990-01-01

    The large-scale or mass cultivation of plant cells is the growth of plant cell suspensions at volumes above those normally produced in shake flasks, that is, above IL. Attempts to grow plant cells in fermenters or bioreactors started in the early 1960s with converted carboys. The area has developed steadily, such that today bioreactors in excess of 5000 L have been used successfully for large-scale plant cell culture (1,2). Much of the early work was carried out using bioreactors designed for microbial culture. It was soon found, however, that although plant cell suspensions appear to be similar in many ways to microbial cultures, there are, in fact, key differences that can have a significant influence on large-scale cultivation. Plant cells are large, 20-40 µM in diameter, and up to 100 PM in length; further, they rarely occur as single cells, but as aggregates of up to 2 mm in diameter (Fig. 1). The individual plant cell soon after division is typically rounded, containing considerable amounts of cytoplasm; however, as it ages, the cell expands and becomes dominated by a large vacuole. In consequence, the overall metabolic activity is low compared with microbial cells, which in turn gives a very slow growth rate (measured in days. PMID:21390630

  6. Production of Zebrafish Offspring from Cultured Female Germline Stem Cells

    PubMed Central

    Wong, Ten-Tsao; Tesfamichael, Abraham; Collodi, Paul

    2013-01-01

    Zebrafish female germline stem cell (FGSC) cultures were generated from a transgenic line of fish that expresses Neo and DsRed under the control of the germ cell specific promoter, ziwi [Tg(ziwi:neo);Tg(ziwi:DsRed)]. Homogeneous FGSC cultures were established by G418 selection and continued to express ziwi for more than 6 weeks along with the germ cell markers nanos3, dnd, dazl and vasa. A key component of the cell culture system was the use of a feeder cell line that was initiated from ovaries of a transgenic line of fish [Tg(gsdf:neo)] that expresses Neo controlled by the zebrafish gonadal soma derived factor (gsdf) promoter. The feeder cell line was selected in G418 and engineered to express zebrafish leukemia inhibitory factor (Lif), basic fibroblast growth factor (Fgf2) and glial-cell-line derived neurotrophic factor (Gdnf). These factors were shown to significantly enhance FGSC growth, survival and germline competency in culture. Results from cell transplantation experiments revealed that the cultured FGSCs were able to successfully colonize the gonad of sterile recipient fish and generate functional gametes. Up to 20% of surviving recipient fish that were injected with the cultured FGSCs were fertile and generated multiple batches of normal offspring for at least 6 months. The FGSC cultures will provide an in vitro system for studies of zebrafish germ cell growth and differentiation and their high frequency of germline transmission following transplantation could form the basis of a stem cell-mediated strategy for gene transfer and manipulation of the zebrafish genome. PMID:23671620

  7. Biosynthesis of storage lipids in plant cell and embryo cultures.

    PubMed

    Weber, N; Taylor, D C; Underhill, E W

    1992-01-01

    The biosynthesis of storage lipids in plant cell and embryo cultures is discussed in the light of their significance in the breeding of agriculturally important oil seed crops. After a short introduction to the biosynthesis of storage lipids, i.e. triacylglycerols and wax esters, this review covers the occurrence and biosynthesis of storage lipids in plant cell and embryo cultures. Plant cells in culture generally contain low levels of both unusual fatty acids and triacylglycerols indicating that these cells are quite different from cells of oil storage tissues. There are a few exceptions to this rule which demonstrate that induction of genes involved in the expression of fatty acid modification and triacylglycerol assembly is possible in plant cell cultures. Such biosynthetically active plant cells may be of particular interest in future studies of storage lipid assembly. Both somatic and gametophytic embryos of oil plants exhibit high capacities for storage lipid biosynthesis and accumulation in vitro compared to cultured plant cells. Above all, the microspore-derived embryo system is recommended to both plant breeders and plant biochemists for the selection and multiplication of plants of superior quality. PMID:1605093

  8. Biosynthesis of highly enriched 13C-lycopene for human metabolic studies using repeated batch tomato cell culturing with 13C-glucose

    PubMed Central

    Moran, Nancy E.; Rogers, Randy B.; Lu, Chi-Hua; Conlon, Lauren E.; Lila, Mary Ann; Clinton, Steven K.; Erdman, John W.

    2013-01-01

    While putative disease-preventing lycopene metabolites are found in both tomato (Solanum lycopersicum) products and in their consumers, mammalian lycopene metabolism is poorly understood. Advances in tomato cell culturing techniques offer an economical tool for generation of highly-enriched 13C-lycopene for human bioavailability and metabolism studies. To enhance the 13C-enrichment and yields of labeled lycopene from the hp-1 tomato cell line, cultures were first grown in 13C-glucose media for three serial batches and produced increasing proportions of uniformly labeled lycopene (14.3 +/? 1.2 %, 39.6 +/? 0.5 %, and 48.9 +/? 1.5% with consistent yields (from 5.8 to 9 mg/L). An optimized 9-day-long 13C-loading and 18-day-long labeling strategy developed based on glucose utilization and lycopene yields, yielded 13C-lycopene with 93% 13C isotopic purity, and 55% of isotopomers were uniformly labeled. Furthermore, an optimized acetone and hexane extraction led to a four-fold increase in lycopene recovery from cultures compared to a standard extraction. PMID:23561155

  9. Feeding lactate for CHO cell culture processes: impact on culture metabolism and performance.

    PubMed

    Li, Jincai; Wong, Chun Loong; Vijayasankaran, Natarajan; Hudson, Terry; Amanullah, Ashraf

    2012-05-01

    Lactate has long been regarded as one of the key metabolites of mammalian cell cultures. High levels of lactate have clear negative impacts on cell culture processes, and therefore, a great amount of efforts have been made to reduce lactate accumulation and/or to induce lactate consumption in the later stage of cultures. However, there is virtually no report on the impact of lactate depletion after initial accumulation. In this work, we observed that glucose uptake rate dropped over 50% at the onset of lactate consumption, and that catabolism of alanine due to lactate depletion led to ammonium accumulation. We explored the impact of feeding lactate as well as pyruvate to the cultures. In particular, a strategy was employed where CO(2) was replaced by lactic acid for culture pH control, which enabled automatic lactate feeding. The results demonstrated that lactate or pyruvate can serve as an alternative or even preferred carbon source during certain stage of the culture in the presence of glucose, and that by feeding lactate or pyruvate, very low levels of ammonia can be achieved throughout the culture. In addition, low levels of pCO(2) were also maintained in these cultures. This was in strong contrast to the control cultures where lactate was depleted during the culture, and ammonia and pCO(2) build-up were significant. Culture growth and productivity were similar between the control and lactate-fed cultures, as well as various product quality attributes. To our knowledge, this work represents the first comprehensive study on lactate depletion and offers a simple yet effective strategy to overcome ammonia and pCO(2) accumulation that could arise in certain cultures due to early depletion of lactate. PMID:22124879

  10. [A comparative study of induction regulation in cytochromes family 1 P450 in cell cultures at different stages of tumor transformation].

    PubMed

    Evteev, V A; Shcherbak, N P; Kobliakov, V A

    2006-01-01

    Lipophilic xenobiotics, including some carcinogenic agents and cytostatics, are metabolized by cytochrome P450 isoforms (CYP). In tumours expression of CYP genes and their inducibility are lower than in a homologous normal tissue. This phenomenon determines the known higher cytostatic stability of tumour cells. To clarify, at which particular stage of tumour transformation the level of family 1 CYP may change, we compared mRNA expression of CYP1A1, CYP1B1 and also of proteins regulated CYP expression: Ah receptor, ARNT and AHRR. For this aim we studied embryonic and fibroblast-like cells, in addition to cells of the same types but immortalized by the Rausher virus, or spontaneously after crisis. Besides, we investigated transformed clones obtained by means of benzo/a/pyrene action on Rausher virus-immortalized cells. Constitutive expression of genes studied in all cell cultures was shown. Benzo/a/anthracene induction increases the mRNA expression of all inducible genes (CYP1A1, CYP1B1, AHRR) in the original embryonic cells, in Rausher virus-immortalized cells, and in transformed clone K2. In both spontaneously immortalized cells and transformed clone K1 only CYP1B1 was induced. In transformed clone K8 no inducible gene was induced. In summary, we have shown that: (1) the ability of immortalized cells to CYP induction is determined not only by their capacity for a non-limited persistence, but also by the nature of immortalization; (2) despite their common genesis, the transformed clones differ in their ability to induce CYP. In addition to Ah receptor and ARNT, some other, yet unknown factors may also take part in CYP induction. PMID:17089626

  11. Primary Bovine Extra-Embryonic Cultured Cells: A New Resource for the Study of In Vivo Peri-Implanting Phenotypes and Mesoderm Formation

    PubMed Central

    Hue, Isabelle; Evain-Brion, Danièle; Fournier, Thierry; Degrelle, Séverine A.

    2015-01-01

    In addition to nourishing the embryo, extra-embryonic tissues (EETs) contribute to early embryonic patterning, primitive hematopoiesis, and fetal health. These tissues are of major importance for human medicine, as well as for efforts to improve livestock efficiency, but they remain incompletely understood. In bovines, EETs are accessible easily, in large amounts, and prior to implantation. We took advantage of this system to describe, in vitro and in vivo, the cell types present in bovine EETs at Day 18 of development. Specifically, we characterized the gene expression patterns and phenotypes of bovine extra-embryonic ectoderm (or trophoblast; bTC), endoderm (bXEC), and mesoderm (bXMC) cells in culture and compared them to their respective in vivo micro-dissected cells. After a week of culture, certain characteristics (e.g., gene expression) of the in vitro cells were altered with respect to the in vivo cells, but we were able to identify “cores” of cell-type-specific (and substrate-independent) genes that were shared between in vitro and in vivo samples. In addition, many cellular phenotypes were cell-type-specific with regard to extracellular adhesion. We evaluated the ability of individual bXMCs to migrate and spread on micro-patterns, and observed that they easily adapted to diverse environments, similar to in vivo EE mesoderm cells, which encounter different EE epithelia to form chorion, yolk sac, and allantois. With these tissue interactions, different functions arose that were detected in silico and corroborated in vivo at D21–D25. Moreover, analysis of bXMCs allowed us to identify the EE cell ring surrounding the embryonic disc (ED) at D14-15 as mesoderm cells, which had been hypothesized but not shown prior to this study. We envision these data will serve as a major resource for the future in the analysis of peri-implanting phenotypes in response to the maternal metabolism and contribute to subsequent studies of placental/fetal development in eutherians. PMID:26070137

  12. Cell Culture on MEMS Platforms: A Review

    PubMed Central

    Ni, Ming; Tong, Wen Hao; Choudhury, Deepak; Rahim, Nur Aida Abdul; Iliescu, Ciprian; Yu, Hanry

    2009-01-01

    Microfabricated systems provide an excellent platform for the culture of cells, and are an extremely useful tool for the investigation of cellular responses to various stimuli. Advantages offered over traditional methods include cost-effectiveness, controllability, low volume, high resolution, and sensitivity. Both biocompatible and bio-incompatible materials have been developed for use in these applications. Biocompatible materials such as PMMA or PLGA can be used directly for cell culture. However, for bio-incompatible materials such as silicon or PDMS, additional steps need to be taken to render these materials more suitable for cell adhesion and maintenance. This review describes multiple surface modification strategies to improve the biocompatibility of MEMS materials. Basic concepts of cell-biomaterial interactions, such as protein adsorption and cell adhesion are covered. Finally, the applications of these MEMS materials in Tissue Engineering are presented. PMID:20054478

  13. Effects of Ethanol and Acetaldehyde on Tight Junction Integrity: In Vitro Study in a Three Dimensional Intestinal Epithelial Cell Culture Model

    PubMed Central

    Elamin, Elhaseen; Jonkers, Daisy; Juuti-Uusitalo, Kati; van IJzendoorn, Sven; Troost, Freddy; Duimel, Hans; Broers, Jos; Verheyen, Fons; Dekker, Jan; Masclee, Ad

    2012-01-01

    Background Intestinal barrier dysfunction and translocation of endotoxins are involved in the pathogenesis of alcoholic liver disease. Exposure to ethanol and its metabolite, acetaldehyde at relatively high concentrations have been shown to disrupt intestinal epithelial tight junctions in the conventional two dimensional cell culture models. The present study investigated quantitatively and qualitatively the effects of ethanol at concentrations detected in the blood after moderate ethanol consumption, of its metabolite acetaldehyde and of the combination of both compounds on intestinal barrier function in a three-dimensional cell culture model. Methods and Findings Caco-2 cells were grown in a basement membrane matrix (Matrigel™) to induce spheroid formation and were then exposed to the compounds at the basolateral side. Morphological differentiation of the spheroids was assessed by immunocytochemistry and transmission electron microscopy. The barrier function was assessed by the flux of FITC-labeled dextran from the basal side into the spheroids' luminal compartment using confocal microscopy. Caco-2 cells grown on Matrigel assembled into fully differentiated and polarized spheroids with a central lumen, closely resembling enterocytes in vivo and provide an excellent model to study epithelial barrier functionality. Exposure to ethanol (10–40 mM) or acetaldehyde (25–200 µM) for 3 h, dose-dependently and additively increased the paracellular permeability and induced redistribution of ZO-1 and occludin without affecting cell viability or tight junction-encoding gene expression. Furthermore, ethanol and acetaldehyde induced lysine residue and microtubules hyperacetylation. Conclusions These results indicate that ethanol at concentrations found in the blood after moderate drinking and acetaldehyde, alone and in combination, can increase the intestinal epithelial permeability. The data also point to the involvement of protein hyperacetylation in ethanol- and acetaldehyde-induced loss of tight junctions integrity. PMID:22563376

  14. Basic toxicology and metabolism studies of 1,5-anhydro- d-fructose using bacteria, cultured mammalian cells, and rodents

    Microsoft Academic Search

    Shukun Yu; Jie Mei; Bo Ahrén

    2004-01-01

    1,5-Anhydro-d-fructose (AF) is a monosaccharide occurring in edible morels, red seaweeds and certain mammalian tissues. It can be formed directly from starch and glycogen in vivo by ?-1,4-glucan lyase (EC 4.2.2.13). In this study, the toxicity, absorption and metabolism of AF using bacteria, mammalian cells, rat and mouse models were examined. In Ames test, AF showed no genotoxicity using five

  15. Cell-specific labeling enzymes for analysis of cell-cell communication in continuous co-culture.

    PubMed

    Tape, Christopher J; Norrie, Ida C; Worboys, Jonathan D; Lim, Lindsay; Lauffenburger, Douglas A; Jørgensen, Claus

    2014-07-01

    We report the orthologous screening, engineering, and optimization of amino acid conversion enzymes for cell-specific proteomic labeling. Intracellular endoplasmic-reticulum-anchored Mycobacterium tuberculosis diaminopimelate decarboxylase (DDC(M.tub-KDEL)) confers cell-specific meso-2,6-diaminopimelate-dependent proliferation to multiple eukaryotic cell types. Optimized lysine racemase (Lyr(M37-KDEL)) supports D-lysine specific proliferation and efficient cell-specific isotopic labeling. When ectopically expressed in discrete cell types, these enzymes confer 90% cell-specific isotopic labeling efficiency after 10 days of co-culture. Moreover, DDC(M.tub-KDEL) and Lyr(M37-KDEL) facilitate equally high cell-specific labeling fidelity without daily media exchange. Consequently, the reported novel enzyme pairing can be used to study cell-specific signaling in uninterrupted, continuous co-cultures. Demonstrating the importance of increased labeling stability for addressing novel biological questions, we compare the cell-specific phosphoproteome of fibroblasts in direct co-culture with epithelial tumor cells in both interrupted (daily media exchange) and continuous (no media exchange) co-cultures. This analysis identified multiple cell-specific phosphorylation sites specifically regulated in the continuous co-culture. Given their applicability to multiple cell types, continuous co-culture labeling fidelity, and suitability for long-term cell-cell phospho-signaling experiments, we propose DDC(M.tub-KDEL) and Lyr(M37-KDEL) as excellent enzymes for cell-specific labeling with amino acid precursors. PMID:24820872

  16. Cultural Language Study: Grade 7.

    ERIC Educational Resources Information Center

    Schwartz, Betty L.; Tappenden, Jacqueline W.

    This course guide, the first in a two-year sequence, is designed to give students an overview of Greek and Roman culture and language from the era of the early Aegean civilizations in Greece and Asia Minor to the Augustan Age in Rome. Six units of study are concerned with the growth and development of Greece and with the metamorphosis of Rome from…

  17. Cell Culture as a Model System for Teaching: Using PC12 Cells

    NSDL National Science Digital Library

    Elizabeth M. Adler (American Association for the Advancement of Science; Associate Editor of Science's STKE REV)

    2006-05-09

    This Teaching Resource provides an introduction to the use of cultured cells as an experimental approach in undergraduate laboratory research and the study of neuronal differentiation in PC12 cells. In addition, a thought experiment with answers is provided that can be used to assess student understanding of (i) the scientific method, (ii) signaling processes involved in cellular differentiation, and (iii) the use of pharmacological agents to manipulate a cell culture system.

  18. Characterisation and Germline Transmission of Cultured Avian Primordial Germ Cells

    PubMed Central

    Macdonald, Joni; Glover, James D.; Taylor, Lorna; Sang, Helen M.; McGrew, Michael J.

    2010-01-01

    Background Avian primordial germ cells (PGCs) have significant potential to be used as a cell-based system for the study and preservation of avian germplasm, and the genetic modification of the avian genome. It was previously reported that PGCs from chicken embryos can be propagated in culture and contribute to the germ cell lineage of host birds. Principal Findings We confirm these results by demonstrating that PGCs from a different layer breed of chickens can be propagated for extended periods in vitro. We demonstrate that intracellular signalling through PI3K and MEK is necessary for PGC growth. We carried out an initial characterisation of these cells. We find that cultured PGCs contain large lipid vacuoles, are glycogen rich, and express the stem cell marker, SSEA-1. These cells also express the germ cell-specific proteins CVH and CDH. Unexpectedly, using RT-PCR we show that cultured PGCs express the pluripotency genes c-Myc, cKlf4, cPouV, cSox2, and cNanog. Finally, we demonstrate that the cultured PGCs will migrate to and colonise the forming gonad of host embryos. Male PGCs will colonise the female gonad and enter meiosis, but are lost from the gonad during sexual development. In male hosts, cultured PGCs form functional gametes as demonstrated by the generation of viable offspring. Conclusions The establishment of in vitro cultures of germline competent avian PGCs offers a unique system for the study of early germ cell differentiation and also a comparative system for mammalian germ cell development. Primary PGC lines will form the basis of an alternative technique for the preservation of avian germplasm and will be a valuable tool for transgenic technology, with both research and industrial applications. PMID:21124737

  19. Prevention and Detection of Mycoplasma Contamination in Cell Culture

    PubMed Central

    Nikfarjam, Laleh; Farzaneh, Parvaneh

    2012-01-01

    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 contamination in cell culture; 3. the importance of mycoplasma tests in cell culture; 4. different methods to identify mycoplasma contamination; 5. the consequences of mycoplasma contamination in cell culture and 6. available methods to eliminate mycoplasma contamination. Awareness about the sources of mycoplasma and pursuing aseptic techniques in cell culture along with reliable detection methods of mycoplasma contamination can provide an appropriate situation to prevent mycoplasma contamination in cell culture. PMID:23508237

  20. Sulforaphane induces DNA single strand breaks in cultured human cells

    Microsoft Academic Search

    Piero Sestili; Marco Paolillo; Monia Lenzi; Evelin Colombo; Luciana Vallorani; Lucia Casadei; Chiara Martinelli; Carmela Fimognari

    2010-01-01

    Sulforaphane (SFR), an isothiocyanate from cruciferous vegetables, possesses growth-inhibiting and apoptosis-inducing activities in cancer cell lines. Recently, SFR has been shown to promote the mitochondrial formation of reactive oxygen species (ROS) in human cancer cell lines. The present study was undertaken to see whether SFR-derived ROS might cause DNA damage in cultured human cells, namely T limphoblastoid Jurkat and human

  1. Culture of Normal Human Epidermal Cells with 3T3 Feeders on Millipore Filters

    Microsoft Academic Search

    Shigeo Kondo; Kazuo Aso; Masayoshi Namba

    1979-01-01

    Human epidermal cells were cultured with lethally irradiated 3T3 cells and grew as large colonies both on plastic and millipore filters. Subcultures were possible. The grown colonies were studied morphologically and histologically. The whole intact crossview of epidermal colonies in paraffin sections was obtained from the cell culture on millipore filters. The epidermal cells derived from younger persons had higher

  2. Shape memory polymers for active cell culture.

    PubMed

    Davis, Kevin A; Luo, Xiaofan; Mather, Patrick T; Henderson, James H

    2011-01-01

    Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat(1-5). In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, T(trans;) [either the melting temperature (T(m;)) or the glass transition temperature (T(g;))]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its T(trans;) while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through T(trans;) under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape. Cells are capable of surveying the mechanical properties of their surrounding environment(6). The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces(7-14). These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture. Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions. PMID:21750496

  3. High Frequency Retrotransposition in Cultured Mammalian Cells

    Microsoft Academic Search

    John V Moran; Susan E Holmes; Thierry P Naas; Ralph J DeBerardinis; Jef D Boeke; Haig H Kazazian

    1996-01-01

    We previously isolated two human L1 elements (L1.2 and LRE2) as the progenitors of disease-producing insertions. Here, we show these elements can actively retrotranspose in cultured mammalian cells. When stably expressed from an episome in HeLa cells, both elements retrotransposed into a variety of chromosomal locations at a high frequency. The retrotransposed products resembled endogenous L1 insertions, since they were

  4. Human airway epithelial cells in culture for studying the molecular mechanisms of the inflammatory response triggered by diesel exhaust particles.

    PubMed

    Marano, F; Boland, S; Bonvallot, V; Baulig, A; Baeza-Squiban, A

    2002-01-01

    Epidemiological studies have shown that particulate air pollution is linked to the increase of morbidity and mortality due to respiratory diseases. Diesel exhaust particles (DEPs), which are the most important part of PM2.5 in Western European and Japanese urban areas, have been suspected. The mechanisms of proinflammatory response induced by DEPS were elucidated using a human epithelial cell line (16-HBE). It has been shown that DEPs can be phagocytosed by HBE cells, inducing the release of cytokines. MAP kinase pathways (i.e., ERK1/2 and P38) were triggered as well as the activation of the nuclear factor NF-kappaB. Reactive oxygen species (ROS) were strongly incriminated in this response because DEPs induce the increase of intracellular hydroperoxides and antioxidants inhibit the release of DEP-induced cytokines, the activation of MAP kinases and NF-kappaB. Organic compounds adsorbed on DEPs seemed to be involved in the response and the production of ROS. Moreover, we have demonstrated that DEPs can activate CYP1A1 in HBE cells. These experimental results give biological plausibility to the epidemiological findings. PMID:12240962

  5. Novel Micropatterned Cardiac Cell Cultures with Realistic Ventricular Microstructure

    PubMed Central

    Badie, Nima; Bursac, Nenad

    2009-01-01

    Systematic studies of cardiac structure-function relationships to date have been hindered by the intrinsic complexity and variability of in vivo and ex vivo model systems. Thus, we set out to develop a reproducible cell culture system that can accurately replicate the realistic microstructure of native cardiac tissues. Using cell micropatterning techniques, we aligned cultured cardiomyocytes at micro- and macroscopic spatial scales to follow local directions of cardiac fibers in murine ventricular cross sections, as measured by high-resolution diffusion tensor magnetic resonance imaging. To elucidate the roles of ventricular tissue microstructure in macroscopic impulse conduction, we optically mapped membrane potentials in micropatterned cardiac cultures with realistic tissue boundaries and natural cell orientation, cardiac cultures with realistic tissue boundaries but random cell orientation, and standard isotropic monolayers. At 2 Hz pacing, both microscopic changes in cell orientation and ventricular tissue boundaries independently and synergistically increased the spatial dispersion of conduction velocity, but not the action potential duration. The realistic variations in intramural microstructure created unique spatial signatures in micro- and macroscopic impulse propagation within ventricular cross-section cultures. This novel in vitro model system is expected to help bridge the existing gap between experimental structure-function studies in standard cardiac monolayers and intact heart tissues. PMID:19413993

  6. ANTHOCYANIN (ACN) STABILITY IN CELL CULTURE MEDIA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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, ...

  7. Isolation and culture of protoplasts from cotton cell cultures

    E-print Network

    Finer, John James

    1981-01-01

    cellulase, and 0. 5X Macerase pec. tinase with a pH of 4. 7, (d) and an agitation rate of 40 rpm. This procedure enabled the conversion of 20. 5X of the callus cells to protoplasts. The protoplasts were plated at 10 to 10 protoplasts per ml. 4 5 About 0... Efficiency Protoplast Plating Procedure 17 18 RESULTS 20 Determination of Exponential Growth Phase 20 Length of Incubation Period Concentration of Enzymes Effect of pH Effect of Mannitol Concentration Effect of Agitation Rate Protoplast Culture...

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

    PubMed Central

    Akopian, Veronika; 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.; McKay, Ronald D. G.; Miyazaki, Takamichi; Nakatsuji, Norio; Oh, Steve K. W.; Pera, Martin F.; Rossant, Janet; Stacey, Glyn N.; Suemori, Hirofumi

    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 propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories. PMID:20186512

  9. Using Haworthia Cultured Cells as an Aid in Teaching Botany

    ERIC Educational Resources Information Center

    Majumdar, Shyamal K.; Castellano, John M.

    1977-01-01

    Callus induction from species of Haworthia can be done quickly in the laboratory with minimal equipment to study tissue dedifferentiation and cellular redifferentiation. It is shown that the cultured cell can also be used to study and evaluate the effects of various mutagens, carcinogens, and pesticides in controlled environments. (Author/MA)

  10. Neonatal rat heart cells cultured in simulated microgravity

    Microsoft Academic Search

    Robert E. Akins; Nancy A. Schroedl; Steve R. Gonda; Charles R. Hartzell

    1997-01-01

    Summary  \\u000a In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit\\u000a gravity constrain cells to propagate, differentiate, and interact in a two-dimensional (2D) plane. Neonatal rat cardiac cells\\u000a in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by nonmyocyte cell types.\\u000a Such cardiac cell cultures respond predictably

  11. Placental-derived stem cells: Culture, differentiation and challenges.

    PubMed

    Oliveira, Maira S; Barreto-Filho, João B

    2015-05-26

    Stem cell therapy is a promising approach to clinical healing in several diseases. A great variety of tissues (bone marrow, adipose tissue, and placenta) are potentially sources of stem cells. Placenta-derived stem cells (p-SCs) are in between embryonic and mesenchymal stem cells, sharing characteristics with both, such as non-carcinogenic status and property to differentiate in all embryonic germ layers. Moreover, their use is not ethically restricted as fetal membranes are considered medical waste after birth. In this context, the present review will be focused on the biological properties, culture and potential cell therapy uses of placental-derived stem cells. Immunophenotype characterization, mainly for surface marker expression, and basic principles of p-SC isolation and culture (mechanical separation or enzymatic digestion of the tissues, the most used culture media, cell plating conditions) will be presented. In addition, some preclinical studies that were performed in different medical areas will be cited, focusing on neurological, liver, pancreatic, heart, muscle, pulmonary, and bone diseases and also in tissue engineering field. Finally, some challenges for stem cell therapy applications will be highlighted. The understanding of the mechanisms involved in the p-SCs differentiation and the achievement of pure cell populations (after differentiation) are key points that must be clarified before bringing the preclinical studies, performed at the bench, to the medical practice. PMID:26029347

  12. Effects of carbon monoxide on cardiac muscle cells in culture

    SciTech Connect

    Nag, A.C.; Chen, K.C.; Cheng, Mei (Oakland Univ., Rochester, MI (USA) General Motors Research Laboratories, Warren, MI (USA))

    1988-09-01

    Embryonic rat cardiac muscle cells grown in the presence of various tensions of CO (5-95%) without the presence of O{sub 2} survived and exhibited reduced cell growth, which was concentration dependent. When cardiac muscle cells were grown in the presence of a mixture of CO (10-20%) and O{sub 2} (10-20%), the growth rate of these cells was comparable to that of the control cells. Cardiac myocytes continued to beat when exposed to varying tensions of CO, except in the case of 95% CO. The cells exposed to different concentrations of CO contained fewer myofibrils of different stages of differentiation compared with the control and the culture exposed to a mixture of 20% O{sub 2} and 20% CO, with cells that contained abundant, highly differentiated myofibrils. There was no significant difference in the structural organization of mitochondria between the control and the surviving experimental cells. It is evident from the present studies that O{sub 2} is required for the optimum in vitro cellular growth of cardiac muscle. Furthermore, CO in combination with O{sub 2} at a concentration of 10 or 20% can produce optimal growth of cardiac muscle cells in culture. To determine maximum labeling index during the labeling period, cells were continuously labeled with ({sup 3}H)thymidine for 24 h before the termination of cultures.

  13. Antibody inhibition of human cytomegalovirus spread in epithelial cell cultures

    PubMed Central

    Cui, Xiaohong; Lee, Ronzo; Adler, Stuart P.; McVoy, Michael A.

    2013-01-01

    Anti-cytomegalovirus (CMV) antibodies reduce the incidence of CMV transmission and ameliorate the severity of CMV-associated disease. Neutralizing activity, measured as the ability of antibodies to prevent entry of cell-free virus, is an important component of natural immunity. However, in vivo CMV amplification may occur mainly via spread between adjacent cells within tissues. Thus, inhibition of cell-to-cell spread may be important when evaluating therapeutic antibodies or humoral responses to infection or immunization. In vitro CMV cell-to-cell spread is largely resistant to antibodies in fibroblast cultures but sensitive in endothelial cell cultures. In the present study antibodies in CMV hyperimmuneglobulin or seropositive human sera inhibited CMV cell-to-cell spread in epithelial cell cultures. Spread inhibition activity was quantitated with a GFP reporter assay employing GFP-tagged epithelialtropic variants of CMV strains Towne or AD169. Measurement of spread inhibition provides an additional parameter for the evaluation of candidate vaccines or immunotherapeutics and to further characterize the role of antibodies in controlling CMV transmission and disease. PMID:23669101

  14. Culture and characterisation of epithelial cells from human pterygia

    PubMed Central

    Di, G; Tedla, N.; Kumar, R.; McCluskey, P.; Lloyd, A.; Coroneo, M.; Wakefield, D.

    1999-01-01

    BACKGROUND/AIMS—Pterygia are a common disorder of the ocular surface. The disease represents a chronic fibrovascular and degenerative process thought to originate at the conjunctival-corneal junction, where altered limbal stem cells are proposed to be the cell of origin. Extensive epidemiological evidence exists to implicate ultraviolet B irradiation in the pathogenesis of pterygia. To date no animal or in vitro culture model has been developed to test such an hypothesis. The aim of this study was to establish and characterise a pure population of epithelial cells derived from pterygium tissue.?METHODS—Tissue specimens were obtained from patients undergoing pterygium excision. Explants were cultured in either serum free or serum supplemented medium. Primary and passaged cells were processed for light microscopy, analysed by flow cytometry, and characterised immunohistochemically using specific antibodies.?RESULTS—In serum free culture, cuboidal cells with typical morphology of epithelial cells migrated from the pterygium explants from 3 days onwards and eventually formed a cohesive monolayer. Passaged cells consisted of 98.4% cytokeratin positive cells and demonstrated immunoreactivity for multiple cytokeratins, including AE1, AE3, AE5, but were negative for AE8. These cells also expressed an epithelial specific antigen, together with vimentin and mucin, as did epithelial cells in sections of pterygia.?CONCLUSIONS—A relatively simple method of isolating pterygium epithelial cells has been established. Cultured pterygium epithelial cells are phenotypically and functionally similar to their in vivo counterparts with respect to keratin, vimentin, and mucin expression. In vitro assays using these cells may aid in elucidating the pathogenesis of pterygia.?? PMID:10460780

  15. Co-culture of buffalo preantral follicles with different somatic cells.

    PubMed

    Ramesh, H S; Gupta, P S P; Nandi, S; Manjunatha, B M; Kumar, V Girish; Ravindra, J P

    2008-10-01

    The effect of co-culture of buffalo preantral follicles (PFs) with different somatic cells, i.e, cumulus, granulosa, ovarian mesenchymal and oviductal epithelial cells was studied. Large PFs (250-450 microm) were isolated by microdissecting the trypsin (1%) digested ovarian cortical slices. Cumulus cells were isolated by repeated pipetting of oocytes, granulosa cells were isolated by aspirating from punctured PFs and ovarian mesenchymal cells were isolated from ovarian cortex by scraping the cortical slices and passing through 20 microm filter. Preantral follicles were cultured in standard culture medium without somatic cells or co-cultured with cumulus cells, granulosa cells, ovarian mesenchymal cells and oviductal epithelial cells for 80 days. The growth rate (microm/day) of the PFs was monitored by measuring follicular diameter on day 0, 30, 60 and 80 days of culture. The viability of PFs was evaluated by trypan blue staining. The results indicated that PFs co-cultured with cumulus, granulosa and ovarian mesenchymal cells had a better development and survivality compared with control and those co-culture with oviductal epithelial cells. Maximum growth and survivality of PFs were achieved when cultured with cumulus cells. It is concluded that inclusion of somatic cells in PF culture media had beneficial effect on the growth of PFs and cumulus cells supported maximum growth and survivality of PFs in vitro of all somatic cells tested. PMID:18298404

  16. Response of cultured tomato cells subjected to excess zinc: role of cell wall in zinc compartmentation

    Microsoft Academic Search

    Aurélie Muschitz; Céline Faugeron; Henri Morvan

    2009-01-01

    The aim of this preliminary study was to evaluate the role of the cell wall in Zn accumulation and tolerance by tomato suspension-cultured\\u000a cells. Growth parameters, Zn distribution and accumulation by tomato cells were determined in function of zinc concentration.\\u000a A particular attention was paid to the variations of the total cell wall material (cell wall carbohydrates, proteins, and\\u000a exopolymers)

  17. Cell Culture Assay for Human Noroviruses [response

    SciTech Connect

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

    2007-07-01

    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  18. Cell cultures derived from early zebrafish embryos differentiate in vitro into neurons and astrocytes

    Microsoft Academic Search

    Chandramallika Ghosh; Yi Liu; Chunguang Ma; Paul Collodi

    1997-01-01

    The zebrafish is a polular nonmammalian model for studies of neural development. We have derived cell cultures, initiated from blastula-stage zebrafish embryos, that differentiate in vitro into neurons and astrocytes. Cultures were initiated in basal nutrient medium supplemented with bovine insulin, trout serum, trout embryo extract and fetal bovine serum. After two weeks in culture the cells exhibited extensive neurite

  19. Method of determining the number of cells in cell culture

    SciTech Connect

    Connolly, D.T.

    1990-06-12

    This patent describes a color-sensitivity method for determining the number of cells in in vitro cell culture at a sensitivity as low as about 100 or about 500 cells. It comprises lysing the cells and incubating the lysate with p-nitrophenyl phosphate at acid pH for a predetermined period of time at a temperature of from about 35{degrees} to about 38{degrees}C. and then measuring the color development at 400 to 420 nanometers and correlating the color development with cell number by comparing with a control standard of known cell number.

  20. Sporopollenin in the cell wall of Chlorella and other algae: Ultrastructure, chemistry, and incorporation of (14)C-acetate, studied in synchronous cultures.

    PubMed

    Atkinson, A W; Gunning, B E; John, P C

    1972-03-01

    Cells of Chlorella fusca var. vacuolata (Cambridge 211/8p) resisted efforts aimed at producing naked protoplasts by enzymatic degradation of the cell wall, and a study of the development and composition of the wall was therefore undertaken. 1. After cytokinesis has produced naked autospores within the mother cell wall, cell wall formation commences outside the autospore plasma membrane with the appearance of small trilaminar plaques. These enlarge while inter-autospore granular material diminishes in quantity, and they eventually fuse to produce a complete trilaminar sheath around each autospore. 2. A microfibrillar, cellulase digestible, layer is deposited between the trilaminar component and the plasma membrane. Meanwhile the corresponding microfibrillar component of the mother cell wall is digested leaving only its resistant trilaminar component. 3. The trilaminar component includes a substance considered to be the polymerized carotenoid, sporopollenin, on the basis of its resistance to extreme extraction procedures including acetolysis, and its infra red absorption spectrum. 4. Two phases of sporopollenin biosynthesis were detected by means of pulse and pulse-chase treatments with (14)C-acetate at intervals during the cell cycle in synchronous cultures. One coincides with the formation of the sporopollenin-containing trilaminar wall component, and the other is 6-8 hours earlier while the cells are in karyokinesis. The former yields labelled sporopollenin directly and the latter probably represents formation of a precursor. 5. Of five other strains of Chlorella tested, only one possesses sporopollenin, and so does one Scenedesmus and two out of three strains of Prototheca. 6. Examination of the wall structure of the above algae suggest a relationship between the presence of sporopollenin and the development of an outer, trilaminar wall component. 7. A survey of the literature gives support to this hypothesis and further suggests that the ability to synthesise sporopollenin is related to the ability to produce secondary carotenoids. 8. The significance of the findings is discussed. PMID:24477346

  1. Teaching Culture. The Long Revolution in Cultural Studies.

    ERIC Educational Resources Information Center

    Aldred, Nannette, Ed.; Ryle, Martin, Ed.

    This book contains 12 papers that trace the connections and tensions between the original aims and forms of cultural studies in Great Britain and Northern Ireland and the current settings, goals, and methodologies of cultural studies. The following papers are included: "Introduction" (Nannette Aldred and Martin Ryle); "Marginal Occupations: Adult…

  2. Splitting culture medium by air-jet and rewetting for the assessment of the wettability of cultured epithelial cell surfaces.

    PubMed

    Tanaka, Nobuyuki; Kondo, Makoto; Uchida, Ryohei; Kaneko, Makoto; Sugiyama, Hiroaki; Yamato, Masayuki; Okano, Teruo

    2013-12-01

    This study found that the phenomenon of rewetting after squeezing culture medium varied in different culture conditions for rat oral mucosal epithelial cells. When culture medium covering over cultured cells was squeezed by an air-jet application, the motion of squeezed culture medium was able to be observed by using a commercially available movie camera. Squeezed width on cells cultured in keratinocyte culture medium (KCM), which contained with fetal bovine serum, was one-sixth of that in FBS-free KCM. This result corresponded to the mucous layer staining statuses of cultured cells in both cases; positive in KCM and negative in FBS-free medium. Furthermore, the gene expression of mucous glycoprotein MUC4 in KCM was 100 times higher than that in FBS-free medium, and the expression of MUC4 protein only showed on the apical surface of cells cultured in KCM. The relative gene expression levels of MUC1, 13, 15, and 16 in both the normal and FBS-free medium were found to be no more than one-thirtieth of that of MUC4 in KCM. The main factor of the wettability difference between KCM and FBS-free medium was speculated to be the difference of MUC4 expression between both media. This method can be a simple technique for testing not only the surface wettability but also the mucous formation of cultured cells. PMID:24008039

  3. Primary culture of avian embryonic heart forming region cells to study the regulation of vertebrate early heart morphogenesis by vitamin A

    PubMed Central

    2014-01-01

    Background Important knowledge about the role of vitamin A in vertebrate heart development has been obtained using the vitamin A-deficient avian in ovo model which enables the in vivo examination of very early stages of vertebrate heart morphogenesis. These studies have revealed the critical role of the vitamin A-active form, retinoic acid (RA) in the regulation of several developmental genes, including the important growth regulatory factor, transforming growth factor-beta2 (TGF?2), involved in early events of heart morphogenesis. However, this in ovo model is not readily available for elucidating details of molecular mechanisms determining RA activity, thus limiting further examination of RA-regulated early heart morphogenesis. In order to obtain insights into RA-regulated gene expression during these early events, a reliable in vitro model is needed. Here we describe a cell culture that closely reproduces the in ovo observed regulatory effects of RA on TGF?2 and on several developmental genes linked to TGF? signaling during heart morphogenesis. Results We have developed an avian heart forming region (HFR) cell based in vitro model that displays the characteristics associated with vertebrate early heart morphogenesis, i.e. the expression of Nkx2.5 and GATA4, the cardiogenesis genes, of vascular endothelial growth factor (VEGF-A), the vasculogenesis gene and of fibronectin (FN1), an essential component in building the heart, and the expression of the multifunctional genes TGF?2 and neogenin (NEO). Importantly, we established that the HFR cell culture is a valid model to study RA-regulated molecular events during heart morphogenesis and that the expression of TGF?2 as well as the expression of several TGF?2-linked developmental genes is regulated by RA. Conclusions Our findings reported here offer a biologically relevant experimental in vitro system for the elucidation of RA-regulated expression of TGF?2 and other genes involved in vertebrate early cardiovascular morphogenesis. PMID:24552295

  4. Nanopillar based electrochemical biosensor for monitoring microfluidic based cell culture

    NASA Astrophysics Data System (ADS)

    Gangadharan, Rajan

    In-vitro assays using cultured cells have been widely performed for studying many aspects of cell biology and cell physiology. These assays also form the basis of cell based sensing. Presently, analysis procedures on cell cultures are done using techniques that are not integrated with the cell culture system. This approach makes continuous and real-time in-vitro measurements difficult. It is well known that the availability of continuous online measurements for extended periods of time will help provide a better understanding and will give better insight into cell physiological events. With this motivation we developed a highly sensitive, selective and stable microfluidic electrochemical glucose biosensor to make continuous glucose measurements in cell culture media. The performance of the microfluidic biosensor was enhanced by adding 3D nanopillars to the electrode surfaces. The microfluidic glucose biosensor consisted of three electrodes---Enzyme electrode, Working electrode, and Counter electrode. All these electrodes were enhanced with nanopillars and were optimized in their respective own ways to obtain an effective and stable biosensing device in cell culture media. For example, the 'Enzyme electrode' was optimized for enzyme immobilization via either a polypyrrole-based or a self-assembled-monolayer-based immobilization method, and the 'Working electrode' was modified with Prussian Blue or electropolymerized Neutral Red to reduce the working potential and also the interference from other interacting electro-active species. The complete microfluidic biosensor was tested for its ability to monitor glucose concentration changes in cell culture media. The significance of this work is multifold. First, the developed device may find applications in continuous and real-time measurements of glucose concentrations in in-vitro cell cultures. Second, the development of a microfluidic biosensor will bring technical know-how toward constructing continuous glucose monitoring devices. Third, the methods used to develop 3D electrodes incorporated with nanopillars can be used for other applications such as neural probes, fuel cells, solar cells etc., and finally, the knowledge obtained from the immobilization of enzymes onto nanostructures sheds some new insight into nanomaterial/biomolecule interactions.

  5. An embryogenic cell suspension culture of Picea glauca (White spruce)

    Microsoft Academic Search

    I. Hakman; L. C. Fowke

    1987-01-01

    A cell suspension culture of Picea glauca (White spruce) which continuously produces somatic embryos has been established. Embryogenic callus derived from cultured zygotic embryos was used to initiate the culture. Numerous embryos at various early stages of development were recognized; they exhibited a meristematic embryonic region and suspensor consisting of elongate, vacuolated cells. The culture also contained clumps of meristematic

  6. Nerve Growth Factor Receptors on Cultured Rat Schwann Cells

    Microsoft Academic Search

    Peter S. DiStefano; Eugene M. Johnson

    1988-01-01

    Neonatal rat Schwann ceils were grown in tissue culture and assayed for NGF receptors with time in culture. NGF receptor levels on freshly prepared Schwann cells (day 0) were low but increased dramatically during the first week in culture. Characterization of 1a51-NGF binding to resuspended cells grown for 4 d in culture revealed that binding was not sat- urable at

  7. Immunological studies in patients with chronic active hepatitis. Cytotoxic activity of lymphocytes to autochthonous liver cells grown in tissue culture.

    PubMed Central

    Paronetto, F; Vernace, S

    1975-01-01

    The cytotoxic activity of lymphocytes against autochthonous liver cells was studied in patients with chronic liver diseases and in controls. Cytotoxicity of lymphocytes was observed in eight of ten patients with chronic active hepatitis, two patients with chronic persistent hepatitis, one patient with primary biliary cirrhosis, one patient with alcoholic hepatitis and carcinoma of the pancreas, and in three of five patients with acute viral hepatitis, but not in seven patients without liver alteration or with miscellaneous liver diseases. Serum was not cytotoxic, but in three patients it decreased the cytotoxicity of lymphocytes. Cytotoxicity was seen in both HBAg-positive and HBAg-negative patients, appears to be influenced by therapy, and does not correlate with autoantibodies. These data support the hypothesis of an aggressive activity of lymphocytes in certain liver diseases. PMID:1204242

  8. Improved Culture-Based Isolation of Differentiating Endothelial Progenitor Cells from Mouse Bone Marrow Mononuclear Cells

    Microsoft Academic Search

    Haruki Sekiguchi; Masaaki Ii; Kentaro Jujo; Ayumi Yokoyama; Nobuhisa Hagiwara; Takayuki Asahara

    2011-01-01

    Numerous endothelial progenitor cell (EPC)-related investigations have been performed in mouse experiments. However, defined characteristics of mouse cultured EPC have not been examined. We focused on fast versus slow adherent cell population in bone marrow mononuclear cells (BMMNCs) in culture and examined their characteristics. After 24 h-culture of BMMNCs, attached (AT) cells and floating (FL) cells were further cultured in

  9. Generation of a large volume of clinically relevant nanometre-sized ultra-high-molecular-weight polyethylene wear particles for cell culture studies

    PubMed Central

    Ingham, Eileen; Fisher, John; Tipper, Joanne L

    2014-01-01

    It has recently been shown that the wear of ultra-high-molecular-weight polyethylene in hip and knee prostheses leads to the generation of nanometre-sized particles, in addition to micron-sized particles. The biological activity of nanometre-sized ultra-high-molecular-weight polyethylene wear particles has not, however, previously been studied due to difficulties in generating sufficient volumes of nanometre-sized ultra-high-molecular-weight polyethylene wear particles suitable for cell culture studies. In this study, wear simulation methods were investigated to generate a large volume of endotoxin-free clinically relevant nanometre-sized ultra-high-molecular-weight polyethylene wear particles. Both single-station and six-station multidirectional pin-on-plate wear simulators were used to generate ultra-high-molecular-weight polyethylene wear particles under sterile and non-sterile conditions. Microbial contamination and endotoxin levels in the lubricants were determined. The results indicated that microbial contamination was absent and endotoxin levels were low and within acceptable limits for the pharmaceutical industry, when a six-station pin-on-plate wear simulator was used to generate ultra-high-molecular-weight polyethylene wear particles in a non-sterile environment. Different pore-sized polycarbonate filters were investigated to isolate nanometre-sized ultra-high-molecular-weight polyethylene wear particles from the wear test lubricants. The use of the filter sequence of 10, 1, 0.1, 0.1 and 0.015 µm pore sizes allowed successful isolation of ultra-high-molecular-weight polyethylene wear particles with a size range of < 100 nm, which was suitable for cell culture studies. PMID:24658586

  10. Cultured bovine brain capillary endothelial cells (BBCEC) - a blood-brain barrier model for studying the binding and internalization of insulin and insulin-like growth factor 1

    SciTech Connect

    Keller, B.T.; Borchardt, R.T.

    1987-05-01

    Cultured bovine brain capillary endothelial cells (BBCEC) have previously been reported by their laboratory as a working model for studying nutrient and drug transport and metabolism at the blood-brain barrier. In the present study, they have utilized this culture system to investigate the binding and internalization of (/sup 125/I)-labelled insulin (INS) and insulin-like growth factor 1(IGF-1) by BBCEC. After 2 hrs at 23/sup 0/C, the specific binding of INS and IGF-1 was 1.6% and 13.6%, respectively. At 37/sup 0/C, the maximum specific binding was 0.9% for INS and 5.8% for IGF-1. Using an acid-wash technique to assess peptide internalization, it was observed that, at 37/sup 0/C, approximately 60% of the bound INS rapidly became resistant to acid treatment, a value which was constant over 2 hr. With IGF-1, a similar proportion of the bound material, 62%, became resistant by 30 min, but subsequently decreased to 45% by 2 hr. Scatchard analysis of competitive binding studies indicated the presence of two binding sites for each protein, having K/sub d/'s of 0.82 nM and 19.2 nM for INS and 0.39 nM and 3.66 nM for IGF-1. Little change in the amount of INS binding was observed over a four-day interval as the cultures became a confluent monolayer. The present report of binding and internalization of these proteins suggests that the BBCEC may utilize a receptor-mediated process to internalize and/or transport (transcytosis) INS and IGF-1 from the circulation.

  11. Effects of cholesterol oxidase on cultured vascular smooth muscle cells.

    PubMed

    Liu, K Z; Maddaford, T G; Ramjiawan, B; Kutryk, M J; Pierce, G N

    1991-11-13

    Cholesterol oxidase (3 beta-hydroxy-steroid oxidase) catalyzes the oxidation of cholesterol to 4-cholesten-3 one and other oxidized cholesterol derivatives. The purpose of the present study was to investigate its effects on cultured vascular smooth muscle cells. Cultured rabbit aortic smooth muscle cells were morphologically altered after exposure to cholesterol oxidase in the presence of culture medium containing 10% fetal calf serum. If fetal calf serum was absent, cells were unaffected by the treatment. The extent of morphological change of the smooth muscle cells was dependent upon the time of exposure to the enzyme and the concentration of cholesterol oxidase employed. After moderate treatment with cholesterol oxidase, cells excluded trypan blue. Further, a specific mitochondrial marker DASPMI (dimethyl aminostyryl-methyl-pyridiniumiodine) which was used as a fluorescent index of cell viability, revealed that cell viability was unchanged after moderate cholesterol oxidase treatment. Nile red, a hydrophobic probe which selectively stains intracellular lipid droplets, was applied to detect the cellular lipid content after treatment with cholesterol oxidase. Cellular nile red fluorescence intensity increased linearly with the time and concentration of cholesterol oxidase treatment. These results demonstrate that cholesterol oxidase alters lipid deposition in the cell and changes cell morphology. The primary site of action of cholesterol oxidase appears to be independent of the cell membrane itself and instead is dependent upon the lipid content in the surrounding culture media. These changes occur prior to the cytotoxic effects of extensive oxidation. Because oxidized cholesterol may play an important role in the pathogenesis of atherosclerosis, our results have implications for intracellular accumulation of lipids in smooth muscle cells during the atherosclerotic lesion. PMID:1770944

  12. Cell cultures as models of cardiac mechanoelectric feedback

    PubMed Central

    Zhang, Yibing; Sekar, Rajesh B.; McCulloch, Andrew D.; Tung, Leslie

    2008-01-01

    Although stretch-activated currents have been extensively studied in isolated cells and intact hearts in the context of mechanoelectric feedback (MEF) in the heart, quantitative data regarding other mechanical parameters such as pressure, shear, bending, etc, are still lacking at the multicellular level. Cultured cardiac cell monolayers have been used increasingly in the past decade as an in vitro model for the studies of fundamental mechanisms that underlie normal and pathological electrophysiology at the tissue level. Optical mapping makes possible multisite recording and analysis of action potentials and wavefront propagation, suitable for monitoring the electrophysiological activity of the cardiac cell monolayer under a wide variety of controlled mechanical conditions. In this paper, we review methodologies that have been developed or could be used to mechanically perturb cell monolayers, and present some new results on the acute effects of pressure, shear stress and anisotropic strain on cultured neonatal rat ventricular myocyte (NRVM) monolayers. PMID:18384846

  13. Eradication of Mycoplasma contaminations from cell cultures.

    PubMed

    Uphoff, Cord C; Drexler, Hans G

    2014-01-01

    Mycoplasma contaminations have a multitude of effects on cultured cell lines that may influence the results of experiments or pollute bioactive substances isolated from the eukaryotic cells. The elimination of mycoplasma contaminations from cell cultures with antibiotics has been proven to be a practical alternative to discarding and re-establishing important or irreplaceable cell lines. Different fluoroquinolones, tetracyclins, pleuromutilins, and macrolides shown to have strong anti-mycoplasma properties are employed for the decontamination. These antibiotics are applied as single treatments, as combination treatment of two antibiotics in parallel or successively, or in combination with a surface-active peptide to enhance the action of the antibiotic. The protocols in this unit allow eradication of mycoplasmas, prevention of the development of resistant mycoplasma strains, and potential cure of heavily contaminated and damaged cells. Consistent and permanent alterations to eukaryotic cells attributable to the treatment have not been demonstrated. Curr. Protoc. Mol. Biol. 106:28.5.1-28.5.12. © 2014 by John Wiley & Sons, Inc. PMID:24733241

  14. Isolation, culture, and transplantation of muscle satellite cells

    PubMed Central

    Motohashi, Norio; Asakura, Yoko; Asakura, Atsushi

    2014-01-01

    Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2–5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors. However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities. PMID:24747722

  15. [Influence of fluid shear stress on cultured vascular endothelial cells].

    PubMed

    Takakuwa, O

    1990-03-01

    Vascular endothelial cells modulate their functions in response to hemodynamic forces such as fluid shear stress. In the present study, we applied shear to cultured bovine aortic endothelial cells (EC) by using a saecially designed apparatus and examined the effects of their homogenate and conditioned medium on such EC and smooth muscle cell (SMC) functions as adhesion, growth, migration or collagen synthesis. Cultured bovine aortic SMC were stimulated to adhere to wells and grow in the presence of EC conditioned medium. This conditioned medium had no effect on EC adhesion and growth. The activities of stimulating SMC adhesion and growth were almost the same in both EC conditioned medium obtained from static cultures and shear-loaded cultures. Studies with filters in a modified Boyden chamber showed that shear-loaded EC homogenate yielded stimulated SMC migration. Also shear-loaded EC cell layer contained increased amount of collagen compared with static EC cell layer. These observations indicate that:(a) EC secrets the substances which stimulate SMC adhesion and growth, but these functions are not affected by shear stress application, (b) EC produces SMC migration stimulators in response to shear stress, and (c) shear stress can enhance EC collagen synthesis. These results are relevant to EC response to hemodynamic forces and its role in the localization of atherosclerotic lesions in vivo. PMID:2365276

  16. An Introductory Undergraduate Course Covering Animal Cell Culture Techniques

    ERIC Educational Resources Information Center

    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…

  17. Apoptosis in Batch Cultures of Chinese Hamster Ovary Cells

    E-print Network

    Sinskey, Anthony J.

    Apoptosis in Batch Cultures of Chinese Hamster Ovary Cells J. Goswami,1 A. J. Sinskey,2 H. Steller of the main problems in the culture of Chinese Hamster Ovary (CHO) cells continues to be the inability. Keywords: cell culture; Chinese Hamster Ovary; apopto- sis; caspase; bcl-2 INTRODUCTION Chinese Hamster

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

    DOEpatents

    An, Yuehuei H. (Charleston, SC); Mironov, Vladimir A. (Mt. Pleasant, SC); Gutowska, Anna (Richland, WA)

    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.

  19. Analysis of ciliogenesis in primary culture mouse tracheal epithelial cells.

    PubMed

    Vladar, Eszter K; Brody, Steven L

    2013-01-01

    Cell biological and molecular characterization of structural and functional ciliary components and regulators of mammalian motile ciliogenesis is made possible by the development of a robust and biologically faithful mouse tracheal epithelial cell (MTEC) culture system and complementary research techniques. Here, we describe the air-liquid interface culture of mouse airway epithelial progenitor cells that undergo motile ciliogenesis de novo. Multiciliated cells differentiate rapidly, and distinct stages of the ciliogenesis pathway can be identified and characterized with centriolar and ciliary immunofluorescence markers. Immunolabeled structures correlate with morphological features previously identified by electron microscopy, facilitating light microscopy analysis. MTEC cultures can be successfully transduced by lentiviral RNAi or epitope-tagged cDNA constructs to perturb gene expression. Also, motile ciliogenesis can be manipulated by drug treatment. Distinct cell populations can be isolated by cell sorting to facilitate comparison among the multiciliated and other cell types in the in vitro differentiated epithelium. The MTEC system uniquely offers the study of ciliogenesis in cells from genetically modified mouse strains. PMID:23522475

  20. Silicon substrate as a novel cell culture device for myoblast cells

    PubMed Central

    2014-01-01

    Background Tissue and organ regeneration via transplantation of cell bodies in-situ has become an interesting strategy in regenerative medicine. Developments of cell carriers to systematically deliver cell bodies in the damage site have fall shorten on effectively meet this purpose due to inappropriate release control. Thus, there is still need of novel substrate to achieve targeted cell delivery with appropriate vehicles. In the present study, silicon based photovoltaic (PV) devices are used as a cell culturing substrate for the expansion of myoblast mouse cell (C2C12 cells) that offers an atmosphere for regular cell growth in vitro. The adherence, viability and proliferation of the cells on the silicon surface were examined by direct cell counting and fluorescence microscopy. Results It was found that on the silicon surface, cells proliferated over 7 days showing normal morphology, and expressed their biological activities. Cell culture on silicon substrate reveals their attachment and proliferation over the surface of the PV device. After first day of culture, cell viability was 88% and cell survival remained above 86% as compared to the seeding day after the seventh day. Furthermore, the DAPI staining revealed that the initially scattered cells were able to eventually build a cellular monolayer on top of the silicon substrate. Conclusions This study explored the biological applications of silicon based PV devices, demonstrating its biocompatibility properties and found useful for culture of cells on porous 2-D surface. The incorporation of silicon substrate has been efficaciously revealed as a potential cell carrier or vehicle in cell growth technology, allowing for their use in cell based gene therapy, tissue engineering, and therapeutic angiogenesis. PMID:24885347

  1. Transport of anti-allergic drugs across the passage cultured human nasal epithelial cell monolayer

    Microsoft Academic Search

    Hongxia Lin; Jin-Wook Yoo; Hwan-Jung Roh; Min-Ki Lee; Suk-Jae Chung; Chang-Koo Shim; Dae-Duk Kim

    2005-01-01

    The purpose of this study was to investigate the nasal absorption characteristics of a series of anti-allergic drugs across the human nasal epithelial cell monolayer, which was passage cultured by the liquid-covered culture (LCC) method on Transwell®. Characterization of this cell culture model was achieved by bioelectric measurements and morphological studies. The passages 2–4 of cell monolayers exhibited the TEER

  2. Impact of static magnetic fields on human myoblast cell cultures.

    PubMed

    Stern-Straeter, Jens; Bonaterra, Gabriel Alejandro; Kassner, Stefan S; Faber, Anne; Sauter, Alexander; Schulz, Johannes D; Hörmann, Karl; Kinscherf, Ralf; Goessler, Ulrich Reinhart

    2011-12-01

    Treatment of skeletal muscle loss due to trauma or tumor ablation therapy still lacks a suitable clinical approach. Creation of functional muscle tissue in vitro using the differentiation potential of human satellite cells (myoblasts) is a promising new research field called tissue engineering. Strong differentiation stimuli, which can induce formation of myofibers after cell expansion, have to be identified and evaluated in order to create sufficient amounts of neo-tissue. The objective of this study was to determine the influence of static magnetic fields (SMF) on human satellite cell cultures as one of the preferred stem cell sources in skeletal muscle tissue engineering. Experiments were performed using human satellite cells with and without SMF stimulation after incubation with a culture medium containing low [differentiation medium (DM)] or high [growth medium (GM)] concentrations of growth factors. Proliferation analysis using the alamarBlue assay revealed no significant influence of SMF on cell division. Real-time RT-PCR of the following marker genes was investigated: myogenic factor 5 (MYF5), myogenic differentiation antigen 1 (MYOD1), myogenin (MYOG), skeletal muscle ?1 actin (ACTA1), and embryonic (MYH3), perinatal (MYH8) and adult (MYH1) skeletal muscle myosin heavy chain. We detected an influence on marker gene expression by SMF in terms of a down-regulation of the marker genes in cell cultures treated with SMF and DM, but not in cell cultures treated with SMF and GM. Immunocytochemical investigations using antibodies directed against the differentiation markers confirmed the gene expression results and showed an enhancement of maturation after stimulation with GM and SMF. Additional calculation of the fusion index also revealed an increase in myotube formation in cell cultures treated with SMF and GM. Our findings show that the effect of SMF on the process of differentiation depends on the growth factor concentration in the culture medium in human satellite cultures. SMF alone enhances the maturation of human satellite cells treated with GM, but not satellite cells that were additionally stimulated with serum cessation. Therefore, further investigations are necessary before consideration of SMF for skeletal muscle tissue engineering approaches. PMID:21837362

  3. Engineering systems for the generation of patterned co-cultures for controlling cell-cell interactions

    PubMed Central

    Kaji, Hirokazu; Camci-Unal, Gulden; Langer, Robert; Khademhosseini, Ali

    2010-01-01

    Background Inside the body, cells lie in direct contact or in close proximity to other cell types in a tightly controlled architecture that often regulates the resulting tissue function. Therefore, tissue engineering constructs that aim to reproduce the architecture and the geometry of tissues will benefit from methods of controlling cell–cell interactions with microscale resolution. Scope of the review We discuss the use of microfabrication technologies for generating patterned co-cultures. In addition, we categorize patterned co-culture systems by cell type and discuss the implications of regulating cell-cell interactions in the resulting biological function of the tissues. Major conclusions Patterned co-cultures are a useful tool for fabricating tissue engineered constructs and for studying cell–cell interactions in vitro, because they can be used to control the degree of homotypic and heterotypic cell–cell contact. In addition, this approach can be manipulated to elucidate important factors involved in cell-matrix interactions. General significance Patterned co-culture strategies hold significant potential to develop biomimetic structures for tissue engineering. It is expected that they would create opportunities to develop artificial tissues in the future. PMID:20655984

  4. Cell Culture-Derived Influenza Vaccines

    Microsoft Academic Search

    Philip R. Dormitzer

    \\u000a Conventional egg-based vaccine manufacture has provided decades of safe and effective influenza vaccines using the technologies\\u000a of the 1930–1960s. Concerns over the vulnerability of the egg supply in the case of a pandemic with a high pathogenicity avian\\u000a influenza strain have spurred the development and licensure of mammalian cell culture-based influenza vaccines, the first\\u000a major technological innovation in influenza vaccine

  5. In vitro co-culture systems for studying molecular basis of cellular interaction between Aire-expressing medullary thymic epithelial cells and fresh thymocytes.

    PubMed

    Yamaguchi, Yoshitaka; Kudoh, Jun; Yoshida, Tetsuhiko; Shimizu, Nobuyoshi

    2014-01-01

    We previously established three mouse cell lines (Aire(+)TEC1, Aire(+)TEC2 and Aire(+)DC) from the medullary thymic epithelial cells (mTECs) and dendritic cells (mDCs). These cells constitutively expressed "autoimmune regulator (Aire) gene" and they exhibited various features of self antigen-presenting cells (self-APCs) present in the thymic medullary region. Here, we confirmed our previous observation that Aire(+) thymic epithelial cells adhere to fresh thymocytes and kill them by inducing apoptosis, thus potentially reproducing in vitro some aspects of the negative selection of T cells in vivo. In this system, a single Aire(+) cell appeared able to kill ?30 thymocytes within 24?hrs. Moreover, we observed that ectopic expression of peripheral tissue-specific antigens (TSAs), and expression of several surface markers involved in mTEC development, increased as Aire(+) cell density increases toward confluency. Thus, these Aire(+) cells appear to behave like differentiating mTECs as if they pass through the developmental stages from intermediate state toward mature state. Surprisingly, an in vitro co-culture system consisting of Aire(+) cells and fractionated sub-populations of fresh thymocytes implied the possible existence of two distinct subtypes of thymocytes (named as CD4(+) killer and CD4(-) rescuer) that may determine the fate (dead or alive) of the differentiating Aire(+)mTECs. Thus, our in vitro co-culture system appears to mimic a part of "in vivo thymic crosstalk". PMID:25326516

  6. Isolation and cell culture propagation of rotaviruses from turkeys and chickens

    Microsoft Academic Search

    M. S. McNulty; G. M. Allan; D. Todd; J. B. McFerran

    1979-01-01

    Summary Rotaviruses were detected by electron microscopy in the faeces of turkey poults and broiler chickens with diarrhoea. Apparently symptomless infection was also observed in broilers. The avian rotaviruses could not be isolated in cell cultures by conventional techniques, but were adapted to serial growth in chick cell cultures following trypsin treatment of the virus and the cells. Immunofluorescence studies

  7. Chondrogenic differentiation of human adipose-derived stem cells in polyglycolic acid mesh scaffolds under dynamic culture conditions

    Microsoft Academic Search

    Nastaran Mahmoudifar; Pauline M. Doran

    2010-01-01

    Chondrogenic differentiation of human adult adipose-derived stem cells was studied in vitro for the development of engineered cartilage tissue. Cells cultured under dynamic conditions in polyglycolic acid (PGA) scaffolds produced substantially higher glycosaminoglycan (GAG) and total collagen levels than cells in pellet cultures. This result reflects the importance of cell attachment and cell–scaffold interactions in stem cell differentiation and chondrogenesis.

  8. Preparation of single cells from aggregated Taxus suspension cultures for population analysis.

    PubMed

    Naill, Michael C; Roberts, Susan C

    2004-06-30

    A method for the isolation of single plant cells from Taxus suspension cultures has been developed for the analysis of single cells via rapid throughput techniques such as flow cytometry. Several cell wall specific enzymes, such as pectinase, pectolyase Y-23, macerozyme, Driselase(R), and cellulase were tested for efficacy in producing single cell suspensions. The method was optimized for single cell yield, viability, time, and representivity of aggregated cell cultures. The best combination for single cell isolation was found to be 0.5% (w/v) pectolyase Y-23 and 0.04% (w/v) cellulase. High viability (>95%) and high yields of single cell aggregates (>90%) were obtained following 4 hours of digestion for four separate Taxus cell lines. In addition, methyl jasmonate elicitation (200 microM) was found to have no effect on three of the four tested Taxus lines. Isolated single cells were statistically similar to untreated cell cultures for peroxidase activity (model cell wall protein) and paclitaxel content (secondary metabolite produced in Taxus cell cultures). In comparison, protoplasts showed marked changes in both peroxidase activity and paclitaxel content as compared to untreated cultures. The use of flow cytometry was demonstrated with isolated cells that were found to have > 99% viability upon staining with fluorescein diacetate. The development of a method for the isolation of single plant cells will allow the study of population dynamics and culture variability on a single cell level for the development of population models of plant cell cultures and secondary metabolism. PMID:15162458

  9. Increased expression of drug-metabolizing enzymes in human hepatocarcinoma FLC-4 cells cultured on micro-space cell culture plates.

    PubMed

    Kobayashi, Kaoru; Yoshida, Akane; Ejiri, Yoko; Takagi, Sachiko; Mimura, Hanaka; Hosoda, Masaya; Matsuura, Tomokazu; Chiba, Kan

    2012-01-01

    Human hepatocellular carcinoma cell lines cultured in a monolayer show negligible activities of drug-metabolizing enzymes such as cytochrome P450s (CYPs) and UDP-glucuronosyltransferases (UGTs). Here, we show that culture of human hepatocellular carcinoma FLC-4 cells on 24-well plates arrayed with uniform micro-sized compartments on the bottom of the plates (micro-space cell culture plates) resulted in increased expression of drug-metabolizing enzymes (CYP1A2, CYP2C9, CYP3A4, UGT1A1, etc.) and nuclear receptors (pregnane X receptor, constitutive androstane receptor, etc.). When cells were treated with a typical CYP3A substrate (triazolam), CYP2C9 substrate (diclofenac) or UGT1A1 substrate (SN-38), large amounts of their metabolites were detected in the medium of cells cultured on micro-space cell culture plates. The formation of metabolites from triazolam, diclofenac and SN-38 was strongly inhibited by co-treatment with a CYP3A inhibitor (ketoconazole), CYP2C9 inhibitor (sulfaphenazole) and UGT1A1 inhibitor (ketoconazole), respectively. On the other hand, formation of metabolites was not observed in the medium of cells cultured in a monolayer. Finally, the cytotoxic effect of aflatoxin B1 was more potent in cells cultured on micro-space cell culture plates than in cells cultured in a monolayer. The results suggest that FLC-4 cells cultured on micro-space cell culture plates are useful for studying drug metabolism and drug-induced hepatotoxicity. PMID:22447115

  10. Cytotoxicity studies of CdSeS/ZnS quantum dots on cell culture in microfluidic system

    NASA Astrophysics Data System (ADS)

    Haczyk, Maja; Grabowska-Jadach, Ilona; Drozd, Marcin; Pietrzak, Mariusz; Malinowska, El?bieta; Brzózka, Zbigniew

    2014-08-01

    Quantum dots (QDs) semi-conducting nanocrystals have found numerous applications in many fields of science. Nowadays one can observe a growing perspective to use them in biomedicine. Thanks to QDs unique fluorescence properties (narrow emission spectra, high extinction coefficients, high quantum yields, photostability) and possibility to form conjugates with bioactive molecules, they can become a chance for better cancer cells imaging in cancer therapy. Therefore there is a need for better understanding of biological interactions between QDs and cancer cells in vitro. For this purpose we performed cytotoxicity tests of CdSeS/ZnS quantum dots stabilized with mercaptopropionic acid (MPA) ligand, on human lung cancer cell line (A549) in vitro in macro- (96-well plate) and micro-scale (a specially designed and fabricated microfluidic device). The results obtained demonstrated a little extent of cytotoxic effect of selected solutions of QDs to A549 cells.

  11. Microfabricated polyester conical microwells for cell culture applications†

    PubMed Central

    Selimovi?, Šeila; Piraino, Francesco; Bae, Hojae; Rasponi, Marco; Redaelli, Alberto

    2012-01-01

    Over the past few years there has been a great deal of interest in reducing experimental systems to a lab-on-a-chip scale. There has been particular interest in conducting high-throughput screening studies using microscale devices, for example in stem cell research. Microwells have emerged as the structure of choice for such tests. Most manufacturing approaches for microwell fabrication are based on photolithography, soft lithography, and etching. However, some of these approaches require extensive equipment, lengthy fabrication process, and modifications to the existing microwell patterns are costly. Here we show a convenient, fast, and low-cost method for fabricating microwells for cell culture applications by laser ablation of a polyester film coated with silicone glue. Microwell diameter was controlled by adjusting the laser power and speed, and the well depth by stacking several layers of film. By using this setup, a device containing hundreds of microwells can be fabricated in a few minutes to analyze cell behavior. Murine embryonic stem cells and human hepatoblastoma cells were seeded in polyester microwells of different sizes and showed that after 9 days in culture cell aggregates were formed without a noticeable deleterious effect of the polyester film and glue. These results show that the polyester microwell platform may be useful for cell culture applications. The ease of fabrication adds to the appeal of this device as minimal technological skill and equipment is required. PMID:21614380

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

    SciTech Connect

    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.

  13. Viral antigen production in cell cultures on microcarriers Bovine parainfluenza 3 virus and MDBK cells.

    PubMed

    Conceição, M M; Tonso, A; Freitas, C B; Pereira, C A

    2007-11-01

    Viral antigens can be obtained from infected mammalian cells cultivated on microcarriers. We have worked out parameters for the production of bovine parainfluenza 3 (PI-3) virus by Mandin-Darby Bovine Kidney (MDBK) cells cultivated on Cytodex 1 microcarriers (MCs) in spinners flasks and bioreactor using fetal bovine serum (FBS) supplemented Eagle minimal essential medium (Eagle-MEM). Medium renewal during the cell culture was shown to be crucial for optimal MCs loading (>90% MCs with confluent cell monolayers) and cell growth (2.5 x 10(6)cells/mL and a micro(x) (h(-1)) 0.05). Since cell cultures performed with lower amount of MCs (1g/L), showed good performances in terms of cell loading, we designed batch experiments with a lower concentration of MCs in view of optimizing the cell growth and virus production. Studies of cell growth with lower concentrations of MCs (0.85 g/L) showed that an increase in the initial cell seeding (from 7 to 40 cells/MC) led to a different kinetic of initial cell growth but to comparable final cell concentrations ((8-10)x10(5)cells/mL at 120 h) and cell loading (210-270 cells/MC). Upon infection with PI-3 virus, cultures showed a decrease in cell growth and MC loading directly related to the multiplicity of infection (moi) used for virus infection. Infected cultures showed also a higher consumption of glucose and production of lactate. The PI-3 virus and PI-3 antigen production among the cultures was not significantly different and attained values ranging from, respectively, 7-9 log(10) TCID(50)/mL and 1.5-2.2 OD. The kinetics of PI-3 virus production showed a sharp increase during the first 24h and those of PI-3 antigen increased after 24h. The differential kinetics of PI-3 virus and PI-3 antigen can be explained by the virus sensitivity to temperature. In view of establishing a protocol of virus production and based on the previous experiments, MDBK cell cultures performed under medium perfusion in a bioreactor of 1.2L were infected and the PI-3 virus production in 12L attained 12 log(10) TCID(50). Other than establishing a protocol for PI-3 production in MDBK cell cultures on Cytodex 1, the experiments are proposed as a basis for approaching the development of a virus production protocol in mammalian cells cultivated on microcarriers in bioreactors. PMID:17920165

  14. Alginate as a cell culture substrate for growth and differentiation of human retinal pigment epithelial cells.

    PubMed

    Heidari, Razeih; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Davari, Maliheh; Nazemroaya, Fatemeh; Bagheri, Abouzar; Deezagi, Abdolkhalegh

    2015-03-01

    The purpose of this study was to evaluate retinal pigment epithelium (RPE) cells' behavior in alginate beads that establish 3D environment for cellular growth and mimic extracellular matrix versus the conventional 2D monolayer culture. RPE cells were encapsulated in alginate beads by dripping alginate cell suspension into CaCl2 solution. Beads were suspended in three different media including Dulbecco's modified Eagle's medium (DMEM)/F12 alone, DMEM/F12 supplemented with 10 % fetal bovine serum (FBS), and DMEM/F12 supplemented with 30 % human amniotic fluid (HAF). RPE cells were cultivated on polystyrene under the same conditions as controls. Cell phenotype, cell proliferation, cell death, and MTT assay, immunocytochemistry, and real-time RT-PCR were performed to evaluate the effect of alginate on RPE cells characteristics and integrity. RPE cells can survive and proliferate in alginate matrixes. Immunocytochemistry analysis exhibited Nestin, RPE65, and cytokeratin expressions in a reasonable number of cultured cells in alginate beads. Real-time PCR data demonstrated high levels of Nestin, CHX10, RPE65, and tyrosinase gene expressions in RPE cells immobilized in alginate when compared to 2D monolayer culture systems. The results suggest that alginate can be used as a reliable scaffold for maintenance of RPE cells' integrity and in vitro propagation of human retinal progenitor cells for cell replacement therapies in retinal diseases. PMID:25502925

  15. Myrosinase-treated glucoerucin is a potent inducer of the Nrf2 target gene heme oxygenase 1 - studies in cultured HT-29 cells and mice.

    PubMed

    Wagner, Anika E; Sturm, Christine; Piegholdt, Stefanie; Wolf, Insa M A; Esatbeyoglu, Tuba; De Nicola, Gina Rosalinda; Iori, Renato; Rimbach, Gerald

    2015-06-01

    In this study, the effect of myrosinase-treated glucoerucin (GER+MYR), which releases the isothiocyanate (ITC) erucin, on heme oxygenase 1 (HO-1) gene expression and Nrf2 signaling was investigated in vitro in cultured cells and in vivo in mice. Treatment of HT-29 cells with GER+MYR resulted in a significant increase in the mRNA and protein levels of nuclear Nrf2 and HO-1. GER+MYR was more potent at enhancing the nuclear Nrf2 levels than were the following myrosinase-treated glucosinolates: sinigrin, glucoraphanin and gluconasturtiin, which are the precursors of allyl-ITC, R-sulforaphane and 2-phenylethyl ITC, respectively. GER+MYR also significantly induced HO-1 gene expression in the mouse intestinal mucosae and liver but not in the brain. Mechanistic studies suggest that GER+MYR induces Nrf2 via ERK1/2-, p38- and JNK-dependent signal transduction pathways. The GER+MYR-mediated increase in HO-1 expression is primarily attributable to p38 signaling. PMID:25776458

  16. Culturing conditions affecting the production of anthocyanin in suspended cell cultures of strawberry

    Microsoft Academic Search

    Kenji Sato; Mamoru Nakayama; Jun-ichi Shigeta

    1996-01-01

    The increase of anthocyanin content in suspended cell cultures of strawberry varied with the increase in the amount of pigmentation in pigmented cells and in number of pigmented cells in a culture. The anthocyanin yield was enhanced by increasing light irradiation, and this may have resulted from increased accumulation of anthocyanin in pigmented cells. The increased anthocyanin yield for the

  17. Culture and Development: A Study.

    ERIC Educational Resources Information Center

    Claxton, Mervyn

    As the nations of the world pursue development, the role of culture is important to consider in development activities. This report examines the link between culture and development and suggests that a people's cultural traditions and practices can be utilized in successful development activities. The investigation functions on the premise that…

  18. Cultural Studies in the English Classroom.

    ERIC Educational Resources Information Center

    Berlin, James A., Ed.; Vivion, Michael J., Ed.

    This book opens up ways of teaching and devising programs which place the students' cultural experiences at the center of language production and consumption. It provides concrete models of cultural studies programs and classrooms for high school and college teachers who would like to try the "cultural studies approach." It also offers a…

  19. Differentiated cultures of primary hamster tracheal airway epithelial cells

    Microsoft Academic Search

    Regina K. Rowe; Steven L. Brody; Andrew Pekosz

    2004-01-01

    Summary  Primary airway epithelial cell cultures can provide a faithful representation of the in vivo airway while allowing for a controlled\\u000a nutrient source and isolation from other tissues or immune cells. The methods used have significant differences based on tissue\\u000a source, cell isolation, culture conditions, and assessment of culture purity. We modified and optimized a method for generating\\u000a tracheal epithelial cultures

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

    PubMed

    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. © 2015 International Society for Advancement of Cytometry. PMID:25929697

  1. Heat-transfer-method-based cell culture quality assay through cell detection by surface imprinted polymers.

    PubMed

    Eersels, Kasper; van Grinsven, Bart; Khorshid, Mehran; Somers, Veerle; Püttmann, Christiane; Stein, Christoph; Barth, Stefan; Diliën, Hanne; Bos, Gerard M J; Germeraad, Wilfred T V; Cleij, Thomas J; Thoelen, Ronald; De Ceuninck, Ward; Wagner, Patrick

    2015-02-17

    Previous work has indicated that surface imprinted polymers (SIPs) allow for highly specific cell detection through macromolecular cell imprints. The combination of SIPs with a heat-transfer-based read-out technique has led to the development of a selective, label-free, low-cost, and user-friendly cell detection assay. In this study, the breast cancer cell line ZR-75-1 is used to assess the potential of the platform for monitoring the quality of a cell culture in time. For this purpose, we show that the proposed methodology is able to discriminate between the original cell line (adherent growth, ZR-75-1a) and a descendant cell line (suspension growth, ZR-75-1s). Moreover, ZR-75-1a cells were cultured for a prolonged period of time and analyzed using the heat-transfer method (HTM) at regular time intervals. The results of these experiments demonstrate that the thermal resistance (Rth) signal decays after a certain number of cell culture passages. This can likely be attributed to a compromised quality of the cell culture due to cross-contamination with the ZR-75-1s cell line, a finding that was confirmed by classical STR DNA profiling. The cells do not express the same functional groups on their membrane, resulting in a weaker bond between cell and imprint, enabling cell removal by mechanical friction, provided by flushing the measuring chamber with buffer solution. These findings were further confirmed by HTM and illustrate that the biomimetic sensor platform can be used as an assay for monitoring the quality of cell cultures in time. PMID:25654744

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

    SciTech Connect

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

    2012-10-12

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

  3. Two- and three-dimensional culture of keratinocyte stem and precursor cells derived from primary murine epidermal cultures.

    PubMed

    Vollmers, Anne; Wallace, Lee; Fullard, Nicola; Höher, Thorsten; Alexander, Matthew D; Reichelt, Julia

    2012-06-01

    In the skin, multipotent keratinocyte stem cells (KSC) are localised in the hair follicle bulge region. Although, KSC can be cultivated and grown in two-dimensional (2D) culture they rapidly lose stem cell markers when isolated from their niche. Currently, there is no KSC culture method available which recapitulates an environment similar to the KSC niche in the hair follicle. Here we describe the successful establishment of an in vitro 3D stem cell culture model developed from clonally growing keratinocyte lines derived from neonatal mice using culture conditions previously established for human keratinocytes. After 20 passages, keratinocyte lines showed a stable ratio of holoclones (stem cells), meroclones (stem and precursor cells) and paraclones (differentiating cells), with approximately 29% holoclones, 54% meroclones and 17% paraclones, and were thus termed keratinocyte stem and precursor cell (KSPC) cultures. In high calcium medium, KSPC cultures grown at the air-liquid interphase differentiated and formed epidermal equivalents. Notably, and in contrast to primary keratinocytes, keratinocytes from KSPC cultures were able to aggregate and form spherical clusters in hanging drops, a characteristic hallmark shared with other stem cell types. Similar to the in vivo situation in the hair follicle bulge, KSPC aggregates also showed low proliferation, down-regulation of keratin 6, absence of keratin 1, and expression of the KSC markers keratin 15, Sox9, NFATc1 and Zfp145. KSPC aggregates therefore provide an optimal in vitro 3D environment for the further characterisation and study of normal and genetically modified KSPC. PMID:21892602

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

    PubMed

    Hunter, Wayne B

    2010-02-01

    A media for the production of cell cultures from hymenopteran species such as honey bee, Apis mellifera L. (Hymenoptera: Apidae) was developed. Multiple bee cell cultures were produced when using bee larvae and pupae as starting material and modified Hert-Hunter 70 media. Cell culture systems for bees solves an impasse that has hindered efforts to isolate and screen pathogens which may be influencing or causing colony collapse disorder of bees. Multiple life stages of maturing larvae to early pupae were used to successfully establish cell cultures from the tissues of the head, thorax, and abdomen. Multiple cell types were observed which included free-floating suspensions, fibroblast-like, and epithelia-like monolayers. The final culture medium, WH2, was originally developed for hemipterans, Asian citrus psyllid, Diaphorina citri, and leafhopper, Homalodisca vitripennis cell cultures but has been shown to work for a diverse range of insect species such as bees. Bee cell cultures had various doubling times at 21-23 degrees C ranging from 9-15 d. Deformed wing virus was detected in the primary explanted tissues, which tested negative by rt-PCR for Israeli acute paralysis virus (IAPV), Kashmir bee virus, acute bee paralysis virus, and black queen cell virus. Culture inoculation with IAPV from an isolate from Florida field samples, was detectable in cell cultures after two subcultures. Cell culture from hymenoptera species, such as bees, greatly advances the approaches available to the field of study on colony collapse disorders. PMID:20033792

  5. Propylene Glycol-Mediated Cell Injury in a Primary Culture of Human Proximal Tubule Cells

    Microsoft Academic Search

    Khandoker M. Morshed; Sushil K. Jain; Kenneth E. McMartin

    1998-01-01

    Propylene glycol (propane-1,2-diol; PD) is a widely used solvent for intravenous drugs. Clinical studies have reported serious side effects, including the development of renal insufficiency in patients receiving PD as drug vehicle. Despite such clinical reports, the data on the toxicity of PD in isolated renal cells are limited. Using primary cultured human proximal tubule (HPT) cells as anin vitromodel,

  6. Cannabinoids induce incomplete maturation of cultured human leukemia cells

    SciTech Connect

    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.

  7. Arraying cell cultures using PEG-DMA micromolding in standard culture dishes.

    PubMed

    Marel, Anna-Kristina; Rappl, Susanne; Piera Alberola, Alicia; Rädler, Joachim Oskar

    2013-05-01

    A robust and effortless procedure is presented, which allows for the microstructuring of standard cell culture dishes. Cell adhesion and proliferation are controlled by three-dimensional poly(ethylene glycol)-dimethacrylate (PEG-DMA) microstructures. The spacing between microwells can be extended to millimeter size in order to enable the combination with robotic workstations. Cell arrays of microcolonies can be studied under boundary-free growth conditions by lift-off of the PEG-DMA layer in which the growth rate is accessible via the evolution of patch areas. Alternatively, PEG-DMA stencils can be used as templates for plasma-induced patterning. PMID:23460347

  8. Propagation and Immortalization of Human Lens Epithelial Cells in Culture

    Microsoft Academic Search

    Usha P. Andley; Johng S. Rhim; Leo T. Chylack; Timothy P. Fleming

    Purpose. To establish primary and immortalized cell cultures of human lens epithelial cells for a model system investigating human lens epithelial physiology and cataract. Methods. Human lens epithelial cells in culture were grown by isolating epithelium fragments from infant human lenses from patients who underwent treatment for retinopathy of prema- turity and by allowing epithelial cells to grow from explants.

  9. Altered sensitivity to colchicine and PHA in human cultured cells

    Microsoft Academic Search

    Y. Chamla; Monique Roumy; Marguerite Lassègues; J. Battin

    1980-01-01

    PHA-stimulated lymphocytes cultivated from a pair of human monozygotic twins yielded mostly tetraploid cells when colchicine was not used to arrest the metaphases. The rate of tetraploidy was also enhanced by colchicine in fibroblasts cultured without PHA. In in situ condition, larger than usual cells were observed. Other defects found in parental lymphocyte cultures included C-anaphase cells and increased cell

  10. Maintenance of primary cell cultures of immunocytes from Cacopsylla sp. psyllids: a new in vitrio tool for the study of pest insects

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Psyllid species are major vectors of plant pathogens, such as phytoplasmas and Liberibacter bacteria, which threaten economic stability of fruit tee crops and vegetable production worldwide. Primary cell cultures of immunocytes have been developed from the three psyllid species, Cacopsylla melanone...

  11. Establishment condition and characterization of heart-derived cell culture in Siberian sturgeon (Acipenser baerii).

    PubMed

    Kim, Min Sung; Nam, Yoon Kwon; Park, Chulhong; Kim, Hyun-Woo; Ahn, Jiyeon; Lim, Jeong Mook; Gong, Seung Pyo

    2014-12-01

    This study was conducted to establish the efficient condition for stable derivation of heart-derived cell culture in Siberian sturgeon (Acipenser baerii). Three factors including isolation methods, cell densities in initial seeding, and basal media were evaluated for the derivation of heart-derived cell culture. As the results, enzymatic isolation was more efficient than mechanical isolation in both cell retrieval and further culture. Total 48 trials of culture employing low and middle cell densities of less than 5.5?×?10(4) cells/cm(2) in initial seeding did not induce cell survivals (0%, 0/48), but the trials in high cell density of more than 5.5?×?10(5) cells/cm(2) could induce cell survival and primary cell attachment on the plate (88.9%, 24 in 27 trials). When all initially attached cell populations were continuously cultured in two different media, only five cell populations that were enzymatically isolated and cultured under Leibovitz's L-15 medium could grow up to more than 40th subculture. Each cell population was stably cultured according to its own growth rate and all showed normal diploid DNA contents. Two morphologically different cell types that has an elongated shape or a round shape were identified in culture, which was subsequently identified that two cell types are considered as a fibroblast (an elongated shape) and a vascular endothelial cell (a round shape) on the basis of the results of gene and protein expression analysis. Additionally, the sufficient number of viable cells could be successfully retrieved after freezing and thawing from all five cell populations suggesting the feasibility of long-term cryopreservation of the cells. The data and cells obtained from this study will contribute to development of in vitro model for basic biological studies using sturgeon species. PMID:25052194

  12. Expression, secretion, and inhibition of angiotensin-converting enzyme in cultured human bronchial epithelial cells

    Microsoft Academic Search

    Misato Takimoto; Hironobu Mitani; Seiji Hori; Masaaki Kimura; Tsutomu Bandoh; Toshikazu Okada

    1999-01-01

    The purpose of this study was to determine whether angiotensin-converting enzyme is present in cultured human bronchial epithelial cells and which types of epithelial cells possess this enzyme. It is well known that serum promotes squamous differentiation of airway epithelial cell culture in vitro. We found that whole-cell homogenates of both basal (serum-untreated) and squamous-differentiated bronchial epithelial cells degraded hippuryl-l-histidyl-l-leucine,

  13. Equipment for large-scale mammalian cell culture.

    PubMed

    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

  14. Immunoassay sensitivity and kinetic enhancement in cell culture media using electrokinetic preconcentration

    E-print Network

    Li, Leon Daliang

    2009-01-01

    The microfluidic cell culture enables the study of cell signaling in previously impossible or impractical ways by allowing the precise spatial and temporal control of the microenvironment to better mimic in vivo conditions. ...

  15. Microfilaments and tropomyosin of cultured mammalian cells: isolation and characterization

    PubMed Central

    Schloss, JA; Goldman, RD

    1980-01-01

    Microfilaments were isolated from cultured mammalian cells, utilizing procedures similar to those for isolation of "native" thin filaments from muscle. Isolated microfilaments from rat embryo, baby hamster kidney (BHK- 21), and Swiss mouse 3T3 cells appeared structurally similar to muscle thin filaments, exhibiting long, 6 nm Diam profiles with a beaded, helical substructure. An arrowhead pattern was observed after reaction of isolated microfilaments with rabbit skeletal muscle myosin subfragment 1. Under appropriate conditions, isolated microfilaments will aggregate into a form that resembles microfilament bundles seen in situ cultured cells. Isolated microfilaments represent a complex of proteins including actin. Some of these components have been tentatively identified, based on coelectrophoresis with purified proteins, as myosin, tropomyosin, and a high molecular weight actin-binding protein. The tropomyosin components of isolated microfilaments were unexpected; polypeptides comigrated on SDS-polyacrylamide gels with both muscle and nonmuscle types of tropomyosin. In order to identify more specifically these subunits, we isolated and partially characterized tropomyosin from three cell types. BHK-21 cell tropomyosin was similar to other nonmuscle tropomyosins, as judged by several criteria. However, tropomyosin isolated from rate embryo and 3T3 cells contained subunits that comigrated with both skeletal muscle and nonmuscle types of myosin, whereas the BHK cell protein consistently contained a minor muscle-like subunit. The array of tropomyosin subunits present in a cell culture was reflected in the polypeptide chain pattern seen on SDS-polyacrylamide gels of microfilaments isolated from that culture. These studies provide a starting point for correlating changes in the ultrastructural organization of microfilaments with alterations in their protein composition. PMID:6893987

  16. Ca2+ homeostasis in Brody's disease. A study in skeletal muscle and cultured muscle cells and the effects of dantrolene an verapamil.

    PubMed Central

    Benders, A A; Veerkamp, J H; Oosterhof, A; Jongen, P J; Bindels, R J; Smit, L M; Busch, H F; Wevers, R A

    1994-01-01

    Brody's disease, i.e., sarcoplasmic reticulum (SR) Ca(2+)-dependent Mg(2+)-ATPase (Ca(2+)-ATPase) deficiency, is a rare inherited disorder of skeletal muscle function. Pseudo-myotonia is the most important clinical feature. SR Ca(2+)-ATPase and Ca2+ homeostasis are examined in m. quadriceps and/or cultured muscle cells of controls and 10 patients suffering from Brody's disease. In both m. quadriceps and cultured muscle cells of patients, the SR Ca(2+)-ATPase activity is decreased by approximately 50%. However, the concentration of SR Ca(2+)-ATPase and SERCA1 are normal. SERCA1 accounts for 83 and 100% of total SR Ca(2+)-ATPase in m. quadriceps and cultured muscle cells, respectively. This implies a reduction of the molecular activity of SERCA1 in Brody's disease. The cytosolic Ca2+ concentration ([Ca2+]i) at rest and the increase of [Ca2+]i after addition of acetylcholine are the same in cultured muscle cells of controls and patients. The half-life of the maximal response, however, is raised three times in the pathological muscle cells. Addition of dantrolene or verapamil after the maximal response accelerates the restoration of the [Ca2+]i in these muscle cells. The differences in Ca2+ handling disappear by administration of dantrolene or verapamil concomitantly with acetylcholine. The reduced Ca2+ re-uptake from the cytosol presumably due to structural modification(s) of SERCA1 may explain the pseudo-myotonia in Brody's disease. Single cell measurements suggest a beneficial effect of dantrolene or verapamil in treating patients suffering from Brody's disease. Images PMID:8040329

  17. Disposable bioreactors for plant micropropagation and mass plant cell culture.

    PubMed

    Ducos, Jean-Paul; Terrier, Bénédicte; Courtois, Didier

    2009-01-01

    Different types of bioreactors are used at Nestlé R&D Centre - Tours for mass propagation of selected plant varieties by somatic embryogenesis and for large scale culture of plants cells to produce metabolites or recombinant proteins. Recent studies have been directed to cut down the production costs of these two processes by developing disposable cell culture systems. Vegetative propagation of elite plant varieties is achieved through somatic embryogenesis in liquid medium. A pilot scale process has recently been set up for the industrial propagation of Coffea canephora (Robusta coffee). The current production capacity is 3.0 million embryos per year. The pre-germination of the embryos was previously conducted by temporary immersion in liquid medium in 10-L glass bioreactors. An improved process has been developed using a 10-L disposable bioreactor consisting of a bag containing a rigid plastic box ('Box-in-Bag' bioreactor), insuring, amongst other advantages, a higher light transmittance to the biomass due to its horizontal design. For large scale cell culture, two novel flexible plastic-based disposable bioreactors have been developed from 10 to 100 L working volumes, validated with several plant species ('Wave and Undertow' and 'Slug Bubble' bioreactors). The advantages and the limits of these new types of bioreactor are discussed, based mainly on our own experience on coffee somatic embryogenesis and mass cell culture of soya and tobacco. PMID:19475375

  18. Peptide Hydrogelation and Cell Encapsulation for 3D Culture of MCF-7 Breast Cancer Cells

    PubMed Central

    Sun, Xiuzhi S.; Nguyen, Thu A.

    2013-01-01

    Three-dimensional (3D) cell culture plays an invaluable role in tumor biology by providing in vivo like microenviroment and responses to therapeutic agents. Among many established 3D scaffolds, hydrogels demonstrate a distinct property as matrics for 3D cell culture. Most of the existing pre-gel solutions are limited under physiological conditions such as undesirable pH or temperature. Here, we report a peptide hydrogel that shows superior physiological properties as an in vitro matrix for 3D cell culture. The 3D matrix can be accomplished by mixing a self-assembling peptide directly with a cell culture medium without any pH or temperature adjustment. Results of dynamic rheological studies showed that this hydrogel can be delivered multiple times via pipetting without permanently destroying the hydrogel architecture, indicating the deformability and remodeling ability of the hydrogel. Human epithelial cancer cells, MCF-7, are encapsulated homogeneously in the hydrogel matrix during hydrogelation. Compared with two-dimensional (2D) monolayer culture, cells residing in the hydrogel matrix grow as tumor-like clusters in 3D formation. Relevant parameters related to cell morphology, survival, proliferation, and apoptosis were analyzed using MCF-7 cells in 3D hydrogels. Interestingly, treatment of cisplatin, an anti-cancer drug, can cause a significant decrease of cell viability of MCF-7 clusters in hydrogels. The responses to cisplatin were dose- and time-dependent, indicating the potential usage of hydrogels for drug testing. Results of confocal microscopy and Western blotting showed that cells isolated from hydrogels are suitable for downstream proteomic analysis. The results provided evidence that this peptide hydrogel is a promising 3D cell culture material for drug testing. PMID:23527204

  19. Chloride and fluid secretion by cultured human polycystic kidney cells

    Microsoft Academic Search

    Darren P Wallace; Jared J Grantham; Lawrence P Sullivan

    1996-01-01

    Chloride and fluid secretion by cultured human polycystic kidney cells. Epithelial cells cultured from the renal cysts of patients with autosomal dominant polycystic kidney disease (ADPKD) secrete fluid via a process stimulated by adenosine 3?,5?-cyclic monophosphate (cAMP). We have investigated the hypothesis that fluid secretion by these cells is dependent on cAMP-mediated chloride secretion. Individual cultured ADPKD cells were suspended

  20. Cytopathogenic Effect of Trichomonas vaginalis on Human Vaginal Epithelial Cells Cultured In Vitro

    Microsoft Academic Search

    R. O. Gilbert; G. Elia; D. H. Beach; SUZANNE KLAESSIG; B. N. Singh

    2000-01-01

    In this study we established human vaginal epithelial cells (hVECs) in culture and evaluated their inter- action with Trichomonas vaginalis parasites to complement previous studies using other cell types. Primary cultures of hVECs were established. Contaminating fibroblasts were separated from epithelial cells by differ- ential trypsinization. Specific antibody staining revealed that over 92% of cells in hVEC monolayers were epithelial

  1. Metabolomic profiling of cultured cancer cells.

    PubMed

    Scoazec, Marie; Durand, Sylvere; Chery, Alexis; Galluzzi, Lorenzo; Kroemer, Guido

    2014-01-01

    Quantitative proteomics approaches have been developed-and now begin to be implemented on a high-throughput basis-to fill-in the large gap between the genomic/transcriptomic setup of (cancer) cells and their phenotypic/behavioral traits, reflecting a significant degree of posttranscriptional regulation in gene expression as well as a robust posttranslational regulation of protein function. However, proteomic profiling assays not only fail to detect labile posttranslational modifications as well as unstable protein-to-protein interactions but also are intrinsically incapable of assessing the enzymatic activity, as opposed to the mere abundance, of a given protein. Thus, determining the abundance of theoretically all the metabolites contained in a cell/tissue/organ/organism may significantly improve the informational value of proteomic approaches. Several techniques have been developed to this aim, including high-performance liquid chromatography (HPLC) coupled to quadrupole time-of-flight (Q-TOF) high-resolution mass spectrometry (HRMS). This approach is particularly advantageous for metabolomic profiling as it offers elevated accuracy and improved sensitivity. Here, we describe a simple procedure to determine the complete complement of intracellular metabolites in cultured malignant cells by HPLC coupled to Q-TOF HRMS. According to this method, (1) cells are collected and processed to minimize contaminations as well as fluctuations in their metabolic profile; (2) samples are separated by HPLC and analyzed on a Q-TOF spectrometer; and (3) data are extracted, normalized, and deconvoluted according to refined mathematical methods. This protocol constitutes a simple approach to determine the intracellular metabolomic profile of cultured cancer cells. With minimal variations (mostly related to sample collection and processing), this method is expected to provide reliable metabolomic data on a variety of cellular samples. PMID:24924132

  2. Hemicelluloses of cell walls of a proso millet cell suspension culture.

    PubMed

    Carpita, N C; Mulligan, J A; Heyser, J W

    1985-10-01

    Cell wall composition of a stable suspension of proso millet (Panicum miliaceum L. cv Abarr) cells is similar to those of tissues and cell suspensions of other graminaceous species. Extraction of hemicelluloses with step-wise increasing concentrations of alkali yields materials that, like those of embryonal cells of maize coleoptiles, comprise mostly glucuronoarabinoxylan, xyloglucan, and small amounts of (1-3),(1-4)-beta-d-glucan. As in the walls of embryonal cells of the maize coleoptile, 5-arabinosyl and 3-arabinosyl comprise much higher proportions of the total hemicellulosic sugars than in walls of developed or elongated cells. Unlike cells of many dicotyledonous species, millet cells do not elongate or undergo observable differentiation during the stationary phase of culture, and consequently, their wall composition is remarkably consistent throughout the culture cycle. The proso millet cell suspension culture constitutes a reasonable model for study of cell wall biogenesis in embryonal cells of a graminaceous species, but because of marked changes in the composition of hemicelluloses in these species during cell enlargement, additional model systems should be sought. PMID:16664435

  3. Fetal Calf Serum Protects Cultured Porcine Corneal Endothelial Cells from Endotoxin-Mediated Cell Damage

    Microsoft Academic Search

    Angela C. Sobottka Ventura; Katrin Engelmann; Matthias Böhnke

    1999-01-01

    In corneal organ culture, a contamination of sterile culture media with endotoxin is frequently found. Thus, we investigated if the presence of endotoxin affects the viability of cultured porcine corneal endothelial cells. Endotoxin in high concentrations caused morphological cell changes in porcine corneal endothelial monolayer cultures, delayed proliferation and decreased cellular esterase activity of porcine corneal endothelial cells in vitro.

  4. High-affinity binding of fibronectin to cultured Kupffer cells

    SciTech Connect

    Cardarelli, P.M.; Blumenstock, F.A.; McKeown-Longo, P.J.; Saba, T.M.; Mazurkiewicz, J.E.; Dias, J.A. (Albany Medical College of Union Univ., NY (USA))

    1990-11-01

    Hepatic Kupffer cells are a major component of the reticuloendothelial or macrophage system. They were the first phagocytic cell type whose phagocytosis was shown to be influenced by plasma fibronectin, a dimeric opsonic glycoprotein. In the current study, the binding of soluble radioiodinated fibronectin purified from rat serum to isolated rat hepatic Kupffer cells was investigated using a cultured Kupffer cell monolayer technique. Binding was specific, since unlabeled purified fibronectin competed in a dose-dependent manner with the 125I-fibronectin for binding to the Kupffer cells. Addition of gelatin enhanced the binding of 125I-fibronectin to Kupffer cells. The phagocytosis of gelatinized-coated red cells by Kupffer cells was increased either by preopsonizing the target particles with purified fibronectin or by the addition of purified fibronectin to the culture medium. In contrast, exposure of the Kupffer cells to medium containing purified fibronectin followed by wash-removal of the fibronectin did not increase the uptake of gelatin-coated red blood cells, even though fibronectin was detected on the surface of the Kupffer cells by immunofluorescence. Trypsinized monolayers expressed decreased capacity to bind 125I-fibronectin as well as fibronectin-coated sheep erythrocytes. The binding of 125I-fibronectin-gelatin complexes was inhibited by excess unlabeled fibronectin. We calculated that specific high-affinity (Kd = 7.46 x 10(-9) M) binding sites for fibronectin exist on Kupffer cells. There are approximately 2,800-3,500 binding sites or putative fibronectin receptors per Kupffer cell. These sites appear to mediate the enhanced phagocytosis of gelatin-coated particles opsonized by fibronectin.

  5. Dynamic 3D micropatterned cell co-cultures within photocurable and chemically degradable hydrogels

    PubMed Central

    Sugiura, Shinji; Cha, Jae Min; Yanagawa, Fumiki; Zorlutuna, Pinar; Bae, Hojae; Khademhosseini, Ali

    2014-01-01

    In this paper we report on the development of dynamically controlled 3D micropatterned cellular co-cultures within photocurable and chemically degradable hydrogels. Specifically, we generated dynamic co-cultures of micropatterned murine embryonic stem (mES) cells with human hepatocellular carcinoma (HepG2) cells within 3D hydrogels. HepG2 cells were used due to their ability to direct the differentiation of mES cells through secreted paracrine factors. To generate dynamic co-cultures, mES cells were first encapsulated within micropatterned photocurable poly(ethylene glycol) (PEG) hydrogels. These micropatterned cell-laden PEG hydrogels were subsequently surrounded by calcium alginate (Ca-Alg) hydrogels containing HepG2 cells. After 4 days, the co-culture step was halted by exposing the system to sodium citrate solution, which removed the alginate gels and the encapsulated HepG2 cells. The encapsulated mES cells were then maintained in the resulting cultures for 16 days and cardiac differentiation was analyzed. We observed that the mES cells that were exposed to HepG2 cells in the co-cultures, generated cells with higher expression of cardiac genes and proteins as well as increased spontaneous beating. Due to its ability to control the 3D microenvironment of cells in a spatially and temporally regulated manner the method presented in this study is useful for a range of cell culture applications related to tissue engineering and regenerative medicine. PMID:24170301

  6. Polyglycolic Acid–Polylactic Acid Scaffold Response to Different Progenitor Cell In Vitro Cultures: A Demonstrative and Comparative X-Ray Synchrotron Radiation Phase-Contrast Microtomography Study

    PubMed Central

    Moroncini, Francesca; Mazzoni, Serena; Belicchi, Marzia Laura Chiara; Villa, Chiara; Erratico, Silvia; Colombo, Elena; Calcaterra, Francesca; Brambilla, Lucia; Torrente, Yvan; Albertini, Gianni; Della Bella, Silvia

    2014-01-01

    Spatiotemporal interactions play important roles in tissue development and function, especially in stem cell-seeded bioscaffolds. Cells interact with the surface of bioscaffold polymers and influence material-driven control of cell differentiation. In vitro cultures of different human progenitor cells, that is, endothelial colony-forming cells (ECFCs) from a healthy control and a patient with Kaposi sarcoma (an angioproliferative disease) and human CD133+ muscle-derived stem cells (MSH 133+ cells), were seeded onto polyglycolic acid–polylactic acid scaffolds. Three-dimensional (3D) images were obtained by X-ray phase-contrast microtomography (micro-CT) and processed with the Modified Bronnikov Algorithm. The method enabled high spatial resolution detection of the 3D structural organization of cells on the bioscaffold and evaluation of the way and rate at which cells modified the construct at different time points from seeding. The different cell types displayed significant differences in the proliferation rate. In conclusion, X-ray synchrotron radiation phase-contrast micro-CT analysis proved to be a useful and sensitive tool to investigate the spatiotemporal pattern of progenitor cell organization on a bioscaffold. PMID:23879738

  7. Transplantation with cultured stem cells derived from the human amniotic membrane for corneal alkali burns: an experimental study.

    PubMed

    Zeng, Wei; Li, Yanwei; Zeng, Guangwei; Yang, Bo; Zhu, Yu

    2014-01-01

    Amniotic membranes (AM) have been used in a wide range of clinical applications. We successfully extracted mesenchymal stem cells (MSCs) from human AM, but little is known about the use and efficacy of human amniotic membrane-derived mesenchymal stem cells (hAM-dMSCs) for the treatment of alkali burns. We utilized hAM-dMSCs transplantation, AM grafting, and their combined use in the treatment of alkali burns. An experimental model in rabbits was devised to analyze the use of these techniques with immunocytochemistry and ELISA. The survival and migration of hAM-dMSCs labeled by SPION in the host were assessed with Prussian blue staining. Compared with the control group, the treated groups demonstrated faster reconstruction of the corneal epithelium, and lower levels of corneal opacification and neovascularization within corneal alkali burns. Furthermore, dark blue-stained particles were detected in the limbus corneae at day 28. These results demonstrated the ability of hAM-dMSCs to enhance epithelial healing and reduce corneal opacification and neovascularization in corneal alkali wounds. PMID:24695478

  8. The Effect of Spaceflight on Bone Cell Cultures

    NASA Technical Reports Server (NTRS)

    Landis, William J.

    1999-01-01

    Understanding the response of bone to mechanical loading (unloading) is extremely important in defining the means of adaptation of the body to a variety of environmental conditions such as during heightened physical activity or in extended explorations of space or the sea floor. The mechanisms of the adaptive response of bone are not well defined, but undoubtedly they involve changes occurring at the cellular level of bone structure. This proposal has intended to examine the hypothesis that the loading (unloading) response of bone is mediated by specific cells through modifications of their activity cytoskeletal elements, and/or elaboration of their extracellular matrices. For this purpose, this laboratory has utilized the results of a number of previous studies defining molecular biological, biochemical, morphological, and ultrastructural events of the reproducible mineralization of a primary bone cell (osteoblast) culture system under normal loading (1G gravity level). These data and the culture system then were examined following the use of the cultures in two NASA shuttle flights, STS-59 and STS-63. The cells collected from each of the flights were compared to respective synchronous ground (1G) control cells examined as the flight samples were simultaneously analyzed and to other control cells maintained at 1G until the time of shuttle launch, at which point they were terminated and studied (defined as basal cells). Each of the cell cultures was assayed in terms of metabolic markers- gene expression; synthesis and secretion of collagen and non-collagenous proteins, including certain cytoskeletal components; assembly of collagen into macrostructural arrays- formation of mineral; and interaction of collagen and mineral crystals during calcification of the cultures. The work has utilized a combination of biochemical techniques (radiolabeling, electrophoresis, fluorography, Western and Northern Blotting, and light microscopic immunofluorescence) and structural methods (conventional and high voltage electron microscopy, inununocytochemistry, stereomicroscopy, and 3D image reconstruction). The studies have provided new knowledge of aspects of bone cell development and structural regulation, extracellular matrix assembly, and mineralization during spaceflight and under normal gravity. The information has contributed to insights into the means in general by which cells respond and adapt to different conditions of gravity (loading). The data may as well have suggested an underlying basis for the observed loss of bone by vertebrates, including man, in microgravity; and these scientific results may have implications for understanding bone loss following fracture healing and extended periods of inactivity such as during long-term bedrest.

  9. Gravity, chromosomes, and organized development in aseptically cultured plant cells

    NASA Technical Reports Server (NTRS)

    Krikorian, Abraham D.

    1993-01-01

    The objectives of the PCR experiment are: to test the hypothesis that microgravity will in fact affect the pattern and developmental progression of embryogenically competent plant cells from one well-defined, critical stage to another; to determine the effects of microgravity in growth and differentiation of embryogenic carrot cells grown in cell culture; to determine whether microgravity or the space environment fosters an instability of the differentiated state; and to determine whether mitosis and chromosome behavior are adversely affected by microgravity. The methods employed will consist of the following: special embryogenically competent carrot cell cultures will be grown in cell culture chambers provided by NASDA; four cell culture chambers will be used to grow cells in liquid medium; two dishes (plant cell culture dishes) will be used to grow cells on a semi-solid agar support; progression to later embryonic stages will be induced in space via crew intervention and by media manipulation in the case of liquid grown cell cultures; progression to later stages in case of semi-solid cultures will not need crew intervention; embryo stages will be fixed at a specific interval (day 6) in flight only in the case of liquid-grown cultures; and some living cells and somatic embryos will be returned for continued post-flight development and 'grown-out.' These will derive from the semi-solid grown cultures.

  10. Cultured leptomeningeal cells secrete cerebrospinal fluid proteins.

    PubMed

    Ohe, Y; Ishikawa, K; Itoh, Z; Tatemoto, K

    1996-09-01

    To extrapolate the function of the leptomeninges, we examined the profile of the proteins secreted from the cultured leptomeningeal cells prepared from 1-2-day-old rats. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the medium conditioned with the cultured cells, 20-25 differentially distinctive protein bands were noted. Through several chromatographic procedures (Sephadex G-75, Mono Q, and 7C8-300), altogether 18 proteins were purified to homogeneity, and the partial amino acid sequence of each protein was determined. Homology search revealed that the major proteins included prostaglandin-D-synthase or beta-trace protein, insulin-like growth factor (IGF)-II, IGF-binding protein-2, apolipoprotein E, beta 2-microglobulin, cystatin C, transferrin, peptidyl-prolyl cis-trans isomerase or cyclophilin C, secreted protein acidic and rich in cysteine, ubiquitin, lysozyme C, extracellular superoxide dismutase, and collagen alpha-1 (III). Most of these proteins are known to be the major brain-derived protein constituents of CSF and are thought to play important roles in certain biological events in the brain. Considering the morphological features, the present findings suggest the importance of the leptomeninges as an origin of such proteins in CSF. PMID:8752101

  11. Insulin concentration is critical in culturing human neural stem cells and neurons

    PubMed Central

    Rhee, Y-H; Choi, M; Lee, H-S; Park, C-H; Kim, S-M; Yi, S-H; Oh, S-M; Cha, H-J; Chang, M-Y; Lee, S-H

    2013-01-01

    Cell culture of human-derived neural stem cells (NSCs) is a useful tool that contributes to our understanding of human brain development and allows for the development of therapies for intractable human brain disorders. Human NSC (hNSC) cultures, however, are not commonly used, mainly because of difficulty with consistently maintaining the cells in a healthy state. In this study, we show that hNSC cultures, unlike NSCs of rodent origins, are extremely sensitive to insulin, an indispensable culture supplement, and that the previously reported difficulty in culturing hNSCs is likely because of a lack of understanding of this relationship. Like other neural cell cultures, insulin is required for hNSC growth, as withdrawal of insulin supplementation results in massive cell death and delayed cell growth. However, severe apoptotic cell death was also detected in insulin concentrations optimized to rodent NSC cultures. Thus, healthy hNSC cultures were only produced in a narrow range of relatively low insulin concentrations. Insulin-mediated cell death manifested not only in all human NSCs tested, regardless of origin, but also in differentiated human neurons. The underlying cell death mechanism at high insulin concentrations was similar to insulin resistance, where cells became less responsive to insulin, resulting in a reduction in the activation of the PI3K/Akt pathway critical to cell survival signaling. PMID:23928705

  12. Callus formation from protoplasts of a maize cell culture

    Microsoft Academic Search

    P. S. Chourey; D. B. Zurawski

    1981-01-01

    A finely dispersed cell suspension culture from the friable callus of the ‘Black Mexican Sweet’ line of maize was obtained. Protoplasts from this cell culture, when grown in a simplified medium described here, showed sustained cell divisions and gave rise to callus.

  13. Cholera toxin stimulation of human mammary epithelial cells in culture

    SciTech Connect

    Stampfer, M.R.

    1982-06-01

    Addition of cholera toxin to human mammary epithelial cultures derived from reduction mammoplasties and primary carcinomas greatly stimulated cell growth and increased the number of times the cells could be successfully subcultured. Other agents known to increase intracellular cAMP levels were also growth stimulatory. The increased growth potential conferred by cholera toxin enhances the usefulness of this cell culture system.

  14. Internalization of swine vesicular disease virus into cultured cells: a comparative study with foot-and-mouth disease virus.

    PubMed

    Martín-Acebes, Miguel A; González-Magaldi, Mónica; Vázquez-Calvo, Angela; Armas-Portela, Rosario; Sobrino, Francisco

    2009-05-01

    We performed a comparative analysis of the internalization mechanisms used by three viruses causing important vesicular diseases in animals. Swine vesicular disease virus (SVDV) internalization was inhibited by treatments that affected clathrin-mediated endocytosis and required traffic through an endosomal compartment. SVDV particles were found in clathrin-coated pits by electron microscopy and colocalized with markers of early endosomes by confocal microscopy. SVDV infectivity was significantly inhibited by drugs that raised endosomal pH. When compared to foot-and-mouth disease virus (FMDV), which uses clathrin-mediated endocytosis, the early step of SVDV was dependent on the integrity of microtubules. SVDV-productive endocytosis was more sensitive to plasma membrane cholesterol extraction than that of FMDV, and differential cell signaling requirements for virus infection were also found. Vesicular stomatitis virus, a model virus internalized by clathrin-mediated endocytosis, was included as a control of drug treatments. These results suggest that different clathrin-mediated routes are responsible for the internalization of these viruses. PMID:19225001

  15. Internalization of Swine Vesicular Disease Virus into Cultured Cells: a Comparative Study with Foot-and-Mouth Disease Virus? †

    PubMed Central

    Martín-Acebes, Miguel A.; González-Magaldi, Mónica; Vázquez-Calvo, Angela; Armas-Portela, Rosario; Sobrino, Francisco

    2009-01-01

    We performed a comparative analysis of the internalization mechanisms used by three viruses causing important vesicular diseases in animals. Swine vesicular disease virus (SVDV) internalization was inhibited by treatments that affected clathrin-mediated endocytosis and required traffic through an endosomal compartment. SVDV particles were found in clathrin-coated pits by electron microscopy and colocalized with markers of early endosomes by confocal microscopy. SVDV infectivity was significantly inhibited by drugs that raised endosomal pH. When compared to foot-and-mouth disease virus (FMDV), which uses clathrin-mediated endocytosis, the early step of SVDV was dependent on the integrity of microtubules. SVDV-productive endocytosis was more sensitive to plasma membrane cholesterol extraction than that of FMDV, and differential cell signaling requirements for virus infection were also found. Vesicular stomatitis virus, a model virus internalized by clathrin-mediated endocytosis, was included as a control of drug treatments. These results suggest that different clathrin-mediated routes are responsible for the internalization of these viruses. PMID:19225001

  16. To grow mouse mammary epithelial cells in culture

    PubMed Central

    1984-01-01

    Normal mouse mammary epithelial cells from Balb/c mice were successfully cultivated on tissue culture plastic with lethally irradiated LA7 feeder cells. The feeder cells also promoted colony formation from single mouse mammary cells, and the fraction of cells that formed colonies was proportional to the density of feeder cells. The mouse mammary cells could be passaged at least 8-12 times as long as new feeder cells were added at each passage. The cells now in culture have doubled in number at least 30 times, but the in vitro lifespan is not yet known. The cultures of mouse cells maintained by this technique never became overgrown with fibroblasts and numerous domes formed in the cultures. PMID:6699079

  17. LATENT VIRAL INFECTION OF CELLS IN TISSUE CULTURE

    PubMed Central

    Bader, John P.; Morgan, Herbert R.

    1961-01-01

    A study of the metabolic requirements for the growth of psittacosis virus in L cells has been extended to the water-soluble vitamins. In a system in which a balanced salt solution was used to deplete the cells of their vitamin constituents, only thiamine was essential for psittacosis virus production. Extended depletion of cells with media deficient in specific vitamins demonstrated that pantothenate, niacin (niacinamide), pyridoxine (pyridoxal), and choline, in addition to thiamine, were essential for maximal growth of psittacosis virus. No requirement for biotin, inositol, folic acid, or riboflavin was demonstrated, although the possibility of incomplete vitamin depletion of the cells has not been eliminated. In most cases in which a specific vitamin requirement was shown the decreased yield of virus was correlated with a delay in the cytopathic effects produced in the cell cultures by psittacosis virus. PMID:13685754

  18. Dissolved oxygen and pH monitoring within cell culture media using a hydrogel microarray sensor

    E-print Network

    Lee, Seung Joon

    2009-05-15

    and control of cell culture processes is required. To do this measurement, multiple sensors must be implemented to monitor various parameters of the cell culture medium. The model analytes used in this study were pH and dissolved oxygen which have...

  19. Biotechnology Apprenticeship for Secondary-Level Students: Teaching Advanced Cell Culture Techniques for Research

    ERIC Educational Resources Information Center

    Lewis, Jennifer R.; Kotur, Mark S.; Butt, Omar; Kulcarni, Sumant; Riley, Alyssa A.; Ferrell, Nick; Sullivan, Kathryn D.; Ferrari, Mauro

    2002-01-01

    The purpose of this article is to discuss "small-group apprenticeships (SGAs)" as a method to instruct cell culture techniques to high school participants. The study aimed to teach cell culture practices and to introduce advanced imaging techniques to solve various biomedical engineering problems. Participants designed and completed experiments…

  20. Can Vero cell co-culture improve in-vitro maturation of bovine oocytes?

    Microsoft Academic Search

    Fariba Moulavi; Sayyed Mortaza Hosseini; Saeid Kazemi Ashtiani; Abdolhossein Shahverdi; Mohammad Hossein Nasr-Esfahani

    2006-01-01

    This study was carried out to evaluate the effect of Vero cell co-culture on developmental competence of immature oocytes. Bovine cumulus–oocyte complexes (COC) were matured in presence or absence of Vero cells. Matured oocytes were inseminated and cultured for up to 9 days. Cleavage percentages were recorded on day 2 after insemination and embryos were evaluated on a daily basis.

  1. Isotopomer Study of Lipogenesis in Human Hepatoma Cells in Culture: Contribution of Carbon and Hydrogen Atoms from Glucose

    Microsoft Academic Search

    W. N. P. Lee; L. O. Byerley; S. Bassilian; H. O. Ajie; I. Clark; J. Edmond; E. A. Bergner

    1995-01-01

    Recent developments in the application of stable isotopes and mass spectrometry have permitted the estimation of precursor enrichment and fractional synthesis of the product through mass isotopomer analysis. Thus, the application of isotopomer analysis in studies with 2H- and 13C-labeled glucose may potentially overcome the limitations of traditional methods which can only estimate the fractional use of carbon and hydrogen

  2. Primary targets in photochemical inactivation of cells in culture

    Microsoft Academic Search

    Kristian Berg; Stuart G. Jones; Kristian Prydz; Johan Moan

    1995-01-01

    The mechanisms of photoinactivation of NHIK 3025 cells in culture sensitized by tetrasulfonated phenylporphines (TPPS4) are described). Ultracentrifugation studies on postnuclear supernatants indicated that the intracellular distribution of TPPS4 resembles that of (beta) -N-acetyl-D-glucosaminidase ((beta) -AGA), a lysosomal marker enzyme, and that the cytosolic content of TPPS4 is below the detection limit of the ultracentrifugation method. Upon light exposure more

  3. Properties of Muscarinic Acetylcholine Receptors in Heart Cell Cultures

    Microsoft Academic Search

    Jonas B. Galper; Thomas W. Smith

    1978-01-01

    The binding of acetylcholine to receptors in the intact heart causes a decrease in the frequency (chronotropic effect) and force (ionotropic effect) of contraction. The studies reported here demonstrate a chronotropic response of cultured embryonic chicken heart cells to the muscarinic agonist carbamoylcholine. This response is markedly decreased after a 3-hr incubation with 0.1 mM carbamoylcholine. In order to determine

  4. Toxoplasma dye test using cell culture derived tachyzoites

    Microsoft Academic Search

    D Ashburn; R Evans; J M W Chatterton; A W L Joss; D O Ho-Yen

    2000-01-01

    Aims—To assess the diagnostic usefulness of Toxoplasma gondii tachyzoites produced by serial passage in HeLa cell culture.Methods—Tachyzoites derived from serial passage in cell culture were used in the dye test. Human sera were also examined to determine their suitability for use as accessory factor. Using the optimum conditions, the dye test using cell culture derived tachyzoites was compared with the

  5. Autocrine and paracrine actions of breast tumor aromatase. A three-dimensional cell culture study involving aromatase transfected MCF7 and T-47D cells

    Microsoft Academic Search

    Xiu-Zhu Sun; Dujin Zhou; Shiuan Chen

    1997-01-01

    Stable aromatase-expressing MCF-7 and T-47D cell lines (i.e. MCF-7aro and T-47Daro) have been prepared by aromatase cDNA transfection and G418 (neomycin) selection. MCF-7aro was further subjected to a clonal purification. Aromatase activity in the transfected MCF-7 and T-47D cell lines was determined to be 73 ± 6 pmol\\/mg\\/h and 48 ± 4 pmol\\/mg\\/h, respectively. It is thought that these cell

  6. Use of different cell lines for in vitro cultures of bovine respiratory syncytial virus.

    PubMed

    Urban-Chmiel, Renata; Wernicki, Andrzej; Majer-Dziedzic, Barbara; Gnat, Sebastian; Puchalski, Andrzej; Dec, Marta

    2014-08-01

    This study compared the use of different cell lines for in vitro cultures of bovine respiratory syncytial virus (BRSV). The BRSV 375 strain and 3 nasal swabs obtained from Simmental calves were used for this study. The culture was performed on 3 cell lines: bovine kidney cells (LLC-PK1), bovine tracheal cells (TBTR) and primary chicken embryo-related cells (CER). A comparative analysis of titres was performed using a microplate agglutination test with human group O erythrocytes and bovine erythrocytes. The presence of BRSV in all cell lines was confirmed using the reverse transcriptase polymerase chain reaction (RT-PCR) method. The first small refractile changes in the LLC-PK1 cells occurred at 48h after infection. Syncytial changes were noted 4 days after incubation. Large refractile cell changes were observed on day 3 of growth in the TBTR culture. Syncytia were observed on the second day after infection in subsequent passages. The cytopathic effect in the CER cells occurred 24h after infection, and syncytia appeared after 3 passages. Changes in syncytia indicate an adaptation of the virus for the infection of cells other than tracheal cells in primary and secondary cultures. The highest viral titre was obtained using the TBTR line. The titres obtained in the LLC-PK1 and CER cultures averaged 10(1.86)/ml. The low virus titres in all culture types suggest the need for research aimed at the optimisation of culture conditions. PMID:24747584

  7. Cell culture quality control by rapid isoenzymatic characterization.

    PubMed

    Halton, D M; Peterson, W D; Hukku, B

    1983-01-01

    Procedures that involve cell cultures require careful quality control to avoid inter- and intraspecies contamination. We have developed an electrophoresis technique that can be used routinely in cell culture laboratories to monitor cell line integrity. The method involves the isoenzymatic separation of nine polymorphic enzymes, three of which can be used for cell line species determinations and seven of which can be used for human cell line characterizations. Examples of how the system has been applied to both inter- and intraspecies identifications are described. The routine application of this protocol would be a valuable asset for laboratories concerned with establishing effective cell culture quality control. PMID:6401685

  8. Seed coat removal improves iron bioavailability in cooked lentils: studies using an in vitro digestion/Caco-2 cell culture model.

    PubMed

    DellaValle, Diane M; Vandenberg, Albert; Glahn, Raymond P

    2013-08-28

    In this study we examined the range of Fe concentration and relative Fe bioavailability of 24 varieties of cooked lentils, as well as the impact of seed coat removal on Fe nutritional as well as antinutrient properties. Relative Fe bioavailability was assessed by the in vitro/Caco-2 cell culture method. While the Fe concentration of the whole lentil was moderately high (72.8 ± 10.8 ?g/g, n = 24), the relative Fe bioavailability was moderate (2.4 ± 1.0 ng of ferritin/mg of protein). Although removing the seed coat reduced the Fe concentration by an average of 16.4 ± 9.4 ?g/g, the bioavailability was significantly improved (+5.3 ± 2.2 ng of ferritin/mg of protein; p < 0.001), and the phytic acid concentration was reduced by 7% (p = 0.04). Like most legume seeds, the lentil seed coat contains a range of polyphenols known to inhibit Fe bioavailability. Thus, along with breeding for high Fe concentration and bioavailability (i.e., biofortification), seed coat removal appears to be a practical way to improve Fe bioavailability of the lentil. PMID:23915260

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

    Microsoft Academic Search

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

    1988-01-01

    Normal differentiation and malignant transformation of human cells are characterized by specific changes in surface antigen phenotype. In the present study, the authors have defined six cell-surface antigens of human sarcomas and normal mesenchymal cells, by using mixed hemadsorption assays and immunochemical methods for the analysis of cultured cells and immunohistochemical staining for the analysis of normal tissues and >

  10. Purification and Culture of Erythroid Progenitor Cells

    Microsoft Academic Search

    Chun-Hua Dai; Amittha Wickrema; Sanford B Krantz

    Purified cells can be utilized for studies involving growth factor regulated development of erythroid progenitor cells. For\\u000a example, Fig. 4 demonstrates the expression pattern of ?-globin and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA during\\u000a differentiation of highly purified human erythroid progenitors. Studies have been performed to delineate the expression of\\u000a Epo receptor mRNA and the roles of each of the growth

  11. Long-term culture of lymphohematopoietic stem cells.

    PubMed Central

    Palacios, R; Bucana, C; Xie, X

    1996-01-01

    Pluripotent hematopoietic stem cells (PHSCs) show self-renewal and give rise to all blood cell types. The extremely low number of these cells in primary hematopoietic organs and the lack of culture systems that support proliferation of undifferentiated PHSCs have precluded the study of both the biology of these cells and their clinical application. We describe here cell lines and clones derived from PHSCs that were established from hematopoietic cells from the fetal liver or bone marrow of normal and p53-deficient mice with a combination of four growth factors. Most cell lines were Sca-1+, c-Kit+, PgP-1+, HSA+, and Lin- (B-220-, Joro 75-, 8C5-, F4/80-, CD4-, CD8-, CD3-, IgM-, and TER 119-negative) and expressed three new surface markers: Joro 177, Joro 184, and Joro 96. They did not synthesize RNA transcripts for several genes expressed at early stages of lymphocyte and myeloid/erythroid cell development. The clones were able to generate lymphoid, myeloid, and erythroid hematopoietic cells and to reconstitute the hematopoietic system of irradiated mice for a long time. The availability of lymphohematopoietic stem cell lines should facilitate the analysis of the molecular mechanisms that control self-renewal and differentiation and the development of efficient protocols for somatic gene therapy. Images Fig. 1 Fig. 2 PMID:8643561

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

    PubMed

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

    2014-12-15

    From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy. PMID:25453259

  13. Isolation and culture of sheep bronchial artery endothelial cells

    Microsoft Academic Search

    S. P. Sanders; S. J. Harrison; W. S. Lee; D. B. Pearse; E. M. Wagner

    1995-01-01

    Methods are described for the isolation of endothelial cells from sheep bronchial artery by treatment with collagenase. Cells obtained from the perfusate of the collagenase-treated vessel are cultured. Identified by their cobblestone morphology, endothelial cell colonies of approximately 50 cells are selected by a cloning cylinder and subcultured using trypsin. Endothelial cells are characterized by the formation of vessel-like tubes

  14. Cell and tissue culture of Miscanthus Sacchariflorus

    SciTech Connect

    Godovikova, V.A.; Moiseyeva, E.A.; Shumny, V.K. [Institute of Cytology and Genetics, Novosibirsk (Russian Federation)

    1995-11-01

    Since recent time search and introduction of new species of plants have paid attention. More perspective are perennial low maintenance landscape plants from genera Phragmites L. and Miscanthus Anderss. known as high speed growing and great amount of cellulose`s containing. Absence of seeds production and limited distribution area prevent from immediately introduction the plants of this species. The main goal of our investigation is the scientific development of the cell and tissue culture methods to get changing clones, salt and cold tolerant plants and their micropogation. At present there are collection of biovariety represented by subspecies, ecotypes and plant regenerants of two species - Miscanthus purpurascens (Anders.) and Miscanthus sacchariflorus (Maxim.). Successful results have been achieved in screening of culture media, prepared on MS base medium and contained a row of tropic components to protect the explant and callus tissue from oxidation and necrosis. Initially the callus was induced from stem segments, apical and nodular meristem of vegetative shoots of elulalia, growing in hydroponic greenhouse. Morphological and cytologic analysis of plant-regenerants have been done.

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

    PubMed

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

    2015-07-01

    The development of fin primary cell cultures for in vitro cellular and physiological studies is hampered by slow cell outgrowth, low proliferation rate, poor viability, and sparse cell characterization. Here, we investigated whether the recycling of fresh explants after a first conventional culture could improve physiological stability and sustainability of the culture. The recycled explants were able to give a supplementary cell culture showing faster outgrowth, cleaner cell layers and higher net cell production. The cells exhibited a highly stabilized profile for marker gene expression including a low cytokeratin 49 (epithelial marker) and a high collagen 1a1 (mesenchymal marker) expression. Added to the cell spindle-shaped morphology, motility behavior, and actin organization, this suggests that the cells bore stable mesenchymal characteristics. This contrast with the time-evolving expression pattern observed in the control fresh explants during the first 2 weeks of culture: a sharp decrease in cytokeratin 49 expression was concomitant with a gradual increase in col1a1. We surmise that such loss of epithelial features for the benefit of mesenchymal ones was triggered by an epithelial to mesenchymal transition (EMT) process or by way of a progressive population replacement process. Overall, our findings provide a comprehensive characterization of this new primary culture model bearing mesenchymal features and whose stability over culture time makes those cells good candidates for cell reprogramming prior to nuclear transfer, in a context of fish genome preservation. PMID:25929521

  16. Treating cell culture media with UV irradiation against adventitious agents: minimal impact on CHO performance.

    PubMed

    Yen, Sandi; Sokolenko, Stanislav; Manocha, Bhavik; Blondeel, Eric J M; Aucoin, Marc G; Patras, Ankit; Daynouri-Pancino, Farnaz; Sasges, Michael

    2014-01-01

    Sterility of cell culture media is an important concern in biotherapeutic processing. In large scale biotherapeutic production, a unit contamination of cell culture media can have costly effects. Ultraviolet (UV) irradiation is a sterilization method effective against bacteria and viruses while being non-thermal and non-adulterating in its mechanism of action. This makes UV irradiation attractive for use in sterilization of cell culture media. The objective of this study was to evaluate the effect of UV irradiation of cell culture media in terms of chemical composition and the ability to grow cell cultures in the treated media. The results showed that UV irradiation of commercial cell culture media at relevant disinfection doses impacted the chemical composition of the media with respect to several carboxylic acids, and to a minimal extent, amino acids. The cumulative effect of these changes, however, did not negatively influence the ability to culture Chinese Hamster Ovary cells, as evaluated by cell viability, growth rate, and protein titer measurements in simple batch growth compared with the same cells cultured in control media exposed to visible light. PMID:25044686

  17. The advantages of three-dimensional culture in a collagen hydrogel for stem cell differentiation.

    PubMed

    Naito, Hiroshi; Yoshimura, Mamiko; Mizuno, Toshihide; Takasawa, Shin; Tojo, Takashi; Taniguchi, Shigeki

    2013-10-01

    We evaluated the advantages of three-dimensional (3D) culture in a collagen hydrogel for stem cell differentiation, including the morphology of differentiated cells, differentiation efficiency of stem cells from aged rat and cells after passaging and freeze/thawing. Rat mesenchymal stem cells (MSCs) from young and aged rats, and MSCs after passaging and freeze/thawing were induced to differentiate into osteoblasts in 3D and 2D cultures, and histological studies were performed. Differentiation efficiency was evaluated by markers of osteoblastic differentiation including Runx2 and osterix gene expressions, osteocalcin secretion and calcium deposition. MSCs were stained positive for alkaline phosphatase in 3D and 2D cultures. However, the morphology of differentiated cells in 3D culture, which was different from that in 2D culture, was similar to that of osteoblasts in vivo. Markers of osteoblastic differentiation in MSCs from aged rats in 3D culture were higher than those in MSCs from young rats in 2D culture. Markers of osteoblastic differentiation in MSCs after passaging and freeze/thawing in 3D culture were higher than those in nonpassaged MSCs in 2D culture. These results indicate that 3D culture in a collagen hydrogel has advantages for the differentiation of MSCs into osteoblasts with a similar phenotype to that of in vivo, when using even MSCs from aged donors or after passaging and freeze/thawing. PMID:23468218

  18. A microfluidic dual-well device for high-throughput single-cell capture and culture.

    PubMed

    Lin, Ching-Hui; Hsiao, Yi-Hsing; Chang, Hao-Chen; Yeh, Chuan-Feng; He, Cheng-Kun; Salm, Eric M; Chen, Chihchen; Chiu, Ing-Ming; Hsu, Chia-Hsien

    2015-06-30

    In vitro culture of single cells facilitates biological studies by deconvoluting complications from cell population heterogeneity. However, there is still a lack of simple yet high-throughput methods to perform single cell culture experiments. In this paper, we report the development and application of a microfluidic device with a dual-well (DW) design concept for high-yield single-cell loading (~77%) in large microwells (285 and 485 ?m in diameter) which allowed for cell spreading, proliferation and differentiation. The increased single-cell loading yield is achieved by using sets of small microwells termed "capture-wells" and big microwells termed "culture-wells" according to their utilities for single-cell capture and culture, respectively. This novel device architecture allows the size of the "culture" microwells to be flexibly adjusted without affecting the single-cell loading efficiency making it useful for cell culture applications as demonstrated by our experiments of KT98 mouse neural stem cell differentiation, A549 and MDA-MB-435 cancer cell proliferation, and single-cell colony formation assay with A549 cells in this paper. PMID:26060987

  19. Rapid single-cell electroporation for labeling organotypic cultures

    E-print Network

    Steinmeyer, Joseph D. (Joseph Daly)

    2010-01-01

    Single-cell electroporation is a technique for transfecting individual cells in tissue culture at relatively high efficiencies, however it is both time-consuming and low-throughput and this limits the number of different ...

  20. Culture of cells from mammalian tissue cryopreserved without cryoprotection 

    E-print Network

    Charles, Lara Nicole

    2009-05-15

    Donor cells for nuclear transfer are usually prepared by the culture of fresh tissue. However, animal carcasses are sometimes frozen without cryoprotectants and if it were possible to obtain live cells from carcasses (tissue) preserved...

  1. CFU-GM Like Colonies Derived from Embryonic Stem Cells Cultured on the Bone Marrow Stromal Cells

    Microsoft Academic Search

    Ali Akbar Movassagh Pour; Mojdeh Salehnia; Ali Akbar Pourfatollah; Masoud Soleimani

    2004-01-01

    The aim of this study was to isolate mouse embryonic stem cells from late blastocyst stage embryos and to use them as a model system for the study of hematopoietic induction outside the embryo by coculturing of embryonic stem cells with bone marrow stromal cells. Blastocyst stage embryos from pregnant NMRI mice were obtained and cultured for 1-2 days in

  2. Ascorbic acid transport into cultured pituitary cells

    Microsoft Academic Search

    E. I. Cullen; V. May; R. A. Eipper

    1986-01-01

    An amidating enzyme designated peptidyl-glycine ..cap alpha..-amidating monooxygenase (PAM) has been studied in a variety of tissues and is dependent on molecular oxygen and stimulated by copper and ascorbic acid. To continue investigating the relationship among cellular ascorbic acid concentrations, amidating ability, and PAM activity, the authors studied ascorbic acid transport in three cell preparations that contain PAM and produce

  3. Cultural Studies Meets Religious Education

    ERIC Educational Resources Information Center

    Parker, Evelyn

    2006-01-01

    In this article, the author discusses how contemporary popular culture influences religious meaning of people in pluralistic society. One such powerful media that influences religious beliefs is the television. Meaning making about faith, how people make meaning, and the nature of those meanings are central concerns of religious education and…

  4. Exposure to cell phone radiation up-regulates apoptosis genes in primary cultures of neurons and astrocytes

    Microsoft Academic Search

    Tian-Yong Zhao; Shi-Ping Zou; Pamela E. Knapp

    2007-01-01

    The health effects of cell phone radiation exposure are a growing public concern. This study investigated whether expression of genes related to cell death pathways are dysregulated in primary cultured neurons and astrocytes by exposure to a working Global System for Mobile Communication (GSM) cell phone rated at a frequency of 1900MHz. Primary cultures were exposed to cell phone emissions

  5. Differentiated Rat Glial Cell Strain in Tissue Culture

    Microsoft Academic Search

    Philippe Benda; James Lightbody; Gordon Sato; Lawrence Levine; William Sweet

    1968-01-01

    Rat glial tumors induced by injections of N-nitrosomethylurea, were plated and propagated in culture. Among a few cell strains obtained, one clone contains S-100 protein, which is unique to brain in vertebrates. Stationary-phase cultures contain approximately ten times more S-100 protein per cell than exponentially growing cells. When injected into newborn rats, cells producing S-100 grew as a glial tumor,

  6. Cell culture quality control by rapid isoenzymatic characterization

    Microsoft Academic Search

    David M. Halton; Ward D. Peterson; Bharati Hukku

    1983-01-01

    Summary  Procedures that involve cell cultures require careful quality control to avoid inter- and intraspecies contamination. We have\\u000a developed and electrophoresis technique that can be used routinely in cell culture laboratories to monitor cell line integrity.\\u000a The method involves the isoenzymatic separation of nine polymorphic enzymes, three of which can be used for cell line species\\u000a determinations and seven of which

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

    NASA Astrophysics Data System (ADS)

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

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

  8. Biotransformation of hyoscyamine into scopolamine in transgenic tobacco cell cultures.

    PubMed

    Moyano, Elisabeth; Palazón, Javier; Bonfill, Mercedes; Osuna, Lidia; Cusidó, Rosa M; Oksman-Caldentey, Kirsi-Marja; Piñol, M Teresa

    2007-04-01

    Hyoscyamine-6beta-hydroxylase (H6H) catalyses the conversion of hyoscyamine into its epoxide scopolamine, a compound with a higher added value in the pharmaceutical market than hyoscyamine. We report the establishment of tobacco cell cultures carrying the Hyoscyamus muticus h6h gene under the control of the promoter CAMV 35S. The cell cultures were derived from hairy roots obtained via genetically modified Agrobacterium rhizogenes carrying the pRi and pLAL21 plasmids. The cultures were fed with hyoscyamine, and 4 weeks later the amount of scopolamine produced was quantified by HPLC. The transgenic cell suspension cultures showed a considerable capacity for the bioconversion of hyoscyamine into scopolamine, and released it to the culture medium. Although the scale-up from shake-flask to bioreactor culture usually results in reduced productivities, our transgenic cells grown in a 5-L turbine stirred tank reactor in a batch mode significantly increased the scopolamine accumulation. PMID:16904229

  9. Enhanced infectivity of bluetongue virus in cell culture by centrifugation.

    PubMed Central

    Sundin, D R; Mecham, J O

    1989-01-01

    The effects of centrifugation of the infection of cell culture with bluetongue virus (BTV) were investigated. Baby hamster kidney cells were infected with BTV with or without centrifugation. Viral antigen was detected by immunofluorescence at 24 h in both centrifuged and noncentrifuged cultures. However, after 24 h of infection, the production of PFU in centrifuged cell cultures was 10- to 20-fold greater than that seen in cultures not centrifuged. In addition, centrifugation enhanced the direct detection of PFU from blood samples collected from a sheep experimentally infected with BTV. Images PMID:2549092

  10. Radiosensitivity of cultured insect cells: I. Lepidoptera

    SciTech Connect

    Koval, T.M.

    1983-10-01

    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 D/sub 0/, 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 D/sub 0/ 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.

  11. Benzoic acid biosynthesis in cell cultures of Hypericum androsaemum.

    PubMed

    Abd El-Mawla, Ahmed M A; Beerhues, Ludger

    2002-03-01

    Biosynthesis of benzoic acid from cinnamic acid has been studied in cell cultures of Hypericum androsaemum L. The mechanism underlying side-chain shortening is CoA-dependent and non-beta-oxidative. The enzymes involved are cinnamate:CoA ligase, cinnamoyl-CoA hydratase/lyase and benzaldehyde dehydrogenase. Cinnamate:CoA ligase was separated from benzoate:CoA ligase and 4-coumarate:CoA ligase, which belong to xanthone biosynthesis and general phenylpropanoid metabolism, respectively. Cinnamoyl-CoA hydratase/lyase catalyzes hydration and cleavage of cinnamoyl-CoA to benzaldehyde and acetyl-CoA. Benzaldehyde dehydrogenase finally supplies benzoic acid. In cell cultures of H. androsaemum, benzoic acid is a precursor of xanthones, which accumulate during cell culture growth and after methyl jasmonate treatment. Both the constitutive and the induced accumulations of xanthones were preceded by increases in the activities of all benzoic acid biosynthetic enzymes. Similar changes in activity were observed for phenylalanine ammonia-lyase and the xanthone biosynthetic enzymes benzoate:CoA ligase and benzophenone synthase. PMID:11882941

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

    PubMed

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

    2015-08-01

    At present, biopsy specimens, cancer cell lines and tissues obtained by gastric surgery are used in the study and analysis of gastric cancer, including the molecular mechanisms and proteomics. However, fibroblasts and other tissue components may interfere with these techniques. Therefore, the present study aimed to develop a procedure for the isolation of viable human gastric epithelial cells from gastric surgical tissues. A method was developed to culture human gastric epithelial cells using fresh, surgically excised tissues and was evaluated using immunocytochemistry, periodic acid?Schiff (PAS) staining and cell viability assays. Low cell growth was observed surrounding the gastric tissue on the seventh day of tissue explant culture. Cell growth subsequently increased, and at 12 days post?explant a high number of pure epithelial cells were detected. The gastric cancer cells exhibited rapid growth with a doubling time of 13?52 h, as compared to normal cells, which had a doubling time of 20?53 h. Immunocytochemical analyses of primary gastric cells revealed positive staining for cytokeratin 18 and 19, which indicated that the culture was comprised of pure epithelial cells and contained no fibroblasts. Furthermore, PAS staining demonstrated that the cultured gastric cells produced neutral mucin. Granulin and carbohydrate antigen 724 staining confirmed the purity of gastric cancer and normal cells in culture. This method of cell culture indicated that the gastric cells in primary culture consisted of mucin?secreting gastric epithelial cells, which may be useful for the study of gastric infection with Helicobacter pylori and gastric cancer. PMID:25937205

  13. Identification and regulation of renin in human cultured mesangial cells.

    PubMed

    Chansel, D; Dussaule, J C; Ardaillou, N; Ardaillou, R

    1987-01-01

    Renin activity was measured in the incubation medium, and the cellular extract of human mesangial cells, which had been cultured in the presence of renin-free human plasma (three kidneys; 4-7 passages). Active renin and total renin obtained after trypsin treatment was estimated by radioimmunoassay of angiotensin I using renin-free human plasma as a substrate. Mesangial cell renin had characteristics similar to those of standard human renin; optimum enzymatic activity at pH 5.8, marked inhibition in the presence of two (monoclonal and polyclonal) human renin-specific antibodies and of SR 42128, a new potent statine-containing renin inhibitory peptide. The synthetic capability of the mesangial cells varied markedly with the original kidney (1-49 and 0.3-0.9 ng X h-1 X mg-1 for total renin in the medium and the cellular extract respectively). Renin was secreted mainly as inactive renin. Prostaglandin E2 (PGE2) and carba-prostaglandin I2 (PGI2) (a stable analogue) produced a dose-dependent (0.1-1.10 microM) increase in renin activity in both the cellular extract and the culture medium. Isoproterenol (200 microM) increased renin activity only in the medium. The effects of these agonists were more marked on inactive than on active renin. These results demonstrate that cultured human mesangial cells synthesize and release renin in a stable manner over a long period of culture, thus providing a useful tool for the in vitro study of renin secretion and its control. PMID:3544867

  14. Biological implications of polydimethylsiloxane-based microfluidic cell culture

    PubMed Central

    Regehr, Keil J.; Domenech, Maribella; Koepsel, Justin T.; Carver, Kristopher C.; Ellison-Zelski, Stephanie J.; Murphy, William L.; Schuler, Linda A.; Alarid, Elaine T.; Beebe, David J.

    2009-01-01

    Polydimethylsiloxane (PDMS) has become a staple of the microfluidics community by virtue of its simple fabrication process and material attributes, such as gas permeability, optical transparency, and flexibility. As microfluidic systems are put toward biological problems and increasingly utilized as cell culture platforms, the material properties of PDMS must be considered in a biological context. Two properties of PDMS were addressed in this study: the leaching of uncured oligomers from the polymer network into microchannel media, and the absorption of small, hydrophobic molecules (i.e. estrogen) from serum-containing media into the polymer bulk. Uncured PDMS oligomers were detectable via MALDI-MS in microchannel media both before and after Soxhlet extraction of PDMS devices in ethanol. Additionally, PDMS oligomers were identified in the plasma membranes of NMuMG cells cultured in PDMS microchannels for 24 hours. Cells cultured in extracted microchannels also contained a detectable amount of uncured PDMS. It was shown that MCF-7 cells seeded directly on PDMS inserts were responsive to hydrophilic prolactin but not hydrophobic estrogen, reflecting its specificity for absorbing small, hydrophobic molecules; and the presence of PDMS floating in wells significantly reduced cellular response to estrogen in a serum-dependent manner. Quantification of estrogen via ELISA revealed that microchannel estrogen partitioned rapidly into the surrounding PDMS to a ratio of approximately 9:1. Pretreatments such as blocking with serum or pre-absorbing estrogen for 24 hours did not affect estrogen loss from PDMS-based microchannels. These findings highlight the importance of careful consideration of culture system properties when determining an appropriate environment for biological experiments. PMID:19606288

  15. Controlled delivery of lipophilic agents to cell cultures for in vitro toxicity and biocompatibility assays.

    PubMed

    Zülli, F; Liechti, C; Suter, F

    2000-08-01

    In this report, we present a novel method for delivering lipophilic compounds to cell cultures. The delivery system is based on a nanoemulsion stabilized by phospholipids. These nanoemulsions are well tolerated by cell cultures, such as TK6 lymphoblastoid cells and can be used to deliver defined amounts of encapsulated lipophilic compounds into cells. We measured the growth inhibition of TK6 lymphoblastoid cells caused by different oils, UV-filters and fragrances to determine the biocompatibility or the toxicity of these compounds in simple cell culture experiments. Our data show that the applied nanoemulsion technology is also very suitable to study biological effects of the UV-A-irradiated compounds in cell culture assays. PMID:18503413

  16. Cytotoxic effects of arthropod venoms on various cultured cells

    Microsoft Academic Search

    Ephraim Cohen; Gary B. Quistad

    1998-01-01

    E. Cohen and G. B. Quistad. Cytotoxic effects of arthropod venoms on various cultured cells. Toxicon36, 353–358, 1998.—The action of arthropod venoms is important to predators in search of prey and to humans as incidental victims or as a source for pharmacologically active compounds. Venoms from 30 arthropods (including 26 spider species) were assessed for cytotoxicity using cultured cells from

  17. Mycophenolic acid antagonizes the activation of cultured human mesangial cells

    Microsoft Academic Search

    Isabelle Dubus; Benoît Vendrely; Isabelle Christophe; Jean-Pierre Labouyrie; Yahsou Delmas; Jacques Bonnet; Christian Combe

    2002-01-01

    Mycophenolic acid antagonizes the activation of cultured human mesangial cells.BackgroundActivation of mesangial cells is observed in several forms of chronic renal disease, and in culture conditions upon stimulation by fetal calf serum (FCS), or agonists such as transforming growth factor beta (TGF-?). Mycophenolate mofetil (MMF), the precursor of mycophenolic acid (MPA), is currently used in organ transplantation and has been

  18. Primary cell cultures from sea urchin ovaries: a new experimental tool.

    PubMed

    Mercurio, Silvia; Di Benedetto, Cristiano; Sugni, Michela; Candia Carnevali, M Daniela

    2014-02-01

    In the present work, primary cell cultures from ovaries of the edible sea urchin Paracentrotus lividus were developed in order to provide a simple and versatile experimental tool for researches in echinoderm reproductive biology. Ovary cell phenotypes were identified and characterized by different microscopic techniques. Although cell cultures could be produced from ovaries at all stages of maturation, the cells appeared healthier and viable, displaying a higher survival rate, when ovaries at early stages of gametogenesis were used. In terms of culture medium, ovarian cells were successfully cultured in modified Leibovitz-15 medium, whereas poor results were obtained in minimum essential medium Eagle and medium 199. Different substrates were tested, but ovarian cells completely adhered only on poly-L-lysine. To improve in vitro conditions and stimulate cell proliferation, different serum-supplements were tested. Fetal calf serum and an originally developed pluteus extract were detrimental to cell survival, apparently accelerating processes of cell death. In contrast, cells cultured with sea urchin egg extract appeared larger and healthier, displaying an increased longevity that allowed maintaining them for up to 1 month. Overall, our study provides new experimental bases and procedures for producing successfully long-term primary cell cultures from sea urchin ovaries offering a good potential to study echinoid oogenesis in a controlled system and to investigate different aspects of echinoderm endocrinology and reproductive biology. PMID:24002666

  19. Simultaneous extraction of proteins and metabolites from cells in culture

    PubMed Central

    Sapcariu, Sean C.; Kanashova, Tamara; Weindl, Daniel; Ghelfi, Jenny; Dittmar, Gunnar; Hiller, Karsten

    2014-01-01

    Proper sample preparation is an integral part of all omics approaches, and can drastically impact the results of a wide number of analyses. As metabolomics and proteomics research approaches often yield complementary information, it is desirable to have a sample preparation procedure which can yield information for both types of analyses from the same cell population. This protocol explains a method for the separation and isolation of metabolites and proteins from the same biological sample, in order for downstream use in metabolomics and proteomics analyses simultaneously. In this way, two different levels of biological regulation can be studied in a single sample, minimizing the variance that would result from multiple experiments. This protocol can be used with both adherent and suspension cell cultures, and the extraction of metabolites from cellular medium is also detailed, so that cellular uptake and secretion of metabolites can be quantified. Advantages of this technique includes:1.Inexpensive and quick to perform; this method does not require any kits.2.Can be used on any cells in culture, including cell lines and primary cells extracted from living organisms.3.A wide variety of different analysis techniques can be used, adding additional value to metabolomics data analyzed from a sample; this is of high value in experimental systems biology.

  20. Ethanolamine metabolism in cultured bovine aortic endothelial cells

    SciTech Connect

    Lipton, B.A.; Davidson, E.P.; Ginsberg, B.H.; Yorek, M.A. (Univ. of Iowa, Iowa City (USA))

    1990-05-05

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

  1. Reconstruction of stratum corneum in organotypically cultured canine keratinocyte-derived CPEK cells.

    PubMed

    Yagihara, Hiroko; Okumura, Toshiki; Shiomi, Eri; Shinozaki, Nao; Kuroki, Shiori; Sasaki, Yu; Ito, Keita; Ono, Kenichiro; Washizu, Tsukimi; Bonkobara, Makoto

    2011-10-01

    The stratum corneum of epidermis is an essential barrier against the external environment and water loss. This study aimed to develop an organotypic culture model that targets the reconstruction of the stratum corneum using canine keratinocyte-derived CPEK cells. The CPEK cells cultured at the air-liquid interface became stratified and formed a stratum corneum-like layer on stratum spinosum- and stratum granulosum-like layers. The CPEK cells in the stratum granulosum-like layer expressed the cornified cell envelope (CCE)-related proteins loricrin and keratinocyte differentiation-associated protein. Organotypically cultured CPEK cells were considered to form a CCE at the stratum granulosum-like layer, allowing the formation of a stratum corneum-like layer. The organotypic culture of CPEK cells could be useful for studying the barrier function of canine stratum corneum. PMID:21559887

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

    NASA Technical Reports Server (NTRS)

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

    2015-01-01

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

  3. Beta-amyloid activated microglia induce cell cycling and cell death in cultured cortical neurons

    Microsoft Academic Search

    Qian Wu; Colin Combs; Steven B Cannady; David S Geldmacher; Karl Herrup

    2000-01-01

    Immunocytochemical studies of postmortem human tissue have shown that the neurons at risk for degeneration in Alzheimer’s are marked by the ectopic expression of several cell cycle components. The current work investigates the roles that ?-amyloid activated microglia might play in leading neurons to re-express cell cycle components. Stable cultures of E16.5 mouse cortical neurons were exposed to ?-amyloid alone,

  4. The effects of three-dimensional cell culture on single myoblasts.

    PubMed

    Marquette, Michele L; Byerly, Diane; Sognier, Marguerite

    2008-01-01

    Identification of cellular and morphological changes in myoblasts during three-dimensional (3D) culture may provide novel insight into skeletal muscle morphogenesis. One particular morphological change that occurs during the transition from monolayer culture to the 3D environment is the appearance of cytoplasmic projections (podia). The purpose of these studies was to determine if: (1) 3D culture increased podia formation in single cells, and (2) podia were F-actin dependent. C2C12 cells were grown in 3D conditions using a rotary cell culture system (RCCS) for 3, 6, and 9 h, fixed, and stained. Analysis of confocal images revealed that podia were significantly more numerous on RCCS cultured cells than those on suspension controls. Further, the podia of RCCS cultured cells decreased in number and increased in length during the time intervals examined. RCCS cultured cells showed no significant changes in viability, Annexin V staining, and activated Caspase 3 expression over time. In contrast, significant decreases in viability of suspension controls occurred. The application of 2 muM Latrunculin A (Lat A), an actin depolymerizing agent, significantly reduced the number of cells with podia. The number of cells with podia recovered with Lat A removal. Changes in viability and apoptosis markers were not significant during Lat A application or washout experiments. These observations reveal that: (1) culture conditions in the RCCS increase the quantity of podia formation; (2) these podia increase in length with time; and (3) F-actin plays a role in podia formation. PMID:18246406

  5. Detection of Pemphigoid Antigen, Pemphigus Antigen, and Keratin Filaments by Indirect Immunofluorescence in Cultured Human Epidermal Cells

    Microsoft Academic Search

    John R. Stanley; Pamela Hawley-Nelson; Miriam Poirier; Stephen I. Katz; Stuart H. Yuspa

    1980-01-01

    In order to determine whether pemphigold antigen is synthesized by epidermal cells, and whether other, normal keratinocyte, antigens are synthesized in culture, primary cultures and subsequent subcultures of human epidermal cells derived from neonatal foreskins were studied for the presence of pemphigoid antigen, pemphigus antigen and keratin filaments using indirect immunofluorescence. Twenty-four hr after plating primary cultures, pemphigoid antigen was

  6. ASSEMBLY OF THE SARCOPLASMIC RETICULUM Localization by Immunofluorescence of Sarcoplasmic Reticulum Proteins in Differentiating Rat Skeletal Muscle Cell Cultures

    Microsoft Academic Search

    ANNELISE O. JORGENSEN; VITAUTS I. KALNINS; ELZBIETA ZUBRZYCKA

    Immunofluorescent staining techniques were used to study the distribution of the Ca 2+ + Mg2+-dependent ATPase and calsequestrin in primary cultures of differen- tiating rat skeletal muscle cells, grown for different periods of time under various culture conditions. In mononucleated myoblasts calsequestrin was detected after 45 h in culture whereas the ATPase was not detected until 60 h. After cell

  7. Neurons on Parafilm: versatile elastic substrates for neuronal cell cultures.

    PubMed

    Yoo, Sang Jin; Nam, Yoonkey

    2012-02-15

    A variety of materials has been applied to neuronal cell culture substrates to improve the efficiency of the culture and to provide pertinent cell growth environment. Here we report the application of Parafilm(®) M ('Parafilm') as a novel substrate for neuronal culture and patterning. Cell culture results show that elastic Parafilm had effects on cell viability, length and number of neurites, and soma spreading. Parafilm was also an effective substrate to obtain patterned neuronal cultures using a conventional micro-contract printing (?CP) technique. Polylysine micropatterns in line or grid forms were readily transferred from PDMS stamp to bare Parafilm surfaces and spatially confined neuronal cultures were successfully maintained for over three weeks. We also demonstrate that batch-processing cell culture substrates can be easily fabricated using a piece of Parafilm. The softness, plasticity, and hydrophobicity were main features that made it attractive for Parafilm to be considered as a practical cell culture platform. The results can be extended to develop an inexpensive and practical neuronal culture substrates in tissue engineering and biochip applications. PMID:22068030

  8. Dual polarization of microglia isolated from mixed glial cell cultures.

    PubMed

    Ju, Lili; Zeng, Hui; Chen, Yun; Wu, Yanhong; Wang, Beibei; Xu, Qunyuan

    2015-09-01

    Microglia are versatile immune effector cells of the CNS and are sensitive to various stimuli. The different methods used to isolate microglia may affect some of their characteristics, such as their polarization state. The influence of cell sorting methods on the polarization state of microglia has never been studied. Mixed glial culture system (MGCS) and magnetic activated cell sorting (MACS) are two methods that are commonly used to purify microglia. This study compares the immunological states between microglia isolated by MGCS and microglia isolated by MACS. We show that microglia isolated by MGCS exhibit a stronger immune-activated state than microglia isolated by MACS. They present an elevated phagocytic ability and high levels of markers associated with classical activation (M1) and alternative activation (M2). In addition, high levels of M1-type and M2-type chemokine (C-C motif) ligand 2 and transforming growth factor-?1 were detected in the culture medium of mixed glial cells. Our results show that microglia isolated by MGCS are in an immune-activated state, whereas microglia isolated by MACS appear to be closer to their primary in vivo state. Therefore, the immune status of microglia, depending on the protocol used to purify them, should be carefully considered in neuropathology research. © 2015 Wiley Periodicals, Inc. PMID:26053151

  9. Comparison of immunogenicity of cell-and egg-passaged viruses for manufacturing MDCK cell culture-based influenza vaccines.

    PubMed

    Shin, Duckhyang; Park, Kuk Jin; Lee, Hyeon; Cho, Eun Young; Kim, Mi Suk; Hwang, Mi Hui; Kim, Soo In; Ahn, Dong Ho

    2015-06-01

    While cell culture-based technology has been recently used for manufacturing influenza vaccines, currently available seed viruses are mostly egg-derived reassortants that are egg-adapted to achieve high virus growth in eggs. For use as viruses for cell culture-based influenza vaccine manufacturing, egg-adapted viral seeds may undergo several passages in manufacturing cell lines. However, the suitability of such cell-passaged viruses for vaccine production remains largely unelucidated. In this study, influenza viruses produced in suspension Madin-Darby canine kidney (MDCK) cell cultures were compared to those produced in embryonated hen's eggs for manufacturing MDCK cell culture-based influenza vaccines through comparability studies of virus productivity and vaccine immunogenicity. The results indicate no change in the amino acid sequence of the main antigens, including hemagglutinin (HA) and neuraminidase (NA), of cell-passaged viruses after three passages in suspension MDCK cells. In lab-scale (3-L) single-use bioreactors, suspension MDCK culture supernatants inoculated with cell-passaged viruses were found to show higher virus productivity, suspension MDCK culture supernatants inoculated with egg-passaged viruses, in respect to the HA titers and HA contents determined by single radial immunodiffusion. Finally, comparable hemagglutination inhibition and influenza-specific IgG titers were determined in the mice immunized with cell culture-based vaccines produced with cell- or egg-passaged viruses. These results indicate that MDCK cell-passaged viruses from egg-adapted viruses, as well as egg-derived seed virus, are suitable for MDCK cell culture-based influenza vaccine production. PMID:25892718

  10. Optical Oxygen Sensors for Applications in Microfluidic Cell Culture

    PubMed Central

    Grist, Samantha M.; Chrostowski, Lukas; 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 alternative to traditional cell culture platforms, there is recent interest in integrating oxygen-sensing mechanisms with microfluidics for cell culture applications. Optical, luminescence-based oxygen sensors, in particular, show great promise in their ability to be integrated with microfluidics and cell culture systems. These sensors can be highly sensitive and do not consume oxygen or generate toxic byproducts in their sensing process. This paper presents a review of previously proposed optical oxygen sensor types, materials and formats most applicable to microfluidic cell culture, and analyzes their suitability for this and other in vitro applications. PMID:22163408

  11. Copper Modulates the Differentiation of Mouse Hematopoietic Progenitor Cells in Culture

    PubMed Central

    Huang, Xiaosong; Pierce, L. Jeanne; Cobine, Paul A.; Winge, Dennis R.; Spangrude, Gerald J.

    2014-01-01

    Copper chelation has been shown to favor the expansion of human hematopoietic stem/progenitor cells in vitro. To further understand the effects of copper modulation on defined subsets of stem cells versus progenitor cells, we extended the studies in a mouse system. We isolated mouse hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) and cultured them with or without the copper chelator tetraethylen-epentamine (TEPA) or CuCl2. Cytokine-stimulated HPC cultures treated with TEPA for 7 days generated about two to three times more total and erythroid colony-forming cells (CFCs) compared to control cultures. In contrast, CuCl2 treatment decreased the CFC numbers. Similar results were seen with HSC after 14, but not 7, days of culture. Transplant studies showed that HPCs cultured for 7 days in TEPA had about twofold higher short-term erythroid repopulation potential compared to control cultures, while CuCl2 decreased the erythroid potential of cultured HPCs compared to control cultures. HSCs cultured with TEPA for 7 days did not exhibit significantly higher repopulation potential in either leukocyte or erythrocyte lineages compared to control cultures in short-term or long-term assays. Based on JC-1 staining, the mitochondrial membrane potential of HPCs cultured with TEPA was lower relative to control cultures. Our data suggest that decreasing the cellular copper content with TEPA results in preferential expansion or maintenance of HPC that are biased for erythroid differentiation in vivo, but does not enhance the maintenance of HSC activity in culture. PMID:19520051

  12. Copper modulates the differentiation of mouse hematopoietic progenitor cells in culture.

    PubMed

    Huang, Xiaosong; Pierce, L Jeanne; Cobine, Paul A; Winge, Dennis R; Spangrude, Gerald J

    2009-01-01

    Copper chelation has been shown to favor the expansion of human hematopoietic stem/progenitor cells in vitro. To further understand the effects of copper modulation on defined subsets of stem cells versus progenitor cells, we extended the studies in a mouse system. We isolated mouse hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) and cultured them with or without the copper chelator tetraethylenepentamine (TEPA) or CuCl(2). Cytokine-stimulated HPC cultures treated with TEPA for 7 days generated about two to three times more total and erythroid colony-forming cells (CFCs) compared to control cultures. In contrast, CuCl(2) treatment decreased the CFC numbers. Similar results were seen with HSC after 14, but not 7, days of culture. Transplant studies showed that HPCs cultured for 7 days in TEPA had about twofold higher short-term erythroid repopulation potential compared to control cultures, while CuCl(2) decreased the erythroid potential of cultured HPCs compared to control cultures. HSCs cultured with TEPA for 7 days did not exhibit significantly higher repopulation potential in either leukocyte or erythrocyte lineages compared to control cultures in short-term or long-term assays. Based on JC-1 staining, the mitochondrial membrane potential of HPCs cultured with TEPA was lower relative to control cultures. Our data suggest that decreasing the cellular copper content with TEPA results in preferential expansion or maintenance of HPC that are biased for erythroid differentiation in vivo, but does not enhance the maintenance of HSC activity in culture. PMID:19520051

  13. Cell Culture Extraction and Purification of Rabies Virus Nucleoprotein

    PubMed Central

    Dastkhosh, Mahshid; Rahimi, Pooneh; Haghighat, Setareh; Biglari, Peyvand; Howaizi, Nader; Saghiri, Reza; Roohandeh, Akram

    2014-01-01

    Background: Rabies is a major zoonotic viral disease and is detected using the World Health Organization standard diagnostic techniques. Rabies detection is preferably done using the fluorescent antibody technique (FAT) that provides reliable diagnosis with almost 100% accuracy for all variant strains, if a proper conjugate is used. Rabies virus nucleoprotein (NP) is the most important protein used in production of a specific diagnostic conjugate. Objectives: The aim of this study was to extract the cell-associated rabies virus NP from infected Baby Hamster Kidney cell clone (BSR) with rabies virus (Pasteur vaccine strain/PV) and purify for a future project to produce an anti-NP conjugate. Materials and Methods: Pasteur vaccine strain (PV) as the standard rabies vaccine strain with a focus-forming dose (FFD) of 105 was inoculated in to the BSR cell culture at a concentration of 106 cells per milliliter. Infected cells were harvested 72 hours after infection and the rabies NP was extracted from these cells by low-speed centrifugation and purification by ultracentrifugation in cesium chloride (CsCl) gradient. For analysis, the purified NP was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: The volume of the lysate was 15 mL and it became 2.5 mL after purification, with a concentration of 3.25 mg/mL. The corresponding band to the cell lysate protein on the SDS-PAGE had a molecular weight of 50 KDa, similar to the molecular weight of NP in rabies virus. Conclusions: The rabies virus NP could be extracted and purified in an appropriate amount from infected cell culture. The results of SDS-PAGE analysis showed that the intact rabies virus NP had been purified properly and thus could be used for further steps to produce the specific diagnostic rabies conjugate. PMID:25485056

  14. Stem cell marker CD271 is expressed by vasculogenic mimicry-forming uveal melanoma cells in three-dimensional cultures

    PubMed Central

    Valyi-Nagy, Klara; Kormos, Bernadett; Ali, Mohamed; Shukla, Deepak

    2012-01-01

    Purpose Cancer stem cells have increased resistance against a variety of anti-tumor treatment modalities. Vasculogenic mimicry (VM) patterns are present in numerous malignant tumor types, represent the formation of perfusion pathways by tumor cells, and their presence in tumors is associated with adverse outcome. Earlier we have shown that VM-forming tumor cells in three-dimensional (3D) uveal melanoma cultures have increased resistance against cytotoxic agents and oncolytic herpes simplex virus-mediated destruction. The purpose of the current study was to explore the possibility that this increased resistance of VM-forming tumor cells is due to a cancer stem cell phenotype. Methods The expression of cancer stem cell marker cluster of differentiation 271 (CD271) was determined in traditional two-dimensional (2D) and 3D cultures of C918 uveal melanoma cells by fluorescent immunocytochemistry. Results We found that the VM-forming tumor cell subpopulation in 3D cultures expressed CD271. In contrast, cells grown in 2D cultures and tumor cell subpopulations not participating in VM formation in 3D cultures were negative for CD271. Conclusions These findings suggest that VM-forming uveal melanoma cells acquire a cancer stem cell-like phenotype that may play a role in the increased therapy resistance of these cells. PMID:22419851

  15. Process control in cell culture technology using dielectric spectroscopy

    Microsoft Academic Search

    C. Justice; A. Brix; D. Freimark; M. Kraume; P. Pfromm; B. Eichenmueller; P. Czermak

    2011-01-01

    In the biopharmaceutical industry, mammalian and insect cells as well as plant cell cultures are gaining worldwide importance to produce biopharmaceuticals and as products themselves, for example in stem cell therapy. These highly sophisticated cell-based production processes need to be monitored and controlled to guarantee product quality and to satisfy GMP requirements. With the process analytical technology (PAT) initiative, requirements

  16. Activation of muscle satellite cells in single-fiber cultures

    Microsoft Academic Search

    Judy Anderson; Orest Pilipowicz

    2002-01-01

    Satellite stem cell activation is the process by which quiescent precursor cells resident on muscle fibers are recruited to cycle and move. Two processes are reported to affect satellite cell activation. In vivo, nitric oxide (NO) produced by NO synthase in fibers (NOS-I?) promotes activation. In cell cultures, hepatocyte growth factor (HGF) is the major activating factor isolated from crushed

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

    SciTech Connect

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

    1988-05-01

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

  18. Study on tissue culture for Gelidium seedling

    NASA Astrophysics Data System (ADS)

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

    1996-06-01

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

  19. Morphological studies on the culture of kidney epithelial cells in a fiber-in-fiber bioreactor design with hollow fiber membranes.

    PubMed

    Fey-Lamprecht, F; Albrecht, W; Groth, T; Weigel, T; Gross, U

    2003-05-01

    A hollow fiber-in-fiber-based bioreactor system was tested for the applicability to host kidney epithelial cells as a model system for a bioartificial kidney. Hollow fibers were prepared from polyacrylonitrile (PAN), polysulfone-polyvinylpyrollidinone (PVP) blend (PSU) and poly(acrylonitrile-N-vinylpyrollidinone) copolymer P(AN-NVP). Hollow fibers with smaller and larger diameters were prepared so that the smaller fitted into the larger, with a distance of 50-100 microm in between. The following material combinations as outer and inner fiber were applied: PAN-PAN; PSU-PSU, PSU-P(AN-NVP). Madin-Darby kidney epithelial cells (MDCK) were seeded in the interfiber space and cultured for a period up to 14 days. Light, scanning, and transmission electron microscopy were used to follow the adhesion and growth of cells, and to characterize their morphology. As a result, we found that MDCK cells were able to grow in the interfiber space in mono- and multilayers without signs of systemic degeneration. Comparison of the different materials showed that PAN and P(AN-NVP) provided the best growth conditions, indicated by a tight attachment of cells on hollow fiber membrane, and subsequent proliferation and development of structural elements of normal epithelia, such as tight junctions and microvilli. In conclusion, the fiber-in-fiber design seems to be an interesting system for the construction of a bioartificial kidney. PMID:12734806

  20. Culture studies of human pluripotent hemopoietic progenitors

    Microsoft Academic Search

    H. A. Messner; A. A. Fauser

    1980-01-01

    Conclusions Culture conditions are described that promote the growth of human pluripotent hemopoietic progenitors and facilitate their quantitation. These primitive cells form mixed colonies that may contain all elements of myeloid differentiation, including granulocytes, erythroblasts, megakaryocytes, and macrophages. Some mixed colonies contain, in addition to mature progeny, early progenitors that can be identified by their ability to form secondary hemopoietic

  1. Functional properties of mixed cystic fibrosis and normal bronchial epithelial cell cultures.

    PubMed

    Dannhoffer, Luc; Blouquit-Laye, Sabine; Regnier, Agathe; Chinet, Thierry

    2009-06-01

    Cystic fibrosis (CF) airway epithelia exhibit altered Cl(-) and Na(+) transport properties and increased IL-8 secretion. In the present study, we examined whether a small proportion of cells with a normal phenotype could normalize the ion transport and IL-8 secretion properties of a CF airway epithelial cell layer. We obtained three types of primary cultures of human bronchial epithelial cells: one composed of 100% non-CF cells, one of 100% CF cells, and one of 10% non-CF and 90% CF cells ("cocultures"). Measurement of the bioelectric properties in Ussing chambers revealed that the cocultures displayed Cl(-) and Na(+) transports similar to those observed in the 100% non-CF cultures and significantly different from CF cultures. IL-8 concentration in the coculture supernatant was not different from non-CF cultures, but was significantly lower than in CF cultures. This study provides evidence that 10% bronchial epithelial cells expressing a normal phenotype are sufficient to functionally correct a primary culture of CF bronchial epithelial cells in vitro. We postulate that 10% cells with a non-CF phenotype can be used as a goal for the design of gene therapy and cell therapy trials for CF lung disease. PMID:19011164

  2. Atypical multinucleated cells form in long-term marrow cultures from patients with Paget's disease.

    PubMed Central

    Kukita, A; Chenu, C; McManus, L M; Mundy, G R; Roodman, G D

    1990-01-01

    Although Paget's disease is the most flagrant example of a primary osteoclast disorder, little is known of osteoclast biology in this disease. In this report we have studied the formation of cells with the osteoclast phenotype in long-term cultures of marrow mononuclear cells derived from patients with Paget's disease, and compared these with similar cells formed in long-term marrow cultures from normal individuals, and with osteoclasts present in pagetic bone. Osteoclasts formed in pagetic marrow cultures resembled osteoclasts present in pagetic bone, but were distinctly different from osteoclasts formed in normal marrow cultures. Osteoclast formation was 10-20-fold greater in pagetic marrow cultures than in normal cultures. The multinucleated cells formed in cultures of pagetic marrow were much larger in size, were hyperresponsive to 1,25(OH)2 vitamin D, had more nuclei per cell, had increased levels of tartrate-resistant acid phosphatase activity and had ultrastructural features which were not seen in multinucleated cells formed from normal marrow mononuclear cells. These pagetic marrow-derived multinucleated cells formed large resorption lacunae on calcified matrices and cross-reacted with monoclonal antibodies which preferentially bind to osteoclasts. The multinucleated cells formed from marrow obtained from uninvolved sites in Paget's patients also displayed these abnormal features. Images PMID:2318982

  3. Modulation of cyclin transcript levels in cultured cells of Arabidopsis thaliana.

    PubMed Central

    Fuerst, R A; Soni, R; Murray, J A; Lindsey, K

    1996-01-01

    Previous studies on the cell cycle of Arabidopsis thaliana have been hindered by the lack of synchronous cell culture systems. We have used liquid callus cultures and a cycloheximide-synchronized suspension culture of Arabidopsis to investigate changes in cyclin transcript levels in response to exogenous auxin, cytokinin, and nutrients, and during the cell cycle. CYCD1 (delta 1) transcript was virtually undetectable in liquid-cultured callus or suspension-culture cells. CYCD2 (delta 2) transcript levels were largely unaffected by the readdition of phytohormones or nitrate to the growth medium, and remained constant throughout the cell cycle in suspension-culture cells. CYCD3 (delta 3) transcript levels were strongly dependent on nitrate, and were induced at the G1/S transition following phytohormone readdition. In synchronized suspension-culture cells, CYCD3 transcript accumulated during the S phase, and remained constant thereafter. These results support the hypothesis that D cyclins function as part of the cellular machinery that integrates diverse signals impinging upon commitment to cell division. In synchronized cells transcripts of the mitotic cyclins CYC1, CYC2, and CYC3 reached a maximum with peak mitotic index, but CYC3 transcript levels increased earlier than those of CYC1 or CYC2. The kinetics of accumulation of CYC transcript levels support their classification as A-type (CYC3) and B-type (CYC1 and CYC2) cyclins, respectively. PMID:8938409

  4. Isolation, culture, and differentiation potential of mouse marrow stromal cells.

    PubMed

    Anjos-Afonso, Fernando; Bonnet, Dominique

    2008-10-01

    This unit describes how to isolate and expand mesenchymal stromal cells (MSCs) from mouse bone marrow. For reasons that are not clear, it has been difficult to isolate these cells (also known as mesenchymal stem cells). Furthermore, different mouse strains seem to have specific requirements for successful extraction and culture of these cells. A general and easy protocol is presented here for isolating stromal cells from different inbred and transgenic mice commonly used in the stem cell biology field. PMID:18972375

  5. Correlation of Rapid Cell Death with Metabolic Changes in Fungus-Infected, Cultured Parsley Cells.

    PubMed Central

    Naton, B.; Hahlbrock, K.; Schmelzer, E.

    1996-01-01

    To study in detail the hypersensitive reaction, one of the major defense responses of plants against microbial infection, we used a model system of reduced complexity with cultured parsley (Petroselinum crispum) cells infected with the phytopathogenic fungus Phytophthora infestans. Experimental conditions were established to maintain maximal viability of the cultured cells during co-cultivation with fungal germlings, and a large proportion of the infected parsley cells responded to fungal infection with rapid cell death, thereby exhibiting major features of the hypersensitive reaction in whole-plant-pathogen interactions. Rapid cell death clearly correlated with termination of further growth and development of the fungal pathogen. Thus, the system fulfilled important prerequisites for investigating cell-death-related metabolic changes in individual infected cells. Using cytochemical methods, we monitored the increase of mitochondrial activity in single infected cells and the intracellular accumulation of reactive oxygen species prior to the occurrence of rapid cell death. We obtained strong correlative evidence for the involvement of these intracellularly accumulating reactive oxygen species in membrane damage and in the resulting abrupt collapse of the cell. PMID:12226400

  6. Development of an Integrated Microfluidic Perfusion Cell Culture System for Real-Time Microscopic Observation of Biological Cells

    PubMed Central

    Lin, Lung; Wang, Shih-Siou; Wu, Min-Hsien; Oh-Yang, Chih-Chin

    2011-01-01

    This study reports an integrated microfluidic perfusion cell culture system consisting of a microfluidic cell culture chip, and an indium tin oxide (ITO) glass-based microheater chip for micro-scale perfusion cell culture, and its real-time microscopic observation. The system features in maintaining both uniform, and stable chemical or thermal environments, and providing a backflow-free medium pumping, and a precise thermal control functions. In this work, the performance of the medium pumping scheme, and the ITO glass microheater were experimentally evaluated. Results show that the medium delivery mechanism was able to provide pumping rates ranging from 15.4 to 120.0 ?L·min?1. In addition, numerical simulation and experimental evaluation were conducted to verify that the ITO glass microheater was capable of providing a spatially uniform thermal environment, and precise temperature control with a mild variation of ±0.3 °C. Furthermore, a perfusion cell culture was successfully demonstrated, showing the cultured cells were kept at high cell viability of 95 ± 2%. In the process, the cultured chondrocytes can be clearly visualized microscopically. As a whole, the proposed cell culture system has paved an alternative route to carry out real-time microscopic observation of biological cells in a simple, user-friendly, and low cost manner. PMID:22164082

  7. Assessment of Long-Term Effects of Nanoparticles in a Microcarrier Cell Culture System

    PubMed Central

    Mrakovcic, Maria; Absenger, Markus; Riedl, Regina; Smole, Claudia; Roblegg, Eva; Fröhlich, Leopold F.; Fröhlich, Eleonore

    2013-01-01

    Nano-sized materials could find multiple applications in medical diagnosis and therapy. One main concern is that engineered nanoparticles, similar to combustion-derived nanoparticles, may cause adverse effects on human health by accumulation of entire particles or their degradation products. Chronic cytotoxicity must therefore be evaluated. In order to perform chronic cytotoxicity testing of plain polystyrene nanoparticles on the endothelial cell line EAhy 926, we established a microcarrier cell culture system for anchorage-dependent cells (BioLevitatorTM). Cells were cultured for four weeks and exposed to doses, which were not cytotoxic upon 24 hours of exposure. For comparison, these particles were also studied in regularly sub-cultured cells, a method that has traditionally been used to assess chronic cellular effects. Culturing on basal membrane coated microcarriers produced very high cell densities. Fluorescent particles were mainly localized in the lysosomes of the exposed cells. After four weeks of exposure, the number of cells exposed to 20 nm polystyrene particles decreased by 60% as compared to untreated controls. When tested in sub-cultured cells, the same particles decreased cell numbers to 80% of the untreated controls. Dose-dependent decreases in cell numbers were also noted after exposure of microcarrier cultured cells to 50 nm short multi-walled carbon nanotubes. Our findings support that necrosis, but not apoptosis, contributed to cell death of the exposed cells in the microcarrier culture system. In conclusion, the established microcarrier model appears to be more sensitive for the identification of cellular effects upon prolonged and repeated exposure to nanoparticles than traditional sub-culturing. PMID:23457616

  8. Cytoskeletal properties and endogenous degradation of glial fibrillary acidic protein and vimentin in cultured human glioma cells

    Microsoft Academic Search

    A. Paetau; I. Virtanen

    1986-01-01

    The cytoskeletal properties and endogenous degradation of intermediate filaments in cultured human glioma cells (U-251MG) were studied using monoclonal antibodies in immunohistochemical and immunochemical methods.

  9. Long-Term Culture of Human Bone Marrow Cells

    Microsoft Academic Search

    Suzanne Gartner; Henry S. Kaplan

    1980-01-01

    A method has been described for the long-term culture of human bone marrow cells in liquid medium. Hematopoiesis, as measured by the production of granulocytic-macrophage progenitor cells (CFUc), continued for at least 20 weeks and was dependent upon the presence of a marrow-derived adherent layer of cells. As in the case of murine marrow liquid cultures, the adherent layer consisted

  10. Albumin and mammalian cell culture: implications for biotechnology applications

    Microsoft Academic Search

    Geoffrey L. Francis

    2010-01-01

    Albumin has a long historical involvement in design of media for the successful culture of mammalian cells, in both the research\\u000a and commercial fields. The potential application of albumins, bovine or human serum albumin, for cell culture is a by-product\\u000a of the physico-chemical, biochemical and cell-specific properties of the molecule. In this review an analysis of these features\\u000a of albumin

  11. Serum-free culture of fractionated bovine bronchial epithelial cells

    Microsoft Academic Search

    Joe D. Beckmann; Hajime Takizawa; Debra Romberger; Mary Illig; Lorene Claassen; Kathleen Rickard; Stephen I. Rennard

    1992-01-01

    Summary  Procedures for the serum-free culture of a density fractionated population of bovine bronchial epithelial cells have been\\u000a established. Epithelial cells dispersed by protease digestion were fractionated by density equilibrium centrifugation, followed\\u000a by plating of the small basal-like population on type I collagen-coated culture dishes. Two or three passages of 1:4 split\\u000a enriched for a population of actively dividing cells, which

  12. Toxic Concentrations of Exogenously Supplied Methylglyoxal in Hybridoma Cell Culture

    Microsoft Academic Search

    Benjamin M. Roy; Tiffany D. Rau; R. Robert Balcarcel

    2004-01-01

    Concentrations at which methylglyoxal, a by-product of cellular metabolism, can be toxic to hybridoma cell cultures were determined\\u000a using exogenously supplied doses. Trypan blue cell counts of 6-well cultures incubated for 24 h with various methylglyoxal\\u000a concentrations revealed inhibition of cell growth at 300 ?M and higher, with a median inhibitory concentration of 490±20 ?M. The primary mode of death was apoptosis, as

  13. Production of Alkaloids in Plant Cell and Tissue Cultures

    Microsoft Academic Search

    Dominique Laurain-Mattar

    A low or no productivity of alkaloids in plant cell cultures can be explained by an insufficient level of cell differentiation.\\u000a The first strategy described in this chapter for improving isoquinoline alkaloid accumulation is organogenesis and somatic\\u000a embryogenesis induced by the addition of exogenous growth regulators in Papaver somniferum and Leucojum aestivum cell cultures. The second strategy described is the

  14. Primary culture of proximal tubular cells from normal rat kidney as an in vitro model to study mechanisms of nephrotoxicity. Toxicity of nephrotoxicants at low concentrations during prolonged exposure.

    PubMed

    Boogaard, P J; Zoeteweij, J P; van Berkel, T J; van't Noordende, J M; Mulder, G J; Nagelkerke, J F

    1990-04-15

    The aim of this study was to set up an in vitro system to study nephrotoxicity of xenobiotics which allows exposure at low concentrations for long periods (1-5 days). A very pure preparation of isolated proximal tubular cells (PTC) from rat kidney (Boogaard et al., Toxicol Appl Pharmacol 101: 135-143, 1989) was brought into primary culture. Cells grew to confluence in 3 days and could be maintained up to 8 days in a modification of Dulbecco's modified Eagle's medium Ham F12 nutrient mixture supplemented with fetal calf serum. Fibroblast growth was completely suppressed by replacement of L-valine by D-valine and of L-arginine by L-ornithine. Polarity was retained: in cells grown on filters organic anions were transported at the basolateral membrane while D-glucose transport was located at the apical membrane. Inhibition of the latter was used to assess the functional integrity of the cells after exposure to nephrotoxins. The newly grown cells expressed gamma-glutamyltranspeptidase activity since incubation with the glutathione-conjugate of 1,1-dichloro-2,2-difluoroethylene (DCDFE) induced cytotoxicity. Both beta-lyase and acylase activities were expressed because the cysteine-S-conjugate and the corresponding mercapturate of DCDFE showed cytotoxicity. Cultured cells showed toxicity on prolonged exposure to very low concentrations of gentamicin, cephaloridine, cisplatin and the cysteine-S-conjugate of chlorotrifluoroethylene. The lowest concentrations at which toxicity can be observed are 1-3 orders of magnitude lower in primary cultures than in freshly isolated PTC in suspension. This indicates that this cell model is suitable to investigate mechanisms of nephrotoxicity in vitro, at prolonged exposure to the low concentrations that are relevant in vivo levels. PMID:2322315

  15. Basal and thyroliberin-stimulated prolactin synthesis in single-cell cultures and in populations of rat pituitary cells.

    PubMed Central

    Gautvik, K M; Fossum, S

    1976-01-01

    1. Newly synthesized prolactin was obtained from cultures of rat pituitary tumour cells (GH4C1 cells) after incubation with [35S]methionine. 2. Radioactive synthesized and secreted prolactin was quantified by an immunoprecipitation method by using disc-gel electrophoresis of the dissolved immunoprecipitate in the presence of sodium dodecyl sulphate. By using a microanalytical modification, hormone synthesis and secretion could also be studied in single-cell cultures. This technique was combined with a cytoimmunofluorescence method in which rhodamine-conjugated antibodies were used for studying intracellular prolactin. 3. The presence of radioactive synthesized and secreted prolactin was demonstrated in nine out of 13 single-cell cultures. Cell cultures containing 10 cells or more and clonal populations originating from one cell always secreted radioactive prolactin. 4. Thyroliberin treatment (2 muM) for 24h increased the extracellular accumulation of radioactive prolactin in five out of seven single-cell cultures and always in populations of cells. 5. The number of cells showing prolactin specific fluorescence increased from 20 to 50% and the intensity of this fluorescence became greater after thyroliberin treatment. 6. Studies of [35S]prolactin secretion from single cells and immunochemical detection of intracellular prolactin showed that some cells in an unsynchronized population did not secret radioactive prolactin or show prolactin specific fluorescence. 7. The quantitative effect of thyroliberin as studied in single-cell cultures suggested that the main if not the only effect was to increase prolactin synthesis in cells already producing hormone. Images PLATE 1 PMID:822844

  16. Indoxyl sulfate promotes apoptosis in cultured osteoblast cells

    PubMed Central

    2013-01-01

    Background Indoxyl sulfate (IS), an organic anion uremic toxin, promotes the progression of renal dysfunction. Some studies have suggested that IS inhibits osteoclast differentiation and suppresses parathyroid hormone (PTH)-stimulated intracellular cAMP production, decreases PTH receptor expression, and induces oxidative stress in primary mouse calvaria osteoblast cell culture. However, the direct effects of IS on osteoblast apoptosis have not been fully evaluated. Hence, we investigated whether IS acts as a bone toxin by studying whether IS induces apoptosis and inhibits differentiation in the cultured osteoblast cell line MC3T3-E1. Methods We assessed the direct effect of IS on osteoblast differentiation and apoptosis in the MC3T3-E1 cell line. We examined caspase-3/7 activity, apoptosis-related proteins, free radical production, alkaline phosphatase activity, and mRNA expression of type 1 collagen and osteonectin. Furthermore, we investigated the uptake of IS via organic anion transport (OAT). Results We found that IS increased caspase activity and induced apoptosis. Production of free radicals increased depending on the concentration of IS. Furthermore, IS inhibited the expression of mRNA type 1 collagen and osteonectin and alkaline phosphatase activity. The expression of OAT, which is known to mediate the cellular uptake of IS, was detected in in the MC3T3-E1 cell line. The inhibition of OAT improved cell viability and suppressed the production of reactive oxygen species. These results suggest that IS is transported in MC3T3-E1 cells via OAT, which causes oxidative stress to inhibit osteoblast differentiation. Conclusions IS acts as a bone toxin by inhibiting osteoblast differentiation and inducing apoptosis. PMID:24289746

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

    PubMed

    Cheng, Boran; He, Zhaobo; Zhao, Libo; Fang, Yuan; Chen, Yuanyuan; He, Rongxiang; Chen, Fangfang; Song, Haibin; Deng, Yuliang; Zhao, Xingzhong; Xiong, Bin

    2014-01-01

    Circulating tumor cells (CTCs) in the blood which have detached from both the primary tumor and any metastases may be considered as a "liquid biopsy" and are expected to replace tumor biopsies in the monitoring of treatment response and determining patient prognosis. Here, we introduce a facile and efficient CTC detection material made of hydroxyapatite/chitosan (HA/CTS), which is beneficial because of its transparency and excellent biological compatibility. Atomic force microscopy images show that the roughness of the HA/CTS nanofilm (HA/CTSNF) substrates can be controlled by changing the HA:CTS ratio. Enhanced local topographic interactions between nano-components on cancer cell membranes, and the antibody coated nanostructured substrate lead to improved CTC capture and separation. This remarkable nanostructured substrate has the potential for CTC culture in situ and merits further analysis. CTCs captured from artificial blood samples were observed in culture on HA/CTSNF substrates over a period of 14 days by using conventional staining methods (hematoxylin eosin and Wright's stain). We conclude that these substrates are multifunctional materials capable of isolating and culturing CTCs for subsequent studies. PMID:24904216

  18. Adaptation to culture of human embryonic stem cells and oncogenesis in vivo

    Microsoft Academic Search

    Duncan E C Baker; Neil J Harrison; Edna Maltby; Kath Smith; Harry D Moore; Pamela J Shaw; Paul R Heath; Hazel Holden; Peter W Andrews

    2007-01-01

    The application of human embryonic stem cells (HESCs) to provide differentiated cells for regenerative medicine will require the continuous maintenance of the undifferentiated stem cells for long periods in culture. However, chromosomal stability during extended passaging cannot be guaranteed, as recent cytogenetic studies of HESCs have shown karyotypic aberrations. The observed karyotypic aberrations probably reflect the progressive adaptation of self-renewing

  19. Degradation of sarcomeric and cytoskeletal proteins in cultured skeletal muscle cells

    Microsoft Academic Search

    Juntipa Purintrapiban; Mei-chuan Wang; Neil E. Forsberg

    2003-01-01

    The goal of this research was to evaluate the roles of calpains and their interactions with the proteasome and the lysosome in degradation of individual sarcomeric and cytoskeletal proteins in cultured muscle cells. Rat L8-CID muscle cells, in which we expressed a transgene calpain inhibitor (CID), were used in the study. L8-CID cells were grown as myotubes after which the

  20. Role of differential physical properties in emergent behavior of 3D cell co-cultures

    NASA Astrophysics Data System (ADS)

    Kolbman, Dan; Das, Moumita

    2015-03-01

    The biophysics of binary cell populations is of great interest in many biological processes, whether the formation of embryos or the initiation of tumors. During these processes, cells are surrounded by other cell types with different physical properties, often with important consequences. For example, recent experiments on a co-culture of breast cancer cells and healthy breast epithelial cells suggest that the mechanical mismatch between the two cell types may contribute to enhanced migration of the cancer cells. Here we explore how the differential physical properties of different cell types may influence cell-cell interaction, aggregation, and migration. To this end, we study a proof of concept model- a three-dimensional binary system of interacting, active, and deformable particles with different physical properties such as elastic stiffness, contractility, and particle-particle adhesion, using Langevin Dynamics simulations. Our results may provide insights into emergent behavior such as segregation and differential migration in cell co-cultures in three dimensions.

  1. Effects of Porphyromonas gingivalis 2561 extracts on osteogenic and osteoclastic cell function in co-culture.

    PubMed

    Loomer, P M; Ellen, R P; Tenenbaum, H C

    1998-11-01

    This study was undertaken to determine the direct effects of extracts derived from Porphyromonas gingivalis on bone formation and mineral resorption in an osteogenic/osteoclastic cell in vitro co-culture model. Osteogenic bone marrow derived stromal cells were isolated from 18-day old embryonic chickens, while osteoclastic cells were isolated from laying white Leghorn hens on calcium deficient diets. Osteoclastic cells (5 x 10(5)) were seeded onto mineral thin films and suspended above osteogenic cells (1 x 10(4)) already plated on the bottoms of tissue culture plate wells. Sonicated P. gingivalis 2561 extracts were prepared from whole bacterial cells and added in varying proportions (0 to 2 microg/ml) to the co-culture growth medium. These co-cultures, and appropriate mono-culture controls, were incubated for a further 4 days. Parameters of bone forming cell activity including alkaline phosphatase activity, calcium and inorganic phosphate accumulation were performed on the osteogenic cells. Mineral substrate resorption by osteoclastic cells was assessed morphometrically. In their respective mono-cultures, the addition of P. gingivalis sonicate to the culture medium had no effect on osteoclastic mineral resorption, but significantly inhibited osteogenesis (up to 45%; P <0.05). In co-cultures, however, the sonicate induced significant increases in mineral resorption (up to 70%; P <0.05), whereas bone forming cell activity was still inhibited, although to a significantly lesser extent than in mono-cultures (up to 25%; P <0.05). These results suggest that P. gingivalis sonicate induced up-regulation of mineral resorption may be mediated via osteogenic cells. PMID:9848536

  2. The Influence of Micronutrients in Cell Culture: A Reflection on Viability and Genomic Stability

    PubMed Central

    Arigony, Ana Lúcia Vargas; de Oliveira, Iuri Marques; Bordin, Diana Lilian; Prá, Daniel; Pêgas Henriques, João Antonio

    2013-01-01

    Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic the in vivo environment, providing in vitro models used to infer cells' responses to different stimuli. This review summarizes and discusses studies of cell-culture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previous in vitro experiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5–10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of cultured cells. Further studies better evaluating micronutrients' roles at a molecular level and influence on the genomic stability of cells are still needed. PMID:23781504

  3. RNAi Microarray Analysis in Cultured Mammalian Cells

    Microsoft Academic Search

    Spyro Mousses; Natasha J. Caplen; Robert Cornelison; Don Weaver; Mark Basik; Sampsa Hautaniemi; Abdel G. Elkahloun; Roberto A. Lotufo; Ashish Choudary; Edward R. Dougherty; Ed Suh; Olli Kallioniemi

    2003-01-01

    RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) is a powerful new tool for analyzing gene knockdown phenotypes in living mammalian cells. To facilitate large-scale, high-throughput functional genomics studies using RNAi, we have developed a microarray-based technology for highly parallel analysis. Specifically, siRNAs in a transfection matrix were first arrayed on glass slides, overlaid with a monolayer of adherent

  4. On the history of hepatitis C virus cell culture systems.

    PubMed

    Lohmann, Volker; Bartenschlager, Ralf

    2014-03-13

    HCV infections are a major global health burden. After the identification of the virus in 1989, insights into viral replication and drug development have long been hampered by the lack of efficient cell culture models. Their establishment was an important prerequisite for the development of selective antivirals. This review describes the initial difficulties to achieve HCV replication in cell culture, finally leading to the establishment of subgenomic replicons and the infectious virus model (HCVcc). The review further summarizes the current state of HCV cell culture systems with respect to available virus isolates, engineered genomes, and cell types allowing efficient HCV propagation. Finally, we comment on how these cell culture models contributed to the development of directly acting antivirals. PMID:24164647

  5. Cryptosporidium parvum DNA replication in cell-free culture

    PubMed Central

    Zhang, L.; Sheoran, A. S.; Widmer, G.

    2009-01-01

    The lack of robust methods for culturing Cryptosporidium parasites remains a major challenge and is hampering efforts to screen for anti-cryptosporidial drugs. In existing culture methods, monolayers of mammalian epithelial cells are inoculated with oocysts. The system supports an initial phase of asexual proliferation of the parasite. For reasons that are not clear, development rapidly declines within 2 to 3 days. The unexpected report of C. parvum culture in the absence of host cells, and the failure of others to reproduce the method, prompted us to apply quantitative PCR to measure changes C. parvum DNA levels in cell-free cultures, and parasite-specific antibodies to identify different life cycle stages. Based on this approach, which has not been applied previously to analyze C. parvum growth in cell-free culture, we found that the concentration of C. parvum DNA increased by about 5-fold over 5 days of culture. Immuno-labelling of cultured organisms revealed morphologically distinct stages, only some of which reacted with Cryptosporidium-specific monoclonal antibodies. These observations are indicative of a modest proliferation of C. parvum in cell-free culture. PMID:19463037

  6. [Research progress in medicinal plant cell suspension culture].

    PubMed

    Wang, Juan; Gao, Wen-Yuan; Yin, Shuang-Shuang; Liu, Hui; Wei, Chang-Long

    2012-12-01

    China consumes and exports traditional Chinese medicinal resources the most in the world. However, we cannot anchor our hope on field production of traditional Chinese medicinal materials and their active ingredients, due to limited land resources. Therefore, the development of biotechnology is of great importance for China to solve the problem of traditional Chinese medicinal resources. Plant cell culture is an important approach for the sustainable development of precious medicinal resources. This essary summarizes the optimization of conditions for medicinal plant cell culture, the regulation of secondary metabolic pathways and cell bioreactor culture, and realizes that the authentic commercial production of more medicinal plants requires efforts from all aspects. PMID:23630994

  7. Proliferation and differentiation of mesenchymal stem cells in a three- dimensional culture system

    NASA Astrophysics Data System (ADS)

    Sun, S.; Hu, J.; Long, M.; Tao, Z.

    Mesenchymal stem cells (MSCs) have multi-differentiation potential and retain the capacity to proliferate and differentiate in vitro, which in turn holds the promise of being able to repair or replace damaged cells or tissues. Since MSCs are rare in amount in vivo, abundant cells usually need be obtained in time in clinic. T herefore, proliferation and differentiation of MSCs in vitro are necessary and important for future applications. Most current studies o MSCs are focused on the cellular andn molecular biology using a two-dimension (2-D) static culture system at unit gravity. The gravity-induced 2D culture of MSCs could potentially not reflect cell-cell- contacts important for proliferation and differentiation of MSCs in vivo. Here we developed a method to proliferate MSCs by using the rotating three-dimensional (3- D) culture system, which can provide low shear, 3-D environment with simulated microgravity. MSCs from human bone marrow were prepared on microcarrier beads and then were seeded in the 3-D culture system. Various rotation conditions were tested to screen out the most suitable one for proliferation of MSCs. 2-D cultures were prepared in routine cell culture dishes as a control. All cultures were uniformly inoculated and medium exchanged at standard intervals. Results were compared with microscopic and immunochemistrical techniques. The differentiation capacity of proliferated MSCs were also tested through induced differentiation experiments. It is found that simulated microgravity and three-dimensional culture condition is an active factor for proliferation of MSCs.

  8. Choosing an Appropriate Modelling Framework for Analysing Multispecies Co-culture Cell Biology Experiments.

    PubMed

    Markham, Deborah C; Simpson, Matthew J; Baker, Ruth E

    2015-04-01

    In vitro cell biology assays play a crucial role in informing our understanding of the migratory, proliferative and invasive properties of many cell types in different biological contexts. While mono-culture assays involve the study of a population of cells composed of a single cell type, co-culture assays study a population of cells composed of multiple cell types (or subpopulations of cells). Such co-culture assays can provide more realistic insights into many biological processes including tissue repair, tissue regeneration and malignant spreading. Typically, system parameters, such as motility and proliferation rates, are estimated by calibrating a mathematical or computational model to the observed experimental data. However, parameter estimates can be highly sensitive to the choice of model and modelling framework. This observation motivates us to consider the fundamental question of how we can best choose a model to facilitate accurate parameter estimation for a particular assay. In this work we describe three mathematical models of mono-culture and co-culture assays that include different levels of spatial detail. We study various spatial summary statistics to explore if they can be used to distinguish between the suitability of each model over a range of parameter space. Our results for mono-culture experiments are promising, in that we suggest two spatial statistics that can be used to direct model choice. However, co-culture experiments are far more challenging: we show that these same spatial statistics which provide useful insight into mono-culture systems are insufficient for co-culture systems. Therefore, we conclude that great care ought to be exercised when estimating the parameters of co-culture assays. PMID:25549623

  9. Growth, ageing and death of a photoautotrophic plant cell culture.

    PubMed

    Peters, W; Ritter, J; Tiller, H; Valdes, O; Renner, U; Fountain, M; Beck, E

    2000-02-01

    Batch cultures of photoautotrophic cell suspensions of Chenopodium rubrum L., growing in an inorganic medium on CO2 under a daily balanced light-dark regime of 16: 8 h could be maintained for approximately 100 d without subcultivation. The long-lived cultures showed an initial cell division phase of 4 weeks, followed by a stationary phase of another 4 weeks, after which ageing and progressive cell death reduced the number of living cells and the cultures usually expired after another 3-4 weeks. These developmental phases of the cell culture were characterised with respect to photosynthetic performance, dark respiration, content of phytohormones and capacity of cell division. Cell division of the majority of the cells finished in the G1- or G0-phase of the cell cycle, caused by a pronounced decline in the endogenous levels of auxin and cytokinins. Supply of these growth factors to resting cells resulted in resumption of cytokinesis, at least by some of the cells. However, responsiveness to the phytohormones declined during the stationary phase, and subcultivation was no longer possible beyond day 60 when the phases of ageing and death commenced. Ageing was characterised by a further decline in the photosynthetic capacity of the cells, by a climacteric enhancement of dark respiration, but also by a slight increase in the level of IAA and cytokinins concomitant with a decrease in ethylene. Similarities and differences between the development of batch-cultured photoautotrophic cells of C. rubrum and that of a leaf are discussed with respect to using the cell culture as a model for a leaf. PMID:10750906

  10. Biocompatibility of alloys used in orthodontics evaluated by cell culture tests.

    PubMed

    Locci, P; Marinucci, L; Lilli, C; Belcastro, S; Staffolani, N; Bellocchio, S; Damiani, F; Becchetti, E

    2000-09-15

    The cytotoxicity of the most common alloys used in orthodontic appliances was determined by cell culture testing. Human gingival fibroblasts were cultured on 304 and 316 stainless steel, on brazing alloy composed of palladium (Pd), copper (Cu), and silver (Ag), and on plastic substrate (control). Studies were carried out with SEM and radiolabeled precursor incorporation. Cells were cultured in MEM without serum but with the addition of (3)H-thymidine to evaluate cell proliferation and (3)H-glucosamine to evaluate glycosaminoglycan (GAG) synthesis and secretion in the culture medium. Moreover, gingival fibroblasts were cultured in the presence of some metal ions generally released by orthodontic appliances to evaluate the cytotoxic effects of single ions. Morphologic observations with SEM and radiolabeled incorporation studies showed that 304 and 316 stainless steel were more biocompatible than the brazing alloy. Among the metal ions tested, Ag and Pd, constituents of the brazing alloy, showed the highest cytotoxicity. PMID:10880103

  11. A novel method for preserving cultured limbal epithelial cells

    PubMed Central

    Utheim, Tor Paaske; Raeder, Sten; Utheim, Øygunn Aass; Cai, Yiqing; Roald, Borghild; Drolsum, Liv; Lyberg, Torstein; Nicolaissen, Bjørn

    2007-01-01

    Aim To investigate organ culture preservation of cultured limbal epithelial cells in order to enhance the availability of tissue?engineered epithelia that are used to treat patients with limbal stem cell deficiency. Methods Limbal epithelial cells were cultured for 3?weeks on intact amniotic membrane fastened to a polyester membrane carrier. The cultured epithelia were stored for 1?week at 23°C in organ culture medium. The preserved epithelia were then examined using a colorimetric cell viability assay, light microscopy and immunohistochemistry. Results The viability of the preserved epithelia was 84% (20%), and no statistically significant difference was found compared with non?preserved epithelia. In general, the cell borders were maintained, the nuclei showed no sign of degeneration, and the original layered structure was preserved. Mild intercellular oedema was occasionally observed. Expression of p63, K19 and vimentin was maintained. Conclusions Cultured limbal epithelial cells can be preserved in organ culture medium for 1?week at room temperature, while maintaining the original layered structure and undifferentiated phenotype. PMID:17124242

  12. Capillary Gas Chromatography of Hexadecylphosphocholine in Caco-2T Cells and Cell Culture Media

    Microsoft Academic Search

    W. F. A. Steelant; E. A. Bruyneel; M. M. Mareel; E. G. Vandeneeckhout

    1995-01-01

    The gas-chromatographic determination of hexadecylphosphocholine (HePC), an experimental antitumor agent of the alkyllysophospholipid group, in Caco-2T cell culture and cell culture media is described. The Caco-2T cells were treated with HePC at a concentration of 40 ?g\\/ml (9.8 ?M) and the uptake of the drug into the cells (calculated per milligram protein) was measured after 48 h culture (37°C, 10%

  13. Inhibition of lymphokine-activated killer cell generation by cultured tumor cell lines in vitro

    Microsoft Academic Search

    Pierre J. Guillou; Peter C. Sedman; Carol W. Ramsden

    1989-01-01

    The co-culture of human peripheral blood mononuclear cells (PBMC) with high concentrations of interleukin 2 normally generates lymphokine-activated killer (LAK) cells capable of indiscriminate lysis of tumor targets. However, the addition of certain cell-line-derived tumor cells to the LAK generation cultures within the first 48 h of culture initiation resulted in the suppression of the LAK cytotoxicity measured after 3–4

  14. Expansion of Endothelial Progenitor Cells in High Density Dot Culture of Rat Bone Marrow Cells

    PubMed Central

    Wang, Ling; Kretlow, James D.; Zhou, Guangdong; Cao, Yilin; Liu, Wei; Zhang, Wen Jie

    2014-01-01

    In vitro expansion of endothelial progenitor cells (EPCs) remains a challenge in stem cell research and its application. We hypothesize that high density culture is able to expand EPCs from bone marrow by mimicking cell-cell interactions of the bone marrow niche. To test the hypothesis, rat bone marrow cells were either cultured in high density (2×105 cells/cm2) by seeding total 9×105 cells into six high density dots or cultured in regular density (1.6×104 cells/cm2) with the same total number of cells. Flow cytometric analyses of the cells cultured for 15 days showed that high density cells exhibited smaller cell size and higher levels of marker expression related to EPCs when compared to regular density cultured cells. Functionally, these cells exhibited strong angiogenic potentials with better tubal formation in vitro and potent rescue of mouse ischemic limbs in vivo with their integration into neo-capillary structure. Global gene chip and ELISA analyses revealed up-regulated gene expression of adhesion molecules and enhanced protein release of pro-angiogenic growth factors in high density cultured cells. In summary, high density cell culture promotes expansion of bone marrow contained EPCs that are able to enhance tissue angiogenesis via paracrine growth factors and direct differentiation into endothelial cells. PMID:25254487

  15. Expansion of endothelial progenitor cells in high density dot culture of rat bone marrow cells.

    PubMed

    Lu, Yang; Gong, Yiyi; Lian, Jie; Wang, Ling; Kretlow, James D; Zhou, Guangdong; Cao, Yilin; Liu, Wei; Zhang, Wen Jie

    2014-01-01

    In vitro expansion of endothelial progenitor cells (EPCs) remains a challenge in stem cell research and its application. We hypothesize that high density culture is able to expand EPCs from bone marrow by mimicking cell-cell interactions of the bone marrow niche. To test the hypothesis, rat bone marrow cells were either cultured in high density (2 × 10(5) cells/cm(2)) by seeding total 9 × 10(5) cells into six high density dots or cultured in regular density (1.6 × 10(4) cells/cm(2)) with the same total number of cells. Flow cytometric analyses of the cells cultured for 15 days showed that high density cells exhibited smaller cell size and higher levels of marker expression related to EPCs when compared to regular density cultured cells. Functionally, these cells exhibited strong angiogenic potentials with better tubal formation in vitro and potent rescue of mouse ischemic limbs in vivo with their integration into neo-capillary structure. Global gene chip and ELISA analyses revealed up-regulated gene expression of adhesion molecules and enhanced protein release of pro-angiogenic growth factors in high density cultured cells. In summary, high density cell culture promotes expansion of bone marrow contained EPCs that are able to enhance tissue angiogenesis via paracrine growth factors and direct differentiation into endothelial cells. PMID:25254487

  16. Closing the Phenotypic Gap between Transformed Neuronal Cell Lines in Culture and Untransformed Neurons

    PubMed Central

    Myers, Tereance A.; Nickerson, Cheryl A.; Kaushal, Deepak; Ott, C. Mark; Bentrup, Kerstin Höner zu; Ramamurthy, Rajee; Nelman-Gonzalez, Mayra; Pierson, Duane L.; Philipp, Mario T.

    2008-01-01

    Studies of neuronal dysfunction in the central nervous system (CNS) are frequently limited by the failure of primary neurons to propagate in vitro. Neuronal cell lines can be substituted for primary cells but they often misrepresent normal conditions. We hypothesized that a 3-dimensional (3-D) cell culture system would drive the phenotype of transformed neurons closer to that of untransformed cells, as has been demonstrated in non-neuronal cell lines. In our studies comparing 3-D versus 2-dimensional (2-D) culture, neuronal SH-SY5Y (SY) cells underwent distinct morphological changes combined with a significant drop in their rate of cell division. Expression of the proto-oncogene N-myc and the RNA binding protein HuD was decreased in 3-D culture as compared to standard 2-D conditions. We observed a decline in the anti-apoptotic protein Bcl-2 in 3-D culture, coupled with increased expression of the pro-apoptotic proteins Bax and Bak. Moreover, thapsigargin (TG)-induced apoptosis was enhanced in the 3-D cells. Microarray analysis demonstrated significantly differing mRNA levels for over 700 genes in the cells of the two culture types, and indicated that alterations in the G1/S cell-cycle progression contributed to the diminished doubling rate in the 3-D-cultured SY cells. These results demonstrate that a 3-D culture approach narrows the phenotypic gap between neuronal cell lines and primary neurons. The resulting cells may readily be used for in vitro research of neuronal pathogenesis. PMID:18672002

  17. Cultured Porcine Coronary Artery Smooth Muscle Cells A New Model With Advanced Differentiation

    Microsoft Academic Search

    Thomas Christen; Marie-Luce Bochaton-Piallat; Pascal Neuville; Sander Rensen; Mireille Redard; Guillaume van Eys; Giulio Gabbiani

    2010-01-01

    Arterial intimal thickening after endothelial injury induced in rodents has proven to be a relatively unreliable model of restenosis for testing clinically useful compounds. The same has been found for cultured rat or rabbit vascular smooth muscle cells (SMCs). To test alternative possibilities, we have studied several differentiation features of porcine coronary artery SMCs, cultured up to the 5th passage

  18. Nutrient modifications for improved growth of Brassica nigra cell suspension cultures

    Microsoft Academic Search

    Stephen J. Molnar

    1988-01-01

    Cell pellet yield of two Brassica nigra suspension cultures was stimulated by amino acid supplements in the growth medium. This could confound the interpretation of amino acid feeding studies involved in characterizing amino acid metabolism mutants. The nutritional requirements of one of the Brassica nigra suspension cultures growing in modified Murashige & Skoog medium were therefore reviewed. Sucrose at 2%

  19. Effect of supplementing terpenoid biosynthetic precursors on the accumulation of bilobalide and ginkgolides in Ginkgo biloba cell cultures

    Microsoft Academic Search

    Seung-Mi Kang; Ji-Yun Min; Yong-Duck Kim; Dong-Jin Park; Ha-Na Jung; Chandrakant S. Karigar; Yeong-Lae Ha; Seon-Won Kim; Myung-Suk Choi

    2006-01-01

    The effect of precursor feeding on the production of bilobalide and ginkgolides was studied with suspension cell cultures of Ginkgo biloba. The precursors greatly influenced the productivity of bilobalide and ginkgolides. Precursor supplementation increased the accumulation of both bilobalide and ginkgolides, and with positive effect on cell growth. The GA accumulation by cell cultures was influenced by precursors upstream in

  20. Composition of the von Willebrand factor storage organelle (Weibel- Palade body) isolated from cultured human umbilical vein endothelial cells

    Microsoft Academic Search

    B. M. Ewenstein; M. J. Warhol; R. I. Handin; J. S. Pober

    1987-01-01

    von Willebrand factor (VWF) is a large, adhesive glycoprotein that is biosynthesized and secreted by cultured endothelial cells (EC). Although these cells constitutively release VWF, they also con- tain a storage pool of this protein that can be rapidly mobilized. In this study, a dense organelle fraction was isolated from cultured umbilical vein endothelial cells by ceturifugation on a self-generated

  1. Transferrin in cultured human term cytotrophoblast cells: synthesis and heterogeneity.

    PubMed

    Verrijt, C E; Kroos, M J; Verhoeven, A J; van Eijk, H G; van Dijk, J P

    1997-08-01

    Transferrin (Tf) mRNA was recently demonstrated in rat and mouse placental tissue. Rat placental cells were shown to secrete transferrin. The cell type with which Tf mRNA was associated was not investigated. We therefore studied the ability of immunopurified human term cytotrophoblast cells in culture to synthesize Tf, by means of pulse-label experiments with 35S-methionine and report that these cells do synthesize Tf. Tf mRNA was demonstrated in the cell lysates by means of RT-PCR. Tf isolated from cytotrophoblast and syncytiotrophoblast cells was shown to be different from both maternal and fetal serum Tf with respect to the distribution of isoforms as demonstrated by means of iso-electric focusing. The iso-electric points were found at lower pH values (pH 5.0-5.4), compared to the iso-electric points of maternal and fetal serum Tf, suggesting a higher degree of sialylation and glycan chain complexity. PMID:9278269

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  3. Effect of Withaferin A on cells in tissue culture

    Microsoft Academic Search

    Batya Shohat

    1973-01-01

    The experiments were performed with embryonal chicken fibroblasts, HeLa cells and H. Ep. 2 (human larynx carcinoma) in hanging drop cultures. They confirm the results obtained with tumor bearing mice. Withaferin A is acting as a mitotic poison and arrests the tumor cells at metaphase. Its effect on cells may be compared to that of colchicine, alkaloids of vinca rosea

  4. Effects of heat stress and mechanical stretch on protein expression in cultured skeletal muscle cells

    Microsoft Academic Search

    K. Goto; R. Okuyama; H. Sugiyama; M. Honda; T. Kobayashi; K. Uehara; T. Akema; T. Sugiura; S. Yamada; Y. Ohira; T. Yoshioka

    2003-01-01

    Effects of heat stress, mechanical stretching or a combination of both on the expression of heat shock proteins (HSPs) and total protein level were studied in a culture system. Rat skeletal muscle cells (L6) were cultured on flexible-bottomed culture plates. They were subjected to one of the four following conditions: (1) 97 h incubation at 37 °C, (2) 1 h incubation at 41 °C

  5. Molecular study of the retrovirus-like transposable element 412, a 20-OH ecdysone responsive repetitive sequence in Drosophila cultured cells.

    PubMed Central

    Micard, D; Couderc, J L; Sobrier, M L; Giraud, G; Dastugue, B

    1988-01-01

    Used at a physiological concentration, the steroid hormone 20-hydroxyecdysone (20-OHE) induces, in Kc cultured Drosophila melanogaster cells, important and specific changes. Modifications occur at morphological and enzymatical levels. Variations in specific protein synthesis are observed. At the molecular level, 20-OHE particularly induces a decrease in expression of the mobile dispersed genetic element 412. This repeated element which belongs to the "copia-like" family is more widely represented in Kc cells (80 fold) compared to fly cells (25 fold). 412 transcripts are heterogeneous in size, essentially polyadenylated and restricted to the nucleus. A minimal concentration of 10(-8) M and a time treatment of 16 hours are necessary to obtain a strong decrease in 412 expression. The decrease is at least an effect on these sequences at the transcriptional level. Structural similarities between the 412 element and the proviral forms of vertebrate retroviruses are strengthened by the characterization of extrachromosomal circular DNA forms revealed by the 412 probe. Quantifying experiments have shown that the steady state level of such forms is not affected by the steroid treatment. Images PMID:2829128

  6. TRANSFORMATION OF SUGAR BEET CELL SUSPENSION CULTURES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A sugar beet transformation method was developed using particle bombardment of short-term suspension cultures of a breeding line FC607. Highly embryogenic suspension cultures derived from leaf callus were bombarded with the uidA (GUS) reporter gene under the control of either the osmotin or protein...

  7. Simple and Novel Three Dimensional Neuronal Cell Culture Using a Micro Mesh Scaffold

    PubMed Central

    Yoo, Sang Jin; Kim, Jongmin; Lee, Chang-Soo

    2011-01-01

    Conventional method of cell culture studies has been performed on two-dimensional substrates. Recently, three-dimensional (3D) cell culture platforms have been a subject of interest as cells in 3D has significant differences in cell differentiation and behavior. Here we report a novel approach of 3D cell culture using a nylon micro mesh (NMM) as a cell culture scaffold. NMM is commonly used in cell culture laboratory, which eliminates the requirement of special technicality for biological laboratories. Furthermore, it is made of a micro-meter thick nylon fibers, which was adequate to engineer in cellular scales. We demonstrate the feasibility of the NMM as a 3D scaffold using E18 rat hippocampal neurons. NMM could be coated with cell adhesive coatings (polylysine or polyelectrolyte) and neurons showed good viability. Cells were also encapsulated in an agarose hydrogel and cultured in 3D using NMM. In addition, the 3D pattern of NMM could be used as a guidance cue for neurite outgrowth. The flexible and elastic properties of NMMs made it easier to handle the scaffold and also readily applicable for large-scale tissue engineering applications. PMID:22110368

  8. A Novel Strain of Porcine Adenovirus Detected in Urinary Bladder Urothelial Cell Culture

    PubMed Central

    Jerman, Urška Dragin; Kolenc, Marko; Steyer, Andrej; Verani?, Peter; Prijatelj, Mateja Poljšak; Kreft, Mateja Erdani

    2014-01-01

    Contamination of cell cultures is the most common problem encountered in cell culture laboratories. Besides the secondary cell contaminations often occurring in the cell laboratories, the contaminations originating from donor animal or human tissue are equally as common, but usually harder to recognize and as such require special attention. The present study describes the detection of porcine adenovirus (PAdV), strain PAdV-SVN1 in cultures of normal porcine urothelial (NPU) cells isolated from urinary bladders of domestic pigs. NPU cell cultures were evaluated by light microscopy (LM), polymerase chain reaction (PCR), and additionally assessed by transmission electron microscopy (TEM). Characteristic ultrastructure of virions revealed the infection with adenovirus. The adenoviral contamination was further identified by the sequence analysis, which showed the highest similarity to recently described PAdV strain PAdV-WI. Additionally, the cell ultrastructural analysis confirmed the life-cycle characteristic for adenoviruses. To closely mimic the in vivo situation, the majority of research on in vitro models uses cell cultures isolated from human or animal tissue and their subsequent passages. Since the donor tissue could be a potential source of contamination, the microbiological screening of the excised tissue and harvested cell cultures is highly recommended. PMID:24960273

  9. Effect of amniotic fluid on the in vitro culture of human corneal endothelial cells.

    PubMed

    Feizi, Sepehr; Soheili, Zahra-Soheila; Bagheri, Abouzar; Balagholi, Sahar; Mohammadian, Azam; Rezaei-Kanavi, Mozhgan; Ahmadieh, Hamid; Samiei, Shahram; Negahban, Kambiz

    2014-05-01

    The present study was designed to evaluate the effects of human amniotic fluid (HAF) on the growth of human corneal endothelial cells (HCECs) and to establish an in vitro method for expanding HCECs. HCECs were cultured in DMEM-F12 supplemented with 20% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using either FBS- or HAF-containing media. Cell proliferation and cell death ELISA assays were performed to determine the effect of HAF on cell growth and viability. The identity of the cells cultured in 20% HAF was determined using immunocytochemistry (ICC) and real-time reverse transcription polymerase chain reaction (RT-PCR) techniques to evaluate the expression of factors that are characteristic of HCECs, including Ki-67, Vimentin, Na+/K+-ATPase and ZO-1. HCEC primary cultures were successfully established using 20% HAF-containing medium, and these cultures demonstrated rapid cell proliferation according to the cell proliferation and death ELISA assay results. The ICC and real time RT-PCR results indicated that there was a higher expression of Na+/K+-ATPase and ZO-1 in the 20% HAF cell cultures compared with the control (20% FBS) (P < 0.05). The 20% HAF-containing medium exhibited a greater stimulatory effect on HCEC growth and could represent a potential enriched supplement for HCEC regeneration studies. PMID:24726921

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

    Microsoft Academic Search

    Axel Blau; Tanja Neumann; Christiane Ziegler; Fabio Benfenati

    2009-01-01

    An imbalance in medium osmolarity is a determinant that affects cell culture longevity. Even in humidified incubators, evaporation\\u000a of water leads to a gradual increase in osmolarity over time. We present a simple replica-moulding strategy for producing\\u000a self-sealing lids adaptable to standard, small-size cell-culture vessels. They are made of polydimethylsiloxane (PDMS), a\\u000a flexible, transparent and biocompatible material, which is gas-permeable

  11. Preparation of Feeder plates for ES cell culture Gelatinize Tissue Culture Plates

    E-print Network

    Oliver, Douglas L.

    Preparation of Feeder plates for ES cell culture Gelatinize Tissue Culture Plates Gelatinize plates with 0.1% gelatin at room temperature for two hours. (150 µl/well of 96 well plate; 12 ml/10 cm; 4 ml/6cm. Plate cells in gelatinized plates (150 µl/well of 96 well plate; 12 ml/10 cm; 4 ml/6cm; 2 ml/well of 6

  12. Cadmium stimulates osteoclast-like multinucleated cell formation in mouse bone marrow cell cultures

    SciTech Connect

    Miyahara, Tatsuro; Takata, Masakazu; Miyata, Masaki; Nagai, Miyuki; Sugure, Akemi; Kozuka, Hiroshi; Kuze, Shougo (Toyama Medical and Pharmaceutical Univ. (Japan))

    1991-08-01

    Most of cadmium (Cd)-treated animals have been reported to show osteoporosis-like changes in bones. This suggests that Cd may promote bone loss by a direct action on bone. It was found that Cd stimulated prostaglandin E{sub 2}(PGE{sub 2}) production in the osteoblast-like cell, MC3T3-E1. Therefore, Cd stimulates bone resorption by increasing PGE{sub 2} production. Recently, several bone marrow cell culture systems have been developed for examining the formation of osteoclast-like multinucleated cells in vitro. As osteoblasts produce PGE{sub 2} by Cd-induced cyclooxygenase and may play an important role in osteoclast formation, the present study was undertaken to clarify the possibility that Cd might stimulate osteoclast formation in a mouse bone marrow culture system.

  13. Caffeine sensitization of cultured mammalian cells and human lymphocytes irradiated with gamma rays and fast neutrons: a study of relative biological effectiveness in relation to cellular repair

    SciTech Connect

    Hannan, M.A.; Gibson, D.P.

    1985-10-01

    The sensitizing effects of caffeine were studied in baby hamster kidney (BHK-21) cells and human lymphocytes following irradiation with gamma rays and fast neutrons. Caffeine sensitization occurred only when log-phase BHK cells and mitogen-stimulated lymphocytes were exposed to the two radiations. Noncycling (confluent) cells of BHK resulted in a shouldered survival curve following gamma irradiation while a biphasic curve was obtained with the log-phase cells. Survival in the case of lymphocytes was estimated by measurement of (TH)thymidine uptake. The relative biological effectiveness (RBE) of fast neutrons was found to be greater at survival levels corresponding to the resistant portions of the survival curves (shoulder or resistant tail). In both cell types, no reduction in RBE was observed when caffeine was present, because caffeine affected both gamma and neutron survival by the same proportion.

  14. A simple eccentric stirred tank mini-bioreactor: mixing characterization and mammalian cell culture experiments.

    PubMed

    Bulnes-Abundis, David; Carrillo-Cocom, Leydi M; Aráiz-Hernández, Diana; García-Ulloa, Alfonso; Granados-Pastor, Marisa; Sánchez-Arreola, Pamela B; Murugappan, Gayathree; Alvarez, Mario M

    2013-04-01

    In industrial practice, stirred tank bioreactors are the most common mammalian cell culture platform. However, research and screening protocols at the laboratory scale (i.e., 5-100 mL) rely primarily on Petri dishes, culture bottles, or Erlenmeyer flasks. There is a clear need for simple-easy to assemble, easy to use, easy to clean-cell culture mini-bioreactors for lab-scale and/or screening applications. Here, we study the mixing performance and culture adequacy of a 30 mL eccentric stirred tank mini-bioreactor. A detailed mixing characterization of the proposed bioreactor is presented. Laser induced fluorescence (LIF) experiments and computational fluid dynamics (CFD) computations are used to identify the operational conditions required for adequate mixing. Mammalian cell culture experiments were conducted with two different cell models. The specific growth rate and the maximum cell density of Chinese hamster ovary (CHO) cell cultures grown in the mini-bioreactor were comparable to those observed for 6-well culture plates, Erlenmeyer flasks, and 1 L fully instrumented bioreactors. Human hematopoietic stem cells were successfully expanded tenfold in suspension conditions using the eccentric mini-bioreactor system. Our results demonstrate good mixing performance and suggest the practicality and adequacy of the proposed mini-bioreactor. PMID:23124589

  15. The Multiparametric Effects of Hydrodynamic Environments on Stem Cell Culture

    PubMed Central

    Kinney, Melissa A.; Sargent, Carolyn Y.

    2011-01-01

    Stem cells possess the unique capacity to differentiate into many clinically relevant somatic cell types, making them a promising cell source for tissue engineering applications and regenerative medicine therapies. However, in order for the therapeutic promise of stem cells to be fully realized, scalable approaches to efficiently direct differentiation must be developed. Traditionally, suspension culture systems are employed for the scale-up manufacturing of biologics via bioprocessing systems that heavily rely upon various types of bioreactors. However, in contrast to conventional bench-scale static cultures, large-scale suspension cultures impart complex hydrodynamic forces on cells and aggregates due to fluid mixing conditions. Stem cells are exquisitely sensitive to environmental perturbations, thus motivating the need for a more systematic understanding of the effects of hydrodynamic environments on stem cell expansion and differentiation. This article discusses the interdependent relationships between stem cell aggregation, metabolism, and phenotype in the context of hydrodynamic culture environments. Ultimately, an improved understanding of the multifactorial response of stem cells to mixed culture conditions will enable the design of bioreactors and bioprocessing systems for scalable directed differentiation approaches. PMID:21491967

  16. Improvement of liver fibrosis by infusion of cultured cells derived from human bone marrow.

    PubMed

    Tanimoto, Haruko; Terai, Shuji; Taro, Takami; Murata, Yasuhiko; Fujisawa, Kouichi; Yamamoto, Naoki; Sakaida, Isao

    2013-12-01

    We develop "autologous bone marrow cell infusion (ABMi) therapy" for the treatment of human decompensated liver cirrhosis and confirm the efficacy and safety of this treatment in multicenter clinical studies. With the goal of further expanding the applications of ABMi, we first cultured human bone marrow cells and then determined whether a cell fraction found to be effective in improving liver fibrosis can be amplified. Cells harvested after two passages (P2 cells) consistently contained approximately 94% mesenchymal stem cells (MSCs); conversely, the cells harvested after only medium change (P0 cells) contained many macrophages. MSCs (2.8?×?10(8)) in P2 cells were harvested from 3.8 × 10(8) bone marrow-derived mononuclear cells after 22 days. DNA-chip analysis also showed during the culturing step that bone marrow-derived cells decreased with macrophage phenotype. The infused 5 × 10(5) P2 cells significantly improved liver fibrosis in the nonobese diabetic/severe combined immunodeficient (NOD-SCID) mouse carbon tetrachloride (CCl4) liver cirrhosis model and induced the expression of matrix metalloproteinase (MMP)-9 and suppressed expressions of alpha smooth muscle actin (?SMA), tumor necrosis factor alpha (TNF?) and transforming growth factor beta (TGF?) in the liver. Cultured human bone marrow-derived cells (P2 cells) significantly inhibited liver fibrosis. The increase of MMP-9 and suppressed activation of hepatic stellate cells (HSCs) through the regulation of humoral factors (TNF? and TGF?) contribute to the improvement of liver fibrosis by MSCs comprising about 94% of P2 cells. MSCs in cultured human bone marrow-derived mono-nuclear cells (BM-MNCs) proliferate sufficiently in cell therapy, so we believe our cultured bone marrow-derived cell therapy can lead to expanded clinical applications and enable outpatient therapy. PMID:24104560

  17. Developing Cultural Sensitivity Through Study Abroad

    Microsoft Academic Search

    Audra Johns; Cheryl W. Thompson

    2010-01-01

    Study abroad experiences, as a strategy to develop cultural sensitivity, are increasingly popular in nursing education. At a college where resources for developing study abroad were limited, establishing a partnership with an organization experienced in sending medical teams abroad was a means by which a successful study abroad was integrated into a community health nursing course. This collaborative relationship provided

  18. Transdetermination and transdifferentiation of neural retinal cells into lens in cell culture.

    PubMed

    Okada, T S; Yasuda, K; Kondoh, H; Nomura, K; Takagi, S; Okuyama, K

    1982-01-01

    Two aspects of transdifferentiation of avian neural retina (NR) cells into lens in cell culture were discussed. First, by means of the transfer experiments of NR cells pre-cultivated in spreading cultures (SpC) longer than 10 days into aggregation cultures (AgC), it was shown that NR cells are "transdetermined" into lens direction, before the phenotypic expression of lens in cells at such earlier stages of SpC. In the second part of this article, we showed that NR-cells which have already expressed some neuronal phenotypes can transdifferentiate into lens. This statement is based upon the results of chimeric cultures consisting of neuronal cell fraction separated from 10-day SpC of quail NR and of the epithelial cell fraction of SpC of chick NR. Lens cells formed in such chimeric cultures were mainly of quail origin. PMID:7111277

  19. Refsum's disease: characterization of the enzyme defect in cell culture

    PubMed Central

    Herndon, James H.; Steinberg, Daniel; Uhlendorf, B. William; Fales, Henry M.

    1969-01-01

    Refsum's disease (heredopathia atactica polyneuritiformis, HAP) is an inherited neurological disorder associated with storage of the branched-chain fatty acid, phytanic acid (3,7,11,15-tetramethylhexadecanoic acid). Cultured fibroblasts derived from skin biopsies of HAP patients did not contain elevated levels of phytanate, yet showed rates of phytanate-C-14C oxidation less than 3% of those seen in cells from control subjects. Cells of control subjects converted phytanate to ?-hydroxyphytanate, to pristanate (the [n-1] homologue of phytanate) and to 4,8,12-trimethyltridecanoate, compounds previously identified as intermediates on the major pathway for phytanate metabolism in animals, providing the first direct evidence that this same oxidative pathway is operative in human cells. None of these breakdown products could be found after incubation of phytanate with HAP cells. Labeled ?-hydroxyphytanate and labeled pristanate were oxidized at normal rates by HAP cells. Oxidation of the latter proceeded at normal rates both when added to the medium at very low tracer levels and at levels 100 times greater. Phytanate was incorporated into and released from lipid esters at normal rates by HAP cells. Elevated levels of free phytanate in the medium were no more toxic to HAP cells than to control cells over the 48- to 72-hr exposures involved in these studies, as evidenced by morphologic criteria and by ability to oxidize labeled palmitate. These findings are consistent with the hypothesis that the cells from HAP patients are deficient in a single enzyme involved in the ?-hydroxylation of phytanate, while the enzymes involved in later steps are present at normal or near-normal levels. PMID:4181593

  20. Anthraquinones from Ophiorrhiza pumila tissue and cell cultures

    Microsoft Academic Search

    Mariko Kitajima; Ute Fischer; Mio Nakamura; Mika Ohsawa; Masahiro Ueno; Hiromitsu Takayama; Matthias Unger; Joachim Stöckigt; Norio Aimi

    1998-01-01

    We have succeeded in initiating and establishing systems of tissue and cell cultures of Ophiorrhiza pumila. Examination of the constituents of the methanol extract of the cultured calli revealed the presence of 11 anthraquinones including two new ones whose structures have been rigorously proved using advanced spectroscopic methods. These findings demonstrated a remarkable difference in the constituents between the wild

  1. Phenotypic characterization of prostate cancer LNCaP cells cultured within a bioengineered microenvironment.

    PubMed

    Sieh, Shirly; Taubenberger, Anna V; Rizzi, Simone C; Sadowski, Martin; Lehman, Melanie L; Rockstroh, Anja; An, Jiyuan; Clements, Judith A; Nelson, Colleen C; Hutmacher, Dietmar W

    2012-01-01

    Biophysical and biochemical properties of the microenvironment regulate cellular responses such as growth, differentiation, morphogenesis and migration in normal and cancer cells. Since two-dimensional (2D) cultures lack the essential characteristics of the native cellular microenvironment, three-dimensional (3D) cultures have been developed to better mimic the natural extracellular matrix. To date, 3D culture systems have relied mostly on collagen and Matrigel™ hydrogels, allowing only limited control over matrix stiffness, proteolytic degradability, and ligand density. In contrast, bioengineered hydrogels allow us to independently tune and systematically investigate the influence of these parameters on cell growth and differentiation. In this study, polyethylene glycol (PEG) hydrogels, functionalized with the Arginine-glycine-aspartic acid (RGD) motifs, common cell-binding motifs in extracellular matrix proteins, and matrix metalloproteinase (MMP) cleavage sites, were characterized regarding their stiffness, diffusive properties, and ability to support growth of androgen-dependent LNCaP prostate cancer cells. We found that the mechanical properties modulated the growth kinetics of LNCaP cells in the PEG hydrogel. At culture periods of 28 days, LNCaP cells underwent morphogenic changes, forming tumor-like structures in 3D culture, with hypoxic and apoptotic cores. We further compared protein and gene expression levels between 3D and 2D cultures upon stimulation with the synthetic androgen R1881. Interestingly, the kinetics of R1881 stimulated androgen receptor (AR) nuclear translocation differed between 2D and 3D cultures when observed by immunofluorescent staining. Furthermore, microarray studies revealed that changes in expression levels of androgen responsive genes upon R1881 treatment differed greatly between 2D and 3D cultures. Taken together, culturing LNCaP cells in the tunable PEG hydrogels reveals differences in the cellular responses to androgen stimulation between the 2D and 3D environments. Therefore, we suggest that the presented 3D culture system represents a powerful tool for high throughput prostate cancer drug testing that recapitulates tumor microenvironment. PMID:22957009

  2. Oxygen levels in thermoplastic microfluidic devices during cell culture

    E-print Network

    Ochs, Christopher J.

    We developed a computational model to predict oxygen levels in microfluidic plastic devices during cell culture. This model is based on experimental evaluation of oxygen levels. Conditions are determined that provide ...

  3. Cellular heredity in haploid cultures of somatic cells, March 1968-April 1981. Final report

    SciTech Connect

    Freed, J.J.

    1982-03-01

    An account is given of the development and application to cell-culture genetics of unique haploid cell lines from frog embryo developed in this laboratory. Since 1968, the main aim of this project has been to develop the haploid cell system for studies of mutagenesis in culture, particularly by ultraviolet radiation. In the course of this work we isolated chromosomally stable cell lines, derived and characterized a number of variants, and adapted cell hybridization and other methods to this material. Particular emphasis was placed on ultraviolet photobiology, including studies of cell survival, mutagenesis, and pathways of repair of uv-damaged DNA. Although at present less widely used for genetic experiments than mammalian cell lines, the frog cells offer the advantages of authentic haploidy and a favorable repertory of DNA repair pathways for study of uv mutagenesis.

  4. Establishing midgut cell culture from Rhynchophorus ferrugineus (Olivier) and toxicity assessment against ten different insecticides.

    PubMed

    Aljabr, Ahmed Mohammed; Rizwan-ul-Haq, Muhammad; Hussain, Abid; Al-Mubarak, Abdullah I; Al-Ayied, Hassan Y

    2014-04-01

    Midgut epithelial cell culture was successfully developed from red palm weevil (Rhynchophorus ferrugineus) during this study and named as RPW-1. Optimum conditions for four different commercial media were also worked out to successfully maintain the culture. Grace's medium was found to be the most effective for RPW-1 culturing which resulted in the highest cell density of 7.5 × 10(6) cells/ml after 72 h of cell seeding with 96% cell viability. It was followed by Schneider's medium and TNM-FH medium where cell densities reached up to 7.4 × 10(6) and 5.9 × 10(6) cells/ml, respectively, after 72 h having 91 and 89% cell viability. Comparatively, Media-199 was least effective for RPW-1 cell culturing. As a whole, temperature at 27°C and pH 6.3 were the best for RPW-1 culturing where the highest cell density and maximum cell viability were noted. Individually, Grace's medium, Schneider's medium, TNM-FH medium, and Media-199 produced better results at 27°C, 27°C, 24°C, and 21°C and pH 6.3, 6.4, 5.3, and 7.1, respectively. The toxicity assay and MTT cell proliferation assay revealed that, out of the ten insecticides used in this study, emamectin benzoate was the most toxic insecticide to RPW-1 cells resulting in 92% cell mortality and 74% cell growth inhibition. Dieldrin was the least potent, causing only 19% cell mortality and 18% cell growth inhibition. PMID:24197670

  5. Studies on the kinetics of Na + \\/H + exchange in OK cells: Introduction of a new device for the analysis of polarized transport in cultured epithelia

    Microsoft Academic Search

    Danuta Krayer-Pawlowska; Corinna Helmle-Kolb; Marshall H. Montrose; Reto Krapf; Heini Murer

    1991-01-01

    Summary The present study describes a new perfusion technique—based on the use of a routine spectrofluorometer—which enables fluorometric evaluation of polarity, regulation and kinetics of Na+\\/H+ exchange at the level of an intact monolayer. Na+\\/ H+ exchange was evaluated in bicarbonate-free solutions in OK (opossum kidney) cells, a renal epithelial cell line. Na+\\/H+ exchange activity was measured by monitoring changes

  6. Interactions between airway epithelial cells and dendritic cells during viral infections using an in vitro co-culture model

    EPA Science Inventory

    Rationale: Historically, single cell culture models have been limited in pathological and physiological relevance. A co-culture model of dendritic cells (DCs) and differentiated human airway epithelial cells was developed to examine potential interactions between these two cell t...

  7. Efficient Culturing and Genetic Manipulation of Human Pluripotent Stem Cells

    Microsoft Academic Search

    Robert T. Schinzel; Tim Ahfeldt; Frank H. Lau; Youn-Kyoung Lee; Alicia Cowley; Tony Shen; Derek Peters; David H. Lum; Chad A. Cowan

    2011-01-01

    Human pluripotent stem cells (hPSC) hold great promise as models for understanding disease and as a source of cells for transplantation therapies. However, the lack of simple, robust and efficient culture methods remains a significant obstacle for realizing the utility of hPSCs. Here we describe a platform for the culture of hPSCs that 1) allows for dissociation and replating of

  8. Plant regeneration in Robinia pseudoacacia from cell suspension cultures

    Microsoft Academic Search

    K. Kanwar; B. Kaushal; S. Abrol; Raj Deepika

    2008-01-01

    A method for plant regeneration in Robinia pseudoacacia L. from cell suspension culture was established. Non regenerative friable callus from hypocotyls and cotyledon explants from\\u000a in vitro raised seedling induced on solid Murashige and Skoog (MS) medium supplemented with 0.05 mg dm?3 2,4-dichlorophenoxyacetic acid (2,4-D) was used for initiation of cell suspension cultures on same MS medium but without\\u000a agar.

  9. In vitro cytotoxicity of macromolecules in different cell culture systems

    Microsoft Academic Search

    Suthummar Choksakulnimitr; Sada Masuda; Hideaki Tokuda; Yoshinobu Takakura; Mitsuru Hashida

    1995-01-01

    The cytotoxic effect of various macromolecules in cultured bovine brain microvessel endothelial cells, mouse peritoneal macrophages, and rat hepatocytes was analyzed by measuring lactate dehydrogenase (LDH) release and by microscopic observations. Polycations, such as protamine, poly-l-lysine and histone, caused high percentage of LDH-release and significant morphological changes in all cultured cells, whereas other polycations, cationized bovine serum albumin (BSA) and

  10. Toxicity of oxygen radicals in cultured pulmonary endothelial cells

    SciTech Connect

    Autor, A.P.; Bonham, A.C.; Thies, R.L.

    1984-01-01

    Superoxide dismutase and catalase, which catalytically remove superoxide radicals and hydrogen perioxide, respectively, each separately protected cultured pulmonary artery endothelial cells from loss of membrane integrity after exposure to oxygen radicals generated either cellularly (polymorphonuclear leukocytes) or chemically (dihydroxyfumarate). Nicotinamide, a precursor of nicotinamide-adenine dinucleotide (NAD) and an inhibitor of ADP-ribose synthetase, also protected cultured endothelial cells from loss of membrane integrity in a concentration-dependent manner after exposure to dihydroxyfumarate.

  11. Cultural Policy in Israel. Studies and Documents on Cultural Policies.

    ERIC Educational Resources Information Center

    Michman, Jozeph

    A survey of cultural policy in Israel, prepared for UNESCO, is one of a series of booklets to show how cultural policies are planned and implemented in various countries. The series provides a guide to these countries which have yet to establish cultural policies to help them profit from past experiences. The historical background of Israel's…

  12. Human umbilical cord blood serum can replace fetal bovine serum in the culture of mesenchymal stem cells.

    PubMed

    Shetty, P; Bharucha, K; Tanavde, V

    2007-03-01

    The potential of mesenchymal stem cells (MSC) to differentiate into different cell types has opened up the possibility of using these cells clinically to treat a variety of disorders. In this study we describe the use of human umbilical cord blood serum (CBS) as a replacement for fetal bovine serum (FBS) for culturing MSC from different sources. MSC from human and swine bone marrow and human umbilical cord blood were cultured in the presence of DMEM/F12 containing either FBS or CBS. Human MSC cultured in presence of FBS or CBS showed typical fibroblast-like morphology, which is characteristic of MSC. 99% of the cells cultured in FBS had a CD73+/CD105+/CD45- phenotype compared to 96% of cells cultured in CBS. Cells cultured in CBS had a significantly higher cell count as compared to cells cultured in FBS. Swine Bone Marrow MSC cultured in the presence of FBS and CBS were morphologically and phenotypically similar. Human umbilical cord blood serum supports the growth of MSC. While no significant differences were observed in the MSC numbers in swine cells cultured in the presence of FBS or CBS, human cells showed a greater proliferation potential in the presence of CBS as compared to FBS. Therefore, CBS can be used as an effective substitute to FBS for developing clinically useful protocols for culturing MSC. PMID:17208468

  13. ACCUMULATION OF PBDE-47 IN PRIMARY CULTURES OF RAT NEOCORTICAL CELLS.

    EPA Science Inventory

    A number of recent studies have examined the neurotoxic actions of PBDEs using in vitro cell culture models. However, there is little data reporting the final concentration of PBDEs in cells after in vitro exposure to these compounds. To address this issue, the present study exam...

  14. Control of galactosylated glycoforms distribution in cell culture system.

    PubMed

    McCracken, Neil A; Kowle, Ronald; Ouyang, Anli

    2014-01-01

    Cell culture process conditions including media components and bioreactor operation conditions have a profound impact on recombinant protein quality attributes. Considerable changes in the distribution of galactosylated glycoforms (G0F, G1F, and G2F) were observed across multiple CHO derived recombinant proteins in development at Eli Lilly and Company when switching to a new chemically defined (CD) media platform condition. In the new CD platform, significantly lower G0F percentages and higher G1F and G2F were observed. These changes were of interest as glycosylation heterogeneity can impact the effectiveness of a protein. A systematic investigation was done to understand the root cause of the change and control strategy for galactosylated glycoforms distribution. It was found that changes in asparagine concentration could result in a corresponding change in G0F, G1F, and G2F distribution. A follow-up study examined a wider range of asparagine concentration and it was found that G0F, G1F, and G2F percentage could be titrated by adjusting asparagine concentration. The observed changes in heterogeneity from changing asparagine concentration are due to resulting changes in ammonium metabolism. Further study ascertained that different integrated ammonium level during the cell culture process could control G0F, G1F, and G2F percentage distribution. A mechanism hypothesis is proposed that integrated ammonium level impacts intracellular pH, which further regulates ?-1, 4 galactosyltransferase activity. PMID:24692242

  15. Cell culture models demonstrate that CFTR dysfunction leads to defective fatty acid composition and metabolism.

    PubMed

    Andersson, Charlotte; Al-Turkmani, M Rabie; Savaille, Juanito E; Alturkmani, Ragheed; Katrangi, Waddah; Cluette-Brown, Joanne E; Zaman, Munir M; Laposata, Michael; Freedman, Steven D

    2008-08-01

    Cystic fibrosis (CF) is associated with fatty acid alterations characterized by low linoleic and docosahexaenoic acid. It is not clear whether these fatty acid alterations are directly linked to cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction or result from nutrient malabsorption. We hypothesized that if fatty acid alterations are a result of CFTR dysfunction, those alterations should be demonstrable in CF cell culture models. Two CF airway epithelial cell lines were used: 16HBE, sense and antisense CFTR cells, and C38/IB3-1 cells. Wild-type (WT) and CF cells were cultured in 10% fetal bovine serum (FBS) or 10% horse serum. Fatty acid levels were analyzed by GC-MS. Culture of both WT and CF cells in FBS resulted in very low linoleic acid levels. When cells were cultured in horse serum containing concentrations of linoleic acid matching those found in human plasma, physiological levels of linoleic acid were obtained and fatty acid alterations characteristic of CF tissues were then evident in CF compared with WT cells. Kinetic studies with radiolabeled linoleic acid demonstrated in CF cells increased conversion to longer and more-desaturated fatty acids such as arachidonic acid. In conclusion, these data demonstrate that CFTR dysfunction is associated with altered fatty acid metabolism in cultured airway epithelial cells. PMID:18441018

  16. Xeno-free culture of human periodontal ligament stem cells.

    PubMed

    Trubiani, Oriana; Diomede, Francesca

    2015-01-01

    The possibility of transplanting adult stem cells into damaged organs has opened a new prospective for the treatment of several human pathologies. Currently, in vitro expansion and culture of mesenchymal stem cells is founded on supplementing cell culture and differentiation medium with fetal calf serum (FCS) or fetal bovine serum (FBS) that contain numerous growth factors inducing cell attachment to plastic surfaces, proliferation, and differentiation. Mesenchymal stem cells (MSCs) cultured with medium containing FCS or FBS are unusable in the cell therapy; in fact the central issues regarding limitations in using animal sera for cell therapy is that its components are highly variable and often unknown and may trigger a xenogenic immune response, immunological reactions, and the potential transmission of prion diseases and zoonoses. Here we describe the culture system protocols for the expansion and production of human Periodontal Ligament Stem Cells (hPDLSCs) using a new xeno-free medium formulation ensuring the maintenance of the stem cells features comprising the multiple passage expansion, mesengenic lineage differentiation, cellular phenotype, and genomic stability, essential elements for conforming to translation to cell therapy. PMID:25326670

  17. Cryopreservation of photosynthetic plant cell suspension cultures

    Microsoft Academic Search

    Ximing Luo; Jack M. Widholm

    1997-01-01

    Attempts were made to cryopreserve in liquid nitrogen six different photomixotrophic suspension cultured lines of five different\\u000a species:Amaranthus powellii Wats.,Datura innoxia Mill.,Glycine max (L.) Merr.,Gossypium hirsutum L. andNicotiana tabacum xNicotiana glutinosa L. fusion hybrid. Only theD. innoxia line, DAT, and theG. max line, SB1, could be successfully recovered as viable, growing, dark green cultures. The successful method utilized a preculture

  18. Human skeletal muscle-derived stem cells retain stem cell properties after expansion in myosphere culture

    SciTech Connect

    Wei, Yan [Department of Otolaryngology, Head and Neck Surgery Charite-Universitaetsmedizin Berlin, Berlin (Germany) [Department of Otolaryngology, Head and Neck Surgery Charite-Universitaetsmedizin Berlin, Berlin (Germany); Department of Otolaryngology, Head and Neck Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guang Zhou (China); Li, Yuan [Department of Otolaryngology, Head and Neck Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guang Zhou (China)] [Department of Otolaryngology, Head and Neck Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guang Zhou (China); Chen, Chao; Stoelzel, Katharina [Department of Otolaryngology, Head and Neck Surgery Charite-Universitaetsmedizin Berlin, Berlin (Germany)] [Department of Otolaryngology, Head and Neck Surgery Charite-Universitaetsmedizin Berlin, Berlin (Germany); Kaufmann, Andreas M. [Clinic for Gynecology CCM/CBF, Charite-Universitaetsmedizin Berlin, Berlin (Germany)] [Clinic for Gynecology CCM/CBF, Charite-Universitaetsmedizin Berlin, Berlin (Germany); Albers, Andreas E., E-mail: andreas.albers@charite.de [Department of Otolaryngology, Head and Neck Surgery Charite-Universitaetsmedizin Berlin, Berlin (Germany)

    2011-04-15

    Human skeletal muscle contains an accessible adult stem-cell compartment in which differentiated myofibers are maintained and replaced by a self-renewing stem cell pool. Previously, studies using mouse models have established a critical role for resident stem cells in skeletal muscle, but little is known about this paradigm in human muscle. Here, we report the reproducible isolation of a population of cells from human skeletal muscle that is able to proliferate for extended periods of time as floating clusters of rounded cells, termed 'myospheres' or myosphere-derived progenitor cells (MDPCs). The phenotypic characteristics and functional properties of these cells were determined using reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry and immunocytochemistry. Our results showed that these cells are clonogenic, express skeletal progenitor cell markers Pax7, ALDH1, Myod, and Desmin and the stem cell markers Nanog, Sox2, and Oct3/4 significantly elevated over controls. They could be maintained proliferatively active in vitro for more than 20 weeks and passaged at least 18 times, despite an average donor-age of 63 years. Individual clones (4.2%) derived from single cells were successfully expanded showing clonogenic potential and sustained proliferation of a subpopulation in the myospheres. Myosphere-derived cells were capable of spontaneous differentiation into myotubes in differentiation media and into other mesodermal cell lineages in induction media. We demonstrate here that direct culture and expansion of stem cells from human skeletal muscle is straightforward and reproducible with the appropriate technique. These cells may provide a viable resource of adult stem cells for future therapies of disease affecting skeletal muscle or mesenchymal lineage derived cell types.

  19. Tissue culture surface characteristics influence the expansion of human bone marrow cells.

    PubMed

    Koller, M R; Palsson, M A; Manchel, I; Maher, R J; Palsson, B O

    1998-11-01

    Human cell therapy applications in tissue engineering, such as the ex vivo production of hematopoietic cells for transplantation, have recently entered the clinic. Although considerable effort has been focused on the development of biological processes to generate therapeutic cells, little has been published on the design and manufacture of devices for implementation of these processes in a robust and reproducible fashion at a clinical scale. In this study, the effect of tissue culture surface chemistry and texture was assessed in human bone marrow (BM) mononuclear cell (MNC) and CD34-enriched cell cultures. Growth and differentiation was assessed by total, progenitor (CFU-GM), stromal (CFU-F), and primitive (LTC-IC) cell output. Tissue culture treated (TCT) plastic significantly increased MNC culture output as compared with non-TCT plastic, whereas CD34-enriched cell cultures gave lower output (than MNC cultures) that was unaffected by TCT plastic. Interestingly, the level of MNC culture output was significantly different on four commercial TCT surfaces, with the best performing surface giving output that was 1.6- to 2.8-fold greater than the worst one. The surface giving the highest output was the best at supporting development of a distinct morphological feature in the adherent layer (i.e. cobblestone area) indicative of primitive cells, and X-ray photoelectron spectroscopy (XPS) was used to characterize this surface. For custom injection molding of culture devices, the use of three different resins resulted in MNC culture output that was equivalent to commercial cultureware controls, whereas CD34-enriched cell cultures were highly sensitive to resins containing additives. When the texture of molded parts was roughened by sandblasting of the tool, MNC culture output was significantly reduced and higher spikes of IL-6 and G-CSF production were observed, presumably due to macrophage activation. In conclusion, the manufacture of BM MNC culture devices for clinical applications was optimized by consideration of plastic resin, surface treatment, and texture of the culture substratum. Although CD34-enriched cells were insensitive to surface treatment, they were considerably more sensitive to biocompatibility issues related to resin selection. The development of robust systems for BM MNC expansion will enable clinical trials designed to test the safety and efficacy of cells produced in this novel tissue engineering application. PMID:9863530

  20. Survival of Mammary Stem Cells in Suspension Culture: Implications for Stem Cell Biology and Neoplasia

    Microsoft Academic Search

    Gabriela Dontu; Max S. Wicha

    2005-01-01

    There is increasing evidence that a variety of neoplasms including breast cancer may result from transformation of normal stem and progenitor cells. In the past, isolation and characterization of mammary stem cells has been limited by the lack of suitable culture systems able to maintain these cells in an undifferentiated state in vitro. We have recently described a culture system

  1. Conditions for initiating Lake Victoria haplochromine (Oreochromis esculentus) primary cell cultures from caudal fin biopsies.

    PubMed

    Filice, Melissa; Lee, C; Mastromonaco, Gabriela F

    2014-10-01

    The global decline of freshwater fishes has created a need to cryopreserve biological materials from endangered species in an effort to conserve the biodiversity within this taxon. Since maternal gametes and embryos from fish are difficult to cryopreserve, somatic cells obtained from caudal fins have become an increasingly popular resource as they contain both maternal and paternal DNA ensuring valuable traits are not lost from the population. Somatic cells stored in cryobanks can be used to supplement endangered populations with genetically valuable offspring with the use of assisted reproductive technologies. However, initiating primary cell cultures from caudal fin biopsies of endangered species can be challenging as standardized protocols have not yet been developed. The objective of this study was to identify culture conditions, including antibiotic supplementation, biopsy size, and culture temperature, suitable for establishing primary cell cultures of ngege (Oreochromis esculentus), a critically endangered African cichlid. Six-millimeter caudal fin biopsies provided sufficient material to develop a primary cell culture when incubated at 25°C using standard fish cell culture medium containing 1× Primocin. Further investigation and application of these culture conditions for other endangered freshwater fishes is necessary. PMID:24985486

  2. Cultured stem cells are sensitive to gravity changes

    Microsoft Academic Search

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

    2008-01-01

    Stem and precursor cells play an important role in development and regeneration. The state of these cells is regulated by biochemical substances, mechanical stimuli and cellular interactions. To estimate gravity effects we used two types of cultured stem cells: human mesenchymal stromal cells (hMSCs) from bone marrow and mice embryonic stem (mESC) line R1. Gravity changes were simulated by long-term

  3. Dynamic 3D cell culture via a chemoselective photoactuated ligand.

    PubMed

    Westcott, Nathan P; Luo, Wei; Goldstein, Jeffrey; Yousaf, Muhammad N

    2014-09-01

    A new strategy to create a dynamic scaffold for three-dimensional (3D) cell experiments based on a photo-activated cell adhesive peptide ligand is described. After polymerization, the inert matrix becomes cell adhesive by chemoselective modification through the conjugation of oxyamine-terminated ligands. Furthermore, spatial and temporal control of cell culture within the 3D matrix was achieved by the use of a biospecific photoprotected peptide and visualized by confocal microscopy. PMID:25280846

  4. Cultural Pluralism Climate Survey Study.

    ERIC Educational Resources Information Center

    Langan, A. Bud; Keeler, Laura

    A campus climate study was conducted at Olympic College (OC), in Washington, to measure student, staff, and faculty perceptions of acceptance, support, and understanding of diverse groups on campus. Specifically, the student and staff survey instruments requested participants' level of agreement or disagreement with respect to 22 statements about…

  5. Peroxidases from cell suspension cultures of Brassica napus.

    PubMed

    Agostini, E; de Forchetti, S M; Tigier, H A

    2000-08-01

    Cell suspension cultures of Brassica napus were obtained under different hormonal conditions, using 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin as growth regulators. They were analyzed as a culture system for peroxidase production in vitro to avoid many of the problems that affect the production from field-grown roots. Total peroxidase specific activities reached a maximum at the end of exponential growth phase of the cultures. Cultures obtained with 4 mg/l of 2,4-D an without kinetin or with 1 mg/l of 2,4-D and the same amount of kinetin produced twice the total activity of root extracts and, in addition, they released peroxidases to the culture medium, which would be advantageous for the commercial production of the enzyme. Peroxidase patterns, obtained by isoelectric focusing of cell extracts and of culture medium of cell suspension cultures, differed from those of root crude extracts from field-grown plants with additional bands of higher isoelectric points. These cultures showed interesting properties and could be considered an alternative source of peroxidases for commercial production and/or to be applied as a model for physiological research. PMID:10979611

  6. Osteogenic and osteoclastic cell interaction: development of a co-culture system.

    PubMed

    Loomer, P M; Ellen, R P; Tenenbaum, H C

    1998-10-01

    The processes involved in the regulation of bone cell metabolism are complex, including those implicated in bone cell coupling. This study was undertaken to develop a model that would permit real-time interaction between osteoclastic cells and osteoblasts in vitro. Osteogenic bone marrow stromal cells were isolated from 18-day-old embryonic chickens, while osteoclastic cells were isolated from laying White Leghorn hens on calcium-deficient diets. Osteoclastic cells (5x10(5)) were seeded onto mineral thin films and suspended above osteogenic cells (1x10(4)) already plated on the bottoms of tissue culture plate wells. The data showed that after 4 days of incubation there was up to a fivefold (P<0.05) reduction in all measured parameters of osteogenesis (mineralization, alkaline phosphatase activity and type I collagen production) in osteogenic cultures grown in the presence of osteoclastic cells. Similarly, osteoclastic cell-induced mineral resorption was reduced up to threefold (P<0.05). Co-culture effects on cellular responses could be manipulated by known antiresorptive agents (e.g., pamidronate) altering either the source or the age of osteoclastic cells. The results indicate that the co-culture model may be useful in the study of bone cell interactions. PMID:9724460

  7. Morphometric and Colorimetrie Analyses of Human Tumor Cell Line Growth and Drug Sensitivity in Soft Agar Culture1

    Microsoft Academic Search

    M. C. Alle; C. M. Pacula-Co; M. L. Hursey; L. R. Rubinstein; M. R. Boy

    1991-01-01

    Previous studies have demonstrated the suitability of image analysis of tetrazolium-stained colonies to assess growth and drug sensitivity of human tumor cells cultivated in soft agar culture. In the present study, the potential utility of colorimetrie analysis to expedite experimental drug evaluations using human tumor cell lines was investigated. The same culture dishes were assessed by image analysis and by

  8. The effects of poly L-lactic acid nanofiber scaffold on mouse spermatogonial stem cell culture

    PubMed Central

    Eslahi, Neda; Hadjighassem, Mahmoud Reza; Joghataei, Mohammad Taghi; Mirzapour, Tooba; Bakhtiyari, Mehrdad; Shakeri, Malak; Pirhajati, Vahid; Shirinbayan, Peymaneh; Koruji, Morteza

    2013-01-01

    Introduction A 3D-nanofiber scaffold acts in a similar way to the extracellular matrix (ECM)/basement membrane that enhances the proliferation and self-renewal of stem cells. The goal of the present study was to investigate the effects of a poly L-lactic acid (PLLA) nanofiber scaffold on frozen-thawed neonate mouse spermatogonial stem cells (SSCs) and testis tissues. Methods The isolated spermatogonial cells were divided into six culture groups: (1) fresh spermatogonial cells, (2) fresh spermatogonial cells seeded onto PLLA, (3) frozen-thawed spermatogonial cells, (4) frozen-thawed spermatogonial cells seeded onto PLLA, (5) spermatogonial cells obtained from frozen-thawed testis tissue, and (6) spermatogonial cells obtained from frozen-thawed testis tissue seeded onto PLLA. Spermatogonial cells and testis fragments were cryopreserved and cultured for 3 weeks. Cluster assay was performed during the culture. The presence of spermatogonial cells in the culture was determined by a reverse transcriptase polymerase chain reaction for spermatogonial markers (Oct4, GFR?-1, PLZF, Mvh(VASA), Itg?6, and Itg?1), as well as the ultrastructural study of cell clusters and SSCs transplantation to a recipient azoospermic mouse. The significance of the data was analyzed using the repeated measures and analysis of variance. Results The findings indicated that the spermatogonial cells seeded on PLLA significantly increased in vitro spermatogonial cell cluster formations in comparison with the control groups (culture of SSCs not seeded on PLLA) (P?0.001). The viability rate for the frozen cells after thawing was 63.00% ± 3.56%. This number decreased significantly (40.00% ± 0.82%) in spermatogonial cells obtained from the frozen-thawed testis tissue. Both groups, however, showed in vitro cluster formation. Although the expression of spermatogonial markers was maintained after 3 weeks of culture, there was a significant downregulation for some spermatogonial genes in the experimental groups compared with those of the control groups. Furthermore, transplantation assay and transmission electron microscopy studies suggested the presence of SSCs among the cultured cells. Conclusion Although PLLA can increase the in vitro cluster formation of neonate fresh and frozen-thawed spermatogonial cells, it may also cause them to differentiate during cultivation. The study therefore has implications for SSCs proliferation and germ cell differentiation in vitro. PMID:24348035

  9. Bovine oviductal epithelial cells: long term culture characterization and impact of insulin on cell morphology.

    PubMed

    Palma-Vera, S; Einspanier, R; Schoen, J

    2014-09-01

    In vitro models that resemble cell function in vivo are needed to understand oviduct physiology. This study aimed to assess cell functions and insulin effects on bovine oviductal epithelial cells (BOECs) cultured in an air-liquid interface. BOECs (n=6) were grown in conditioned Ham's F12, DMEM or Ham's F12/DMEM with 10% fetal calf serum (FCS) for 3 weeks. After selecting the most suitable medium (Ham's F12), increasing insulin concentrations (1 ng/mL, 20 ng/mL and 5 ?g/mL) were applied, and cell morphology and trans-epithelial electrical resistance (TEER; n=4) were evaluated after 3 and 6 weeks. Keratin immunohistochemistry and mRNA expression of oviductal glycoprotein 1 (OVGP1) and progesterone receptor (PGR) were conducted (n=4) to assess cell differentiation. BOECs grown without insulin supplementation or with 1 ng/mL of insulin displayed polarization and secretory activity. However, cells exhibited only 50% of the height of their in vivo counterparts. Cultures supplemented with 20 ng/mL insulin showed the highest quality, but the 5 ?g/mL concentration induced massive growth. TEER correlated negatively with insulin concentration (r=-0.459; p=0.009). OVGP1 and PGR transcripts were still detectable after 3 and 6 weeks. Cellular localization of keratins closely resembled that of BOECs in vivo. Cultures showed heterogeneous expression of PGR and OVGP1 in response to estradiol (10 pg/mL). In summary, BOECs grown for long term in an air-liquid interface expressed markers of cell differentiation. Additionally, insulin supplementation (20 ng/mL) improved the cell morphology in vitro. PMID:25152518

  10. Differential adhesion of Pseudomonas aeruginosa to human respiratory epithelial cells in primary culture.

    PubMed Central

    Plotkowski, M C; Chevillard, M; Pierrot, D; Altemayer, D; Zahm, J M; Colliot, G; Puchelle, E

    1991-01-01

    Human nasal polyps in outgrowth culture were used to study the Pseudomonas aeruginosa adhesion to respiratory cells. By scanning electron microscopy, P. aeruginosa were seen associated with ciliated cells, but by transmission electron microscopy, bacteria were never seen at the interciliary spaces or attached along cilia, but were identified trapped at the extremities of cilia, usually as bacterial aggregates. A fibronectin-containing fibrillar material was seen associated with aggregated bacteria. By time-lapse video microscopy, bacteria were seen to aggregate in the culture medium following their addition to the culture wells. Progressively, these aggregates were trapped by cilia or attached to migrating cells of a lower cell layer that protruded beneath the upper layer cells, at the outgrowth periphery. P. aeruginosa adhesion to these lower cell layer migrating cells was significantly higher than to ciliated or nonciliated cells of the upper cell layer. Migrating cells were intensely labeled by the complexes Con A and arachis hypogea agglutinin (PNA)-FITC, in contrast to the other cells. The percentage of PNA-labeled cells with attached bacteria was significantly higher than that without bacteria. These results suggest that changes of cell surface glycoconjugates related with cell migration may favor P. aeruginosa adhesion to respiratory cells. Images PMID:1904070

  11. Epstein-Barr Virus Infection in Ex Vivo Tonsil Epithelial Cell Cultures of Asymptomatic Carriers

    PubMed Central

    Pegtel, Dirk M.; Middeldorp, Jaap; Thorley-Lawson, David A.

    2004-01-01

    Epstein-Barr virus (EBV) is found frequently in certain epithelial pathologies, such as nasopharyngeal carcinoma and oral hairy leukoplakia, indicating that the virus can infect epithelial cells in vivo. Recent studies of cell lines imply that epithelial cells may also play a role in persistent EBV infection in vivo. In this report, we show the establishment and characterization of an ex vivo culture model of tonsil epithelial cells, a likely site for EBV infection in vivo. Primary epithelial-cell cultures, generated from tonsil explants, contained a heterogeneous mixture of cells with an ongoing process of differentiation. Keratin expression profiles were consistent with the presence of cells from both surface and crypt epithelia. A small subset of cells could be latently infected by coculture with EBV-releasing cell lines, but not with cell-free virus. We also detected viral-DNA, -mRNA, and -protein expression in cultures from EBV-positive tonsil donors prior to in vitro infection. We conclude that these cells were either already infected at the time of explantation or soon after through cell-to-cell contact with B cells replicating EBV in the explant. Taken together, these findings suggest that the tonsil epithelium of asymptomatic virus carriers is able to sustain EBV infection in vivo. This provides an explanation for the presence of EBV in naso- and oropharyngeal pathologies and is consistent with epithelial cells playing a role in the egress of EBV during persistent infection. PMID:15507648

  12. Growth inhibition and chromosomal instability of cultured marsupial (opossum) cells after treatment with DNA polymerase ? inhibitor.

    PubMed

    Takemura, Masaharu; Kazama, Tomoko; Sakuma, Kurumi; Mizushina, Yoshiyuki; Oshima, Teruyoshi

    2011-01-01

    The DNA replication mechanism has been well established for eutherian mammals (placental mammals such as humans, mice, and cattle), but not, to date, for metatherian mammals (marsupials such as kangaroos, koalas, and opossums). In this study, we found that dehydroaltenusin, a selective inhibitor of mammalian (eutherian) DNA polymerase ?, clearly suppressed the growth of metatherian (opossum and rat kangaroo) cultured cells. In cultured opossum (OK) cells, dehydroaltenusin also suppressed the progression of DNA replication. These results suggest that dehydroaltenusin inhibits metatherian as well as eutherian DNA replication. Dehydroaltenusin treatment of OK cells engendered fluctuations in the numbers of chromosomes in the OK cells as well as inhibition of cell growth and DNA replication. This suggests that partial inhibition of DNA replication by dehydroaltenusin causes chromosomal instability in cultured cells. PMID:21737927

  13. Image-based monitoring system for green algal Haematococcus pluvialis (Chlorophyceae) cells during culture.

    PubMed

    Ohnuki, Shinsuke; Nogami, Satoru; Ota, Shuhei; Watanabe, Koichi; Kawano, Shigeyuki; Ohya, Yoshikazu

    2013-11-01

    The green microalga Haematococcus pluvialis accumulates the red pigment astaxanthin accompanied by morphological changes under stress conditions, including nutrient depletion, continuous light and high temperature. To investigate the physiological state of the algal cells, we developed the digital image-processing software called HaematoCalMorph. The software automatically outputs 25 single-cell measurements of cell morphology and pigments based on color, bright-field microscopic images. Compared with manual inspection, the output values of cell shape were reliable and reproducible. The estimated pigment content fits the values calculated by conventional methods. Using a random forests classifier, we were able to distinguish flagellated cells from immotile cells and detect their transient appearance in culture. By performing principal components analysis, we also successfully monitored time-dependent morphological and colorimetric changes in culture. Thus, combined with multivariate statistical techniques, the software proves useful for studying cellular responses to various conditions as well as for monitoring population dynamics in culture. PMID:24058152

  14. Isolation of cultured endothelial progenitor cells in vitro from PBMCs and CD133(+) enriched cells.

    PubMed

    Zheng, Weihong; Wan, Yafeng; Ma, Xiaopeng; Li, Xingrui; Yang, Zhifang; Yin, Qian; Yi, Jilin

    2010-02-01

    Two isolation methods for sorting of endothelial progenitor cells (EPCs): from peripheral blood mononuclear cells (PBMCs) and CD133(+) enriched cells were compared, by defining the cell morphology, phenotype, reproductive activities and function in vitro, to provide a reference for clinical application of EPCs. PBMCs from healthy subjects were used either directly for cell culture or for CD133(+) sorting. The two groups of cells were cultured in complete medium 199 (M199) for 7 to 14 days and the phenotypes of EPCs were analyzed by FACS. The proliferation of differentiated EPCs was studied by MTT assay, and the VEGF concentration was measured using an ELISA kit. ECM gel experiment and migration assay were performed in vivo. The results showed that PBMCs produced more colony-forming units (CFU) than CD133(+) enriched cells from the same volume of blood (P<0.01). From day 7 to 14, the two groups showed decreased expression of hematopoietic stem cell markers and increased level of endothelial markers, but CD144(+) cells in CD133(+) group were less than in PBMCs group (P<0.01). PBMCs group secreted more VEGF than CD133(+) group on the day 7 (P<0.01). As compared with CD133(+) group, PBMCs group had more potent potential of proliferation and vascularization in vitro. It was concluded that CD133(+) sorted cells showed a lower capacity of differentiation, secretion, proliferation and vascularization in vitro, suggesting that CD133-negative cells may be a preferential way to get EPCs for clinical therapy. PMID:20155450

  15. Relations Between the Stage of Cell Maturation and Lactate Transporter Activities in Rat Neonatal Muscle Cells in Culture

    Microsoft Academic Search

    M. Beaudry; N. Mouaffak; T. Darribere; K. El Abida; M. Rieu; R. Mengual

    2000-01-01

    .   Lactate transport was investigated in newborn rat muscle cells in culture. The aim was to study the lactate transport function\\u000a at two stages of cell differentiation in culture: (i) during the proliferative phase characterized by myoblasts and myotubes\\u000a (MyB\\/MyT2) obtained after 2–3 seedings, (ii) when myotubes (MyT1) grow old in culture after 8–9 seedings. In both developmental\\u000a stages MyB\\/MyT2,

  16. Induction of antibody to foot-and-mouth disease virus in presensitized mouse spleen cell cultures.

    PubMed Central

    Collen, T; McCullough, K C; Doel, T R

    1984-01-01

    Cultures of spleen cells from immunized mice were stimulated in vitro by soluble preparations of purified foot-and-mouth disease virus. Virus-specific antibody, as detected by an enzyme-linked immunosorbent assay, was produced by immune spleen cells but not by normal, nonimmune cells. The optimal specific response was obtained with 1 microgram of virus per ml of culture; as the virus concentration was increased, the production of specific antibody was reduced. For very low concentrations of virus (less than 0.01 microgram per culture), there was tentative evidence of suppression of the specific antibody response. The levels of specific antibody induced were dependent on the source and number of plastic-adherent cells present in the cultures. We intend to use this model system to study further the basis of immunity to foot-and-mouth disease virus. PMID:6092687

  17. Changes in basal cell subpopulations and tissue differentiation in human epidermal cultures treated with epidermal growth factor and cholera toxin

    Microsoft Academic Search

    P. K. A. Jensen; J. O. R. Nørgård; L. Bolund

    1985-01-01

    Summary  Cell kinetic studies on cultured human epidermal cells have indicated that cycling basal cells may be divided into at least\\u000a two subpopulations that seem to differ with respect to the rate of DNA replication. The present study was undertaken in order\\u000a to elucidate the biological significance of these subpopulations.\\u000a \\u000a The proliferation characteristics of cultured basal cells were changed by the

  18. Endocytosis in soybean protoplasts and synchronous suspension cultures of soybean cells

    E-print Network

    Sui, Xiaomei

    1994-01-01

    The double phosphate starvation method for inducing cell division synchrony in suspension cultures was applied to a rapidly growing suspension culture of soybean (Glycine max (L.) Merr.) cells. The synchrony of the culture was confirmed by changes...

  19. The antioxidant effect of the Malaysian Gelam honey on pancreatic hamster cells cultured under hyperglycemic conditions.

    PubMed

    Batumalaie, Kalaivani; Qvist, Rajes; Yusof, Kamaruddin Mohd; Ismail, Ikram Shah; Sekaran, Shamala Devi

    2014-05-01

    Type 2 diabetes consists of progressive hyperglycemia, insulin resistance, and pancreatic ?-cell failure which could result from glucose toxicity, inflammatory cytokines, and oxidative stress. In the present study, we investigate the effect of pretreatment with Gelam honey (Melaleuca spp.) and the individual flavonoid components chrysin, luteolin, and quercetin, on the production of reactive oxygen species (ROS), cell viability, lipid peroxidation, and insulin content in hamster pancreatic cells (HIT-T15 cells), cultured under normal and hyperglycemic conditions. Phenolic extracts from a local Malaysian species of Gelam honey (Melaleuca spp.) were prepared using the standard extraction methods. HIT-T15 cells were cultured in 5 % CO2 and then preincubated with Gelam honey extracts (20, 40, 60, and 80 ?g/ml) as well as some of its flavonoid components chrysin, luteolin, and quercetin (20, 40, 60, and 80 ?M), prior to stimulation by 20 and 50 mM of glucose. The antioxidative effects were measured in these cultured cells at different concentrations and time point by DCFH-DA assay. Pretreatment of cells with Gelam honey extract or the flavonoid components prior to culturing in 20 or 50 mM glucose showed a significant decrease in the production of ROS, glucose-induced lipid peroxidation, and a significant increase in insulin content and the viability of cells cultured under hyperglycemic condition. Our results show the in vitro antioxidative property of the Gelam honey and the flavonoids on the ?-cells from hamsters and its cytoprotective effect against hyperglycemia. PMID:23584372

  20. Isolation and Culture of Skeletal Muscle Myofibers as a Means to Analyze Satellite Cells

    PubMed Central

    Shefer, Gabi; Yablonka-Reuveni, Zipora

    2012-01-01

    Summary Myofibers are the functional contractile units of skeletal muscle. Mononuclear satellite cells located between the basal lamina and the plasmalemma of the myofiber are the primary source of myogenic precursor cells in postnatal muscle. This chapter describes protocols used in our laboratory for isolation, culturing and immunostaining of single myofibers from mouse skeletal muscle. The isolated myofibers are intact and retain their associated satellite cells underneath the basal lamina. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. Myofibers are cultured in dishes coated with Vitrogen collagen and satellite cells remain associated with the myofibers undergoing proliferation and differentiation on the myofiber surface. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL). Different from the FDB myofibers, the longer EDL myofibers tend to tangle and break if cultured together; therefore, EDL myofibers are cultured individually. These myofibers are cultured in dishes coated with Matrigel. The satellite cells initially remain associated with the myofiber and later migrate away to its vicinity, resulting in extensive cell proliferation and differentiation. These culture protocols allow studies on the interplay between the myofiber and its associated satellite cells. PMID:15361669

  1. Challenges of Culturing Human Norovirus in Three-Dimensional Organoid Intestinal Cell Culture Models

    PubMed Central

    Papafragkou, Efstathia; Hewitt, Joanne; Park, Geun Woo; Greening, Gail; Vinjé, Jan

    2013-01-01

    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 ?-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. PMID:23755105

  2. Spatially monitoring oxygen level in 3D microfabricated cell culture systems using optical oxygen sensing beads

    PubMed Central

    Wang, Lin; Acosta, Miguel A.; Leach, Jennie B.; Carrier, Rebecca L.

    2013-01-01

    Capability of measuring and monitoring local oxygen concentration at the single cell level (tens of microns scale) is often desirable but difficult to achieve in cell culture. In this study, biocompatible oxygen sensing beads were prepared and tested for their potential for real-time monitoring and mapping of local oxygen concentration in 3D micro-patterned cell culture systems. Each oxygen sensing bead is composed of a silica core loaded with both an oxygen sensitive Ru(Ph2phen3)Cl2 dye and oxygen insensitive Nile blue reference dye, and a poly-dimethylsiloxane (PDMS) shell rendering biocompatibility. Human intestinal epithelial Caco-2 cells were cultivated on a series of PDMS and type I collagen based substrates patterned with micro-well arrays for 3 or 7 days, and then brought into contact with oxygen sensing beads. Using an image analysis algorithm to convert florescence intensity of beads to partial oxygen pressure in the culture system, tens of microns-size oxygen sensing beads enabled the spatial measurement of local oxygen concentration in the microfabricated system. Results generally indicated lower oxygen level inside wells than on top of wells, and local oxygen level dependence on structural features of cell culture surfaces. Interestingly, chemical composition of cell culture substrates also appeared to affect oxygen level, with type-I collagen based cell culture systems having lower oxygen concentration compared to PDMS based cell culture systems. In general, results suggest that oxygen sensing beads can be utilized to achieve real-time and local monitoring of micro-environment oxygen level in 3D microfabricated cell culture systems. PMID:23443975

  3. Induction of vascular endothelial phenotype and cellular proliferation from human cord blood stem cells cultured in simulated microgravity

    NASA Astrophysics Data System (ADS)

    Chiu, Brian; Z-M Wan, Jim; Abley, Doris; Akabutu, John

    2005-05-01

    Recent studies have demonstrated that stem cells derived from adult hematopoietic tissues are capable of trans-differentiation into non-hematopoietic cells, and that the culture in microgravity ( ?g) may modulate the proliferation and differentiation. We investigated the application of ?g to human umbilical cord blood stem cells (CBSC) in the induction of vascular endothelial phenotype expression and cellular proliferation. CD34+ mononuclear cells were isolated from waste human umbilical cord blood samples and cultured in simulated ?g for 14 days. The cells were seeded in rotary wall vessels (RWV) with or without microcarrier beads (MCB) and vascular endothelial growth factor was added during culture. Controls consisted of culture in 1 G. The cell cultures in RWV were examined by inverted microscopy. Cell counts, endothelial cell and leukocyte markers performed by flow-cytometry and FACS scan were assayed at days 1, 4, 7 and at the termination of the experiments. Culture in RWV revealed significantly increased cellular proliferation with three-dimensional (3D) tissue-like aggregates. At day 4, CD34+ cells cultured in RWV bioreactor without MCB developed vascular tubular assemblies and exhibited endothelial phenotypic markers. These data suggest that CD34+ human umbilical cord blood progenitors are capable of trans-differentiation into vascular endothelial cell phenotype and assemble into 3D tissue structures. Culture of CBSC in simulated ?g may be potentially beneficial in the fields of stem cell biology and somatic cell therapy.

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

    NASA Astrophysics Data System (ADS)

    Frampton, John

    Two-dimensional (2-D) neural cell culture systems have served as physiological models for understanding the cellular and molecular events that underlie responses to physical and chemical stimuli, control sensory and motor function, and lead to the development of neurological diseases. However, the development of three-dimensional (3-D) cell culture systems will be essential for the advancement of experimental research in a variety of fields including tissue engineering, chemical transport and delivery, cell growth, and cell-cell communication. In 3-D cell culture, cells are provided with an environment similar to tissue, in which they are surrounded on all sides by other cells, structural molecules and adhesion ligands. Cells grown in 3-D culture systems display morphologies and functions more similar to those observed in vivo, and can be cultured in such a way as to recapitulate the structural organization and biological properties of tissue. This thesis describes a hydrogel-based culture system, capable of supporting the growth and function of several neural cell types in 3-D. Alginate hydrogels were characterized in terms of their biomechanical and biochemical properties and were functionalized by covalent attachment of whole proteins and peptide epitopes. Methods were developed for rapid cross-linking of alginate hydrogels, thus permitting the incorporation of cells into 3-D scaffolds without adversely affecting cell viability or function. A variety of neural cell types were tested including astrocytes, microglia, and neurons. Cells remained viable and functional for longer than two weeks in culture and displayed process outgrowth in 3-D. Cell constructs were created that varied in cell density, type and organization, providing experimental flexibility for studying cell interactions and behavior. In one set of experiments, 3-D glial-endothelial cell co-cultures were used to model blood-brain barrier (BBB) structure and function. This co-culture system was designed for use as a tool to predict the transport and processing that occurs prior to drug uptake in the central nervous system (CNS), and to predict BBB permeability. Electrochemical techniques and immunohistochemistry were used to validate this model and provide detailed information about cellular organization and function. Electrochemical impedance spectroscopy (EIS) provided evidence that endothelial cells cultured in the presence of astrocytes formed tight junctions capable of occluding the flow of electrical current. In a second series of experiments, a microglia-astrocyte co-culture system was developed to assess the effects of glial cells on electrode impedance recorded from neural prosthetic devices in vitro. Impedance measurements were compared with confocal images to determine the effects of glial cell density and cell type on electrode performance. The results indicate that EIS data can be used to model components of the reactive cell responses in brain tissue, and that impedance measurements recorded in vitro can be compared to measurements recorded in vivo. Taken together, these results demonstrate that alginate hydrogels can be used for the creation of 3-D neural cell scaffolds, and that such cell scaffolds can be used to model a variety of three-dimensional neural tissues in vitro, that cannot be studied in 2-D cultures.

  5. 3D Hepatic Cultures Simultaneously Maintain Primary Hepatocyte and Liver Sinusoidal Endothelial Cell Phenotypes

    PubMed Central

    Kim, Yeonhee; Rajagopalan, Padmavathy

    2010-01-01

    Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes) and non-parenchymal (liver sinusoidal endothelial, LSEC) cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs) were cultured in a layered three-dimensional (3D) configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM), which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1) demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism, detoxification and signaling pathways in vitro. PMID:21103392

  6. Skeletal Muscle Satellite Cells: Background and Methods for Isolation and Analysis in a Primary Culture System

    PubMed Central

    Danoviz, Maria Elena; Yablonka-Reuveni, Zipora

    2012-01-01

    Summary Repair of adult skeletal muscle depends on satellite cells, myogenic stem cells located between the basal lamina and the plasmalemma of the myofiber. Standardized protocols for the isolation and culture of satellite cells are key tools for understanding cell autonomous and extrinsic factors that regulate their performance. Knowledge gained from such studies can contribute important insights to developing strategies for the improvement of muscle repair following trauma and in muscle wasting disorders. This chapter provides an introduction to satellite cell biology and further describes the basic protocol used in our laboratory to isolate and culture satellite cells from adult skeletal muscle. The cell culture conditions detailed herein support proliferation and differentiation of satellite cell progeny and the development of reserve cells, which are thought to reflect the in vivo self-renewal ability of satellite cells. Additionally, this chapter describes our standard immunostaining protocol that allows the characterization of satellite cell progeny by the temporal expression of characteristic transcription factors and structural proteins associated with different stages of myogenic progression. While emphasis is given here to the isolation and characterization of satellite cells from mouse hindlimb muscles, the protocols are suitable for other muscle types (such as diaphragm and extraocular muscles) and for muscles from other species, including chicken and rat. Altogether, the basic protocols described are straightforward and facilitate the study of diverse aspects of skeletal muscle stem cells. PMID:22130829

  7. Isolation and Culture of Human Spermatogonial Stem Cells Derived from Testis Biopsy

    PubMed Central

    Goharbakhsh, Leila; Mohazzab, Arash; Salehkhou, Sheida; Heidari, Mahnaz; Zarnani, Amir Hassan; Parivar, Kazem; Akhondi, Mohammad Mehdi

    2013-01-01

    Background In cancer patients, chemo and radiotherapy can cause infertility by damaging spermatogenesis process. This process is based on self-renewal and differentiation of a rare population of the testicular cells called Spermatogonial Stem Cells (SSCs). Scientists have tried to isolate, enrich and culture Human spermatogonial stem cells, hoping to resolve infertility problems in cancer recovered patients in the future. Methods Spermatogonial stem cells were isolated and purified from human testicular biopsies sample consisting of at least 500,000 and at most 2,000,000 cells. Two enzymatic digestion steps were performed. Enriching methods, differential plating, and specific culture in serum-free medium with added growth factors: human GDNF, bFGF, EGF and LIF was performed on coated dishes. Results Human spermatogonial stem cell clusters were observed after 7 to 10 days in specific culture, then after several passages and successful expanding duration of 52 days, the cells were evaluated by three layer immunocytochemistry test (LSAB) to stain GPR125 protein as a surface marker in human spermatogonial stem cells. Conclusion In current study human spermatogonial stem cell were isolated and expanded with the least manipulations in comparison with the other usual isolation methods like florescent or magnetic activated cell sorting. In contrast to the other SSCs isolation and culture methods, this system is based on the testicular biopsies against large samples, thus suggested method in this study is closer to clinical usage in the future. PMID:23626877

  8. Automatic detection of cellular necrosis in epithelial cell cultures

    NASA Astrophysics Data System (ADS)

    Santos, Andres; Ramiro, Cristina; Desco, Manuel; Malpica, Norberto; Tejedor, Alberto; Torres, Ana; Ledesma-Carbayo, Maria J.; Castilla, Manuela; Garcia-Barreno, Pedro

    2001-07-01

    Automatic discrimination and quantification of alive and dead cells in phase contrast microscopy images allows in vivo analysis of the viability of cultured cells without staining. Unsupervised segmentation, based on texture analysis, classifies each image region into three groups: live cells, necrotic cells and background. The segmentation is based on three discriminant functions, built using a total of 12 parameters derived from the histogram and the co-occurrence matrix. These parameters were selected performing a discriminant analysis on a training set that included images from three different cultures. Once images are automatically segmented, the approximate number of live and dead cells is obtained by dividing each area by the average size of each cell type. The number and percentage of live and necrotic cells have been obtained for primary cellular cultures in intervals of 48 hr. during two weeks. The results have been compared with the figures given by an experienced human observer, showing a very good correlation (Pearson's coefficient 0.95, kappa 0.87). A reliable and easy-to-use tool has been developed. It provides quantitative results on phase contrast microscopy images of cell cultures, with preliminary results showing accuracy similar to that provided by an expert, allowing to count a higher number of fields.

  9. Stem-cell culture on patterned bio-functional surfaces

    Microsoft Academic Search

    A. Ruiz; L. Buzanska; L. Ceriotti; F. Bretagnol; S. Coecke; P. Colpo; F. Rossi

    2008-01-01

    Bio-functional surfaces have been created by printing proteins on antifouling surfaces in a customised geometry. Human umbilical cord neural stem cells incubated on the samples readily attach to the protein defined domains, where they have been monitored during 21 days of culture. The stability of the pattern varies with the density of cells anchored to the microstamped proteins. Highly packed

  10. Culture and characterisation of epithelial cells from human pterygia

    Microsoft Academic Search

    Nick Di Girolamo; Nicodemus Tedla; Rakesh K Kumar; Peter McCluskey; Andrew Lloyd; Minas T Coroneo; Denis Wakefield

    1999-01-01

    BACKGROUND\\/AIMSPterygia are a common disorder of the ocular surface. The disease represents a chronic fibrovascular and degenerative process thought to originate at the conjunctival-corneal junction, where altered limbal stem cells are proposed to be the cell of origin. Extensive epidemiological evidence exists to implicate ultraviolet B irradiation in the pathogenesis of pterygia. To date no animal or in vitro culture

  11. Propagation of infectious salmon anaemia (ISA) virus in cell culture

    E-print Network

    Boyer, Edmond

    Propagation of infectious salmon anaemia (ISA) virus in cell culture BH Dannevig K Falk CMcL Press Summary ― A long-term cell line supporting growth of the infectious salmon anaemia (ISA) virus has Atlantic salmon, and exhibited macrophage-like enzyme reactivities. By means of transmission experiments

  12. Characterization of the glomerular endothelial cell in culture.

    PubMed

    Nitta, K; Uchida, K; Yumura, W; Nihei, H

    1993-08-01

    Because of difficulties associated with the culture, cloning and propagation of glomerular endothelial cells (GENs), the biological properties of these cells remain largely unknown. We modified the methods established by Ballermann to propagate GENs from adult bovine kidney. We found that the addition of insulin, transferrin and selenium into the standard culture media was an important step in promoting the propagation of the first clone from a single cell and in maintaining the viability of the cells. These cells expressed factor VIII-related antigen and took up acetylated-LDL, but did not contain the Weibel-Palade body, unlike endothelial cells derived from large vessels. Furthermore, GENs were compared with aortic endothelial cells (AECs) to investigate the differences in culture conditions. Compared with AECs, GENs required a higher concentration of serum and the supplementation of growth factor to maintain their biological activity. In addition, GENs were very susceptible to trypsinization and produced prostaglandin E2 as a major cyclooxygenase product, whereas AECs produced PGI2. These findings suggest that GENs will be easily obtained from adult bovine kidney in culture and provide useful information on the functional properties of these cells under physiological and pathophysiological conditions. PMID:8254997

  13. Metabolism of different PCB congeners in plant cell cultures

    Microsoft Academic Search

    Annelen Wilken; Claudia Bock; Maria Bokern; Hans Harms

    1995-01-01

    The metabolism of 10 different congeners of polychlorinated biphenyls (PCBs) was tested in cell cultures of 12 different plant species. The rate of metabolism was determined through comparison with cells inactivated by treatment with perchloric acid. The metabolism of defined PCB congeners was strongly dependent on the plant species. In particular, two Fabaceae species exhibited high capacities for metabolizing different

  14. Continuous Recording of Cell Number in Logarithmic and Synchronized Cultures

    Microsoft Academic Search

    T. W. James; Norman G. Anderson

    1963-01-01

    An instrument for the continuous recording of cell number has been developed and is being used to record changes in populations of logarithmic and synchronized cultures of protozoan flagellates. A Coulter cell counter is used in conjunction with a counting chamber that is fitted with a flexible polyethylene aperture (75 mu in diameter). This aperture rarely becomes blocked and appears

  15. Enzymatic measurement of phosphatidic acid in cultured cells.

    PubMed

    Morita, Shin-ya; Ueda, Kazumitsu; Kitagawa, Shuji

    2009-09-01

    In this work, we developed a novel enzymatic method for measuring phosphatidic acid (PA) in cultured cells. The enzymatic reaction sequence of the method involves hydrolysis of PA to produce glycerol-3-phosphate (G3P), which is then oxidized by G3P oxidase to generate hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide reacted with Amplex Red to produce highly fluorescent resorufin. We found that lipase from Pseudomonas sp. can completely hydrolyze PA to G3P and FAs. The calibration curve for PA measurement was linear between 20 and 250 microM, and the detection limit was 5 microM (50 pmol in the reaction mixture). We also modified the method for the enzymatic measurement of lysophosphatidic acid. By this new method, we determined the PA content in the lipid extract from HEK293 cells. The cellular content of PA was decreased with increasing cell density but not correlated with the proliferation rate. The diacylglycerol kinase inhibitor R59949 markedly reduced the cellular PA content, suggesting the diacylglycerol kinase activity was involved in a large part of the PA production in HEK293 cells. This novel method for PA quantification is simple, rapid, specific, sensitive, and high-throughput and will help to study the biological functions of PA and its related enzymes. PMID:19369695

  16. Enzymatic measurement of phosphatidic acid in cultured cells

    PubMed Central

    Morita, Shin-ya; Ueda, Kazumitsu; Kitagawa, Shuji

    2009-01-01

    In this work, we developed a novel enzymatic method for measuring phosphatidic acid (PA) in cultured cells. The enzymatic reaction sequence of the method involves hydrolysis of PA to produce glycerol-3-phosphate (G3P), which is then oxidized by G3P oxidase to generate hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide reacted with Amplex Red to produce highly fluorescent resorufin. We found that lipase from Pseudomonas sp. can completely hydrolyze PA to G3P and FAs. The calibration curve for PA measurement was linear between 20 and 250 µM, and the detection limit was 5 µM (50 pmol in the reaction mixture). We also modified the method for the enzymatic measurement of lysophosphatidic acid. By this new method, we determined the PA content in the lipid extract from HEK293 cells. The cellular content of PA was decreased with increasing cell density but not correlated with the proliferation rate. The diacylglycerol kinase inhibitor R59949 markedly reduced the cellular PA content, suggesting the diacylglycerol kinase activity was involved in a large part of the PA production in HEK293 cells. This novel method for PA quantification is simple, rapid, specific, sensitive, and high-throughput and will help to study the biological functions of PA and its related enzymes. PMID:19369695

  17. Primary targets in photochemical inactivation of cells in culture

    NASA Astrophysics Data System (ADS)

    Berg, Kristian; Jones, Stuart G.; Prydz, Kristian; Moan, Johan

    1995-01-01

    The mechanisms of photoinactivation of NHIK 3025 cells in culture sensitized by tetrasulfonated phenylporphines (TPPS4) are described). Ultracentrifugation studies on postnuclear supernatants indicated that the intracellular distribution of TPPS4 resembles that of (beta) -N-acetyl-D-glucosaminidase ((beta) -AGA), a lysosomal marker enzyme, and that the cytosolic content of TPPS4 is below the detection limit of the ultracentrifugation method. Upon light exposure more than 90% of TPPS4 was lost from the lysosomal fractions, due to lysosomal rupture. The content of TPPS4 in the postnuclear supernatants was reduced by 30 - 40% upon exposure to light. This is most likely due to binding of TPPS4 to the nuclei, which were removed from the cell extracts before ultracentrifugation, after photochemical treatment. The unpolymerized form of tubulin seems to be an important target for the photochemical inactivation of NHIK 3025 cells. Since TPPS4 is mainly localized in lysosomes it was assumed that a dose of light disrupting a substantial number of lysosomes followed by microtubule depolymerization by nocodazole would enhance the sensitivity of the cells to photoinactivation. This was confirmed by using a colony-forming assay. The increased phototoxic effect exerted by such a treatment regime could be explained by an enhanced sensitivity of tubulin to light. Another cytosolic constituent, lactate dehydrogenase, was not photoinactivated by TPPS4 and light.

  18. Whole bone marrow cell culture: A convenient protocol for the in vitro expansion of endothelial progenitor cells

    PubMed Central

    LIU, JUN-FENG; DU, ZHONG-DONG; CHEN, ZHI; HE, ZHI-XU

    2014-01-01

    The number and function of endothelial progenitor cells (EPCs) may be a predictive factor for the severity and outcome of cardiovascular disease. However, the manipulation of bone marrow mononuclear cell (BMMC) cultures for EPCs is an elaborate and difficult procedure in small experimental animals. The present study aimed to assess the feasibility of whole bone marrow cell (WBMC) culture for expanding EPCs in small experimental animals. C57BL/6 mice (age, 3–4 weeks; weight, 9.47±0.76 g) were used as the experimental animals, and WBMCs were isolated from the femora and tibiae and cultured in endothelial cell growth medium-2. A BMMC culture for EPCs was used as a control. EPC growth, phenotype and functions were assessed in vitro and in vivo. The results demonstrated that EPCs were easily obtained from a WBMC culture in vitro. The cells exhibited similar growth and biological characteristics when compared with the EPCs derived from the traditional BMMC culture system. Thus, the cells were able to simultaneously bind to lectin and cause phagocytosis of acetylated-low density lipoproteins. In addition, the cells exhibited high expression levels of cluster of differentiation 34 and fetal liver kinase 1, and possessed similar functional properties to BMMC-derived EPCs, including vascular network formation, proliferation, adhesion and migration abilities in vitro. Thus, WBMC-derived EPCs can improve the outcome of pulmonary vascular disease when transplanted into a monocrotaline-induced pulmonary hypertension mouse model. The results of the present study indicated that the WBMC culture system is a more convenient and effective method of obtaining and expanding EPCs compared with BMMC culture, with the advantage of a simplified procedure. PMID:25120604

  19. Angiotensin II binding to cultured bovine adrenal chromaffin cells: identification of angiotensin II receptors

    SciTech Connect

    Boyd, V.L.; Printz, M.P.

    1986-03-05

    Physiological experiments have provided evidence that angiotensin II stimulates catecholamine secretion from the adrenal gland. Their laboratory and others have now shown by receptor autoradiography the presence of angiotensin II receptors (AIIR) in bovine and rat adrenal medulla. In order to extend these studies they have undertaken to define AIIR on cultured bovine adrenal chromaffin cells. Cells were isolated using the method of Levitt including cell enrichment with Percoll gradient centrifugation. Primary cultures of bovine adrenal medullary cells were maintained in DME/F12 medium containing 10% FCS. Cells were characterized by immunocytochemistry for Met- and Leu-enkephalin, PNMT, DBH and Chromagranin A. Cultured cells bind with high affinity and specificity (/sup 125/I)-ANG II yielding a K/sub D/ of 0.74 nM and B/sub max/ of 24,350 sites/cell. After Percoll treatment values of .77 nm and 34,500 sites/cell are obtained. K/sub D/ values are in close agreement with that obtained in adrenal slices by Healy. Competition studies identify a rank order of binding by this receptor similar to that of other tissues. They conclude that cultured chromaffin cells provide a suitable model system for the investigation and characterization of the ANG II receptor and for cellular studies of its functional significance.

  20. Spontaneous Electrical Activity of Cultured Interstitial Cells of Cajal from Mouse Urinary Bladder

    PubMed Central

    Kim, Sun-Ouck; Jeong, Han-Seong; Jang, Sujeong; Wu, Mei-Jin; Park, Jong Kyu; Jiao, Han-Yi; Jun, Jae Yeoul

    2013-01-01

    Interstitial cells of Cajal (ICCs) from the urinary bladder regulate detrusor smooth muscle activities. We cultured ICCs from the urinary bladder of mice and performed patch clamp and intracellular Ca2+ ([Ca2+]i) imaging to investigate whether cultured ICCs can be a valuable tool for cellular functional studies. The cultured ICCs displayed two types of spontaneous electrical activities which are similar to those recorded in intact bladder tissues. Spontaneous electrical activities of cultured ICCs were nifedipine-sensitive. Carbachol and ATP, both excitatory neurotransmitters in the urinary bladder, depolarized the membrane and increased the frequency of spike potentials. Carbachol increased [Ca2+]i oscillations and basal Ca2+ levels, which were blocked by atropine. These results suggest that cultured ICCs from the urinary bladder retain rhythmic phenotypes similar to the spontaneous electrical activities recorded from the intact urinary bladder. Therefore, we suggest that cultured ICCs from the urinary bladder may be useful for cellular and molecular studies of ICCs. PMID:24381503

  1. The pesticide methoxychlor decreases myotube formation in cell culture by slowing myoblast proliferation.

    PubMed

    Steffens, Bradley W; Batia, Lyn M; Baarson, Chad J; Choi, Chang-Kun Charles; Grow, Wade A

    2007-08-01

    We studied the effect of the estrogenic pesticide methoxychlor (MXC) on skeletal muscle development using C2C12 cell culture. Myoblast cultures were exposed to various concentrations of MXC at various times during the process of myoblast fusion into myotubes. We observed that MXC exposure decreased myotube formation. In addition, we observed myoblasts with cytoplasmic vacuoles in cultures exposed to MXC. Because cytoplasmic vacuoles can be characteristic of cell death, apoptosis assays and trypan blue exclusion assays were performed. We found no difference in the frequency of apoptosis or in the frequency of cell death for cultures exposed to MXC and untreated cultures. Collectively, these results indicate that MXC exposure decreases myotube formation without causing cell death. In contrast, when cell proliferation was assessed, untreated cultures had a myoblast proliferation rate 50% greater than cultures exposed to MXC. We conclude that MXC decreases myotube formation at least in part by slowing myoblast proliferation. Furthermore, we suggest that direct exposure to MXC could affect skeletal muscle development in animals or humans, in addition to the defects in reproductive development that have previously been reported. PMID:17314029

  2. Continuous culture growth of photoautotrophic cell suspensions from Chenopodium rubrum.

    PubMed

    Hüsemann, W

    1983-04-01

    The construction and operation of a continuous culture system for the propagation of cell suspensions from Chenopodium rubrum under photoautotrophic conditions has been described. A dilution rate of 0.16/day gave an equilibrium culture density of 1,100,000 cells/ml and a mean doubling time of 150 hours. During continuous culture steady state conditions with respect to nutrient uptake, cell protein and chlorophyll content, starch accumulation, in vitro activities of enzymes related to different metabolic pathways could be established. Photosynthetic CO2 assimilation of steady state cells was about 100 mol CO2/mg chlorophyll x hour. Dark CO2 fixation was 3% of the light values. PMID:24257948

  3. Hydrodynamic effects on cells in agitated tissue culture reactors

    NASA Technical Reports Server (NTRS)

    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.

  4. Human bone cell cultures in biocompatibility testing. Part I: osteoblastic di!erentiation of serially passaged human bone marrow cells cultured in a-MEM and in DMEM

    Microsoft Academic Search

    M. J. Coelho; A. Trigo Cabral; M. H. Fernandes

    2000-01-01

    Well-characterised human osteoblastic bone marrow cell cultures are a useful in vitro tool to analyse bone tissue\\/biomaterials interactions. In this work, human bone marrow was cultured in experimental conditions described to favour osteoblastic di!erenti- ation and, serially passaged cells were cultured in two widely used culture media, minimum essential medium Eagle, alpha modi\\

  5. Voltage and time-dependent chloride currents in chick skeletal muscle cells grown in tissue culture

    Microsoft Academic Search

    Joy A. Steele

    1989-01-01

    Membrane chloride currents in chick skeletal muscle cells grown in tissue culture were studied by use of the whole cell variation of the patch electrode voltage clamp technique. Small diameter myoballs were obtained by adding colchicine to the growth media. To isolate the currents through the chloride channels, the currents through the sodium, calcium and potassium channels were minimized. With

  6. Effect of pneumolysin on rat brain ciliary function: comparison of brain slices with cultured ependymal cells.

    PubMed

    Hirst, R A; Rutman, A; Sikand, K; Andrew, P W; Mitchell, T J; O'Callaghan, C

    2000-03-01

    This study compares two models for examining ependymal ciliary function: rat brain slices cut from the fourth ventricle and primary ependymal cells in culture. The cilia from both preparations were very reproducible; each preparation had cilia beating at a constant frequency of between 38 and 44 Hz. With the brain slices, ciliary stasis occurred after 5 d in culture. However, ependymal cells had fully functional cilia for up to 48 d in culture. The pneumococcal toxin, pneumolysin, caused a dose-dependent inhibition of cilia beat frequency within 15 min in both models. There were no significant differences in the mean log 50% inhibitory concentration (pIC50) slice = 0.65 +/- 0.05, equivalent to 4.4 hemolytic units (HU)/mL; cells = 0.57 +/- 0.14, equivalent to 3.7 HU/mL. There were also no significant differences in the mean Hill slope factors for the curves (slice = 1.4 +/- 0.05; cells = 1.6 +/- 0.4). These data demonstrate that both models can be used to examine the acute (15-min) effects of pneumolysin on cilia beat frequency. The main advantage of the primary ependymal culture model is that considerably more cultured ependymal cells (approximately 70%) are available, compared with the number of ependymal cells on the brain slices (approximately 2%), thus reducing the number of animals used. A pure ependymal culture was not achieved (approximately 30% of the cells were not ciliated). The increased survival time of the ependymal cells compared with the brain slices make cultured ependymal cells more useful for examining long-term ciliary function, whereas brain slices may be more useful for examining the interactions between ependymal and other nearby cells. PMID:10709739

  7. Hepatitis E Virus Produced from Cell Culture Has a Lipid Envelope

    PubMed Central

    Qi, Ying; Zhang, Feng; Zhang, Li; Harrison, Tim J.; Huang, Weijin; Zhao, Chenyan; Kong, Wei; Jiang, Chunlai; Wang, Youchun

    2015-01-01

    The absence of a productive cell culture system hampered detailed analysis of the structure and protein composition of the hepatitis E virion. In this study, hepatitis E virus from a robust HEV cell culture system and from the feces of infected monkeys at the peak of virus excretion was purified by ultra-centrifugation. The common feature of the two samples after ultracentrifugation was that the ORF2 protein mainly remained in the top fractions. The ORF2 protein from cell culture system was glycosylated, with an apparent molecular weight of 88 kDa, and was not infectious in PLC/PRF/5 cells. The ORF2 protein in this fraction can bind to and protect HEV RNA from digestion by RNase A. The RNA-ORF2 product has a similar sedimentation coefficient to the virus from feces. The viral RNA in the cell culture supernatant was mainly in the fraction of 1.15g/cm3 but that from the feces was mainly in the fraction of 1.21 g/cm3. Both were infectious in PLC/PRF/5 cells. And the fraction in the middle of the gradient (1.06g/cm3) from the cell culture supernatant,but not that from the feces, also has ORF2 protein and HEV RNA but was not infectious in PLC/PRF/5.The infectious RNA-rich fraction from the cell culture contained ORF3 protein and lipid but the corresponding fraction from feces had no lipid and little ORF3 protein. The lipid on the surface of the virus has no effect on its binding to cells but the ORF3 protein interferes with binding. The result suggests that most of the secreted ORF2 protein is not associated with HEV RNA and that hepatitis E virus produced in cell culture differs in structure from the virus found in feces in that it has a lipid envelope. PMID:26161670

  8. Tumor necrosis factor-alpha (TNF-alpha) concentrations from whole blood cultures correlate with isolated peripheral blood mononuclear cell cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many cellular immune assays are impractical because they require labor-intensive isolation of cells from their natural environment. The objectives of this study were to determine the relationship between cell culture supernatant TNF-alpha from isolated peripheral blood mononuclear cells (PBMC) and w...

  9. Glucocorticoid actions on L6 muscle cells in culture

    SciTech Connect

    Max, S.R.; Konagaya, M.; Konagaya, Y.

    1986-05-01

    Glucocorticoids exert striking catabolic effects on skeletal muscle. The mechanism of these effects remains poorly understood. They employed L6 muscle cells in culture to ascertain whether intracellular glucocorticoid receptors are involved. Studies in vitro permit exploration of glucocorticoid effects in the absence of other hormonal influences. L6 myoblasts were induced to form differentiated myotubes by growth in 1% serum. L6 myotubes were found to possess a high-affinity, limited capacity intracellular glucocorticoid receptor (apparent K/sub D/ = 5 x 10/sup -10/ M; B/sub max/ = 711 pmols/g protein) with ligand specificity similar to that of glucocorticoid receptors from classical glucocorticoid target tissues. Further, (/sup 3/H) triamcinolone acetonide specific binding to L6 cell homogenates was blocked by a glucocorticoid antagonist, RU38486 (11..beta..-(4-dimethyl-aminophenyl)-17..beta..-hydroxy-17..cap alpha..-(prop-l-ynyl)-estra-4,9-dien-3-one). Dexamethasone (10/sup -5/M) caused a 10-fold increase in the activity of gluatmine synthetase in L6 myotubes; this increase was prevented by RU38486. Similarly, dexamethasone (10/sup -5/M) caused a 20% decrease in (/sup 12/C) leucine incorporation into protein. This effect also was blocked by RU38486. Thus, induction of glutamine synthetase and diminution of protein synthesis by dexamethasone require intracellular glucocorticoid receptors. L6 cells should prove particularly valuable for further studies of glucocorticoid actions on skeletal muscle.

  10. Three-dimensional culture of differentiated endometrial stromal cells to oligodendrocyte progenitor cells (OPCs) in fibrin hydrogel.

    PubMed

    Asmani, Mohammad Nabi; Ai, Jafar; Amoabediny, Ghasem; Noroozi, Abbas; Azami, Mahmoud; Ebrahimi-Barough, Somayeh; Navaei-Nigjeh, Mona; Ai, Armin; Jafarabadi, Mina

    2013-12-01

    Neural tissue engineering is one of the most promising strategies for treatment of nerve tissue injuries. Three-dimensional (3D) environment mimics in vivo conditions for cells. 3D distribution and growth of the cells within the scaffold are both important for neural tissue engineering. In this study, endometrial stromal cell-derived oligodendrocyte progenitor cells (EnSC-derived OPCs) were cultured in fibrin gel and cell differentiation and viability were evaluated after 8 days of post-culture. The structural and mechanical characteristics of fibrin gel-like scaffold were examined with rheological analysis. EnSCs were isolated from donor tissue and were induced to OPCs with growth factors (FGF2/EGF/PDGF-AA) for 12 days, then were cultured in fibrin gel with Triiodothyronine (T3) medium for another 8 days. The viability of cells was analyzed using MTT assay for a period of 8 days culturing in a fibrin matrix. Structure of fibrin matrix and cell morphology was analyzed with SEM. TEM, immunostaining and quantitative RT-PCR was performed for OPCs markers after cell culturing in fibrin matrix. Cell viability is enhanced in fibrin matrix after 8 days. SEM and TEM show that cells are in good integration with nano-fibers. Moreover, immunohistochemistry and quantitative RT-PCR of OPCs differentiation markers showed that Olig2, Sox10, PDGFRa, CNP, and A2B5 are expressed after 8 days culturing within fibrin matrix. Fibrin can provide a suitable 3-D scaffold for EnSCs differentiated cells for the regeneration of CNS. PMID:24038753

  11. Factors influencing the interaction of Candida albicans with fibroblast cell cultures.

    PubMed Central

    Merkel, G J; Phelps, C L

    1988-01-01

    The interaction of Candida albicans clinical isolates with primary and established fibroblast cultures was studied. The intent was to determine whether yeast adherence and invasion of nonendothelial cell monolayer cultures could be quantitated reproducibly and whether this system could be used for future studies on yeast pathogenesis. Our results demonstrated that specific interactions between the yeast cells and fibroblasts only occurred at 37 degrees C and correlated with the germination process. Fluorescent-antibody staining indicated that invasion or tight associations between the germinating yeast cells and mammalian cells occurred after less than 3 h of incubation. Yeast adherence was estimated radiometrically and trypsin-resistant interaction with individual mammalian cells (infection) was measured microscopically after inoculated monolayer cells were detached with trypsin. We demonstrated that both types of association were time dependent at 37 degrees C; neither was affected by the concentration of glucose used to grow the yeast cells. Primary and established fibroblast cell lines were equally susceptible to infection, but primary cells appeared to have more yeast-binding sites. Fibroblasts maintained in confluent culture for an extended period of time also appeared to have more binding sites, and while not quantitatively more susceptible to infection, the older cells were more susceptible to infection-related cell death. An established kidney epithelial cell line (MDCK) was not susceptible to either type of yeast interaction, indicating that the yeast-fibroblast associations were specific. Images PMID:3278982

  12. Nitric Oxide Inhibits Migration of Cultured Endothelial Cells

    Microsoft Academic Search

    Ying-Tung Lau; Wei-Ching Ma

    1996-01-01

    Endothelial cell migration is an important event in both physiological and pathophysiological processes. Although nitric oxide (NO) plays a critical role in regulating vascular functions, it is not known whether NO modulates migration of endothelial cells. We show here that chemically-derived NO inhibited the serum-induced migration of cultured human umbilical vein endothelial cells (HUVEC) in a time- and dose-dependent manner.

  13. Fatty acid composition in native and cultured human endothelial cells

    Microsoft Academic Search

    M. Lagarde; B. Sicard; M. Guichardant; O. Felisi; M. Dechavanne

    1984-01-