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Sample records for cell culture models

  1. In silico characterization of cell-cell interactions using a cellular automata model of cell culture.

    Science.gov (United States)

    Kihara, Takanori; Kashitani, Kosuke; Miyake, Jun

    2017-07-14

    Cell proliferation is a key characteristic of eukaryotic cells. During cell proliferation, cells interact with each other. In this study, we developed a cellular automata model to estimate cell-cell interactions using experimentally obtained images of cultured cells. We used four types of cells; HeLa cells, human osteosarcoma (HOS) cells, rat mesenchymal stem cells (MSCs), and rat smooth muscle A7r5 cells. These cells were cultured and stained daily. The obtained cell images were binarized and clipped into squares containing about 10 4 cells. These cells showed characteristic cell proliferation patterns. The growth curves of these cells were generated from the cell proliferation images and we determined the doubling time of these cells from the growth curves. We developed a simple cellular automata system with an easily accessible graphical user interface. This system has five variable parameters, namely, initial cell number, doubling time, motility, cell-cell adhesion, and cell-cell contact inhibition (of proliferation). Within these parameters, we obtained initial cell numbers and doubling times experimentally. We set the motility at a constant value because the effect of the parameter for our simulation was restricted. Therefore, we simulated cell proliferation behavior with cell-cell adhesion and cell-cell contact inhibition as variables. By comparing growth curves and proliferation cell images, we succeeded in determining the cell-cell interaction properties of each cell. Simulated HeLa and HOS cells exhibited low cell-cell adhesion and weak cell-cell contact inhibition. Simulated MSCs exhibited high cell-cell adhesion and positive cell-cell contact inhibition. Simulated A7r5 cells exhibited low cell-cell adhesion and strong cell-cell contact inhibition. These simulated results correlated with the experimental growth curves and proliferation images. Our simulation approach is an easy method for evaluating the cell-cell interaction properties of cells.

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

    Science.gov (United States)

    Marquette, Michele L.; Sognier, Marguerite A.

    2013-01-01

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

  3. Agent-Based Computational Modeling of Cell Culture ...

    Science.gov (United States)

    Quantitative characterization of cellular dose in vitro is needed for alignment of doses in vitro and in vivo. We used the agent-based software, CompuCell3D (CC3D), to provide a stochastic description of cell growth in culture. The model was configured so that isolated cells assumed a “fried egg shape” but became increasingly cuboidal with increasing confluency. The surface area presented by each cell to the overlying medium varies from cell-to-cell and is a determinant of diffusional flux of toxicant from the medium into the cell. Thus, dose varies among cells for a given concentration of toxicant in the medium. Computer code describing diffusion of H2O2 from medium into each cell and clearance of H2O2 was calibrated against H2O2 time-course data (25, 50, or 75 uM H2O2 for 60 min) obtained with the Amplex Red assay for the medium and the H2O2-sensitive fluorescent reporter, HyPer, for cytosol. Cellular H2O2 concentrations peaked at about 5 min and were near baseline by 10 min. The model predicted a skewed distribution of surface areas, with between cell variation usually 2 fold or less. Predicted variability in cellular dose was in rough agreement with the variation in the HyPer data. These results are preliminary, as the model was not calibrated to the morphology of a specific cell type. Future work will involve morphology model calibration against human bronchial epithelial (BEAS-2B) cells. Our results show, however, the potential of agent-based modeling

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

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

    2016-01-01

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

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    Microfluidic biochips offer a promising alternative to a conventional biochemical laboratory. There are two technologies for the microfluidic biochips: droplet-based and flow-based. In this paper we are interested in flow-based microfluidic biochips, where the liquid flows continuously through pre......-defined micro-channels using valves and pumps. We present an approach to the system-level modeling and simulation of a cell culture microfluidic biochip called ProCell, Programmable Cell Culture Chip. ProCell contains a cell culture chamber, which is envisioned to run 256 simultaneous experiments (viewed...

  6. Cell-free DNA in a three-dimensional spheroid cell culture model

    DEFF Research Database (Denmark)

    Aucamp, Janine; Calitz, Carlemi; Bronkhorst, Abel J.

    2017-01-01

    Background Investigating the biological functions of cell-free DNA (cfDNA) is limited by the interference of vast numbers of putative sources and causes of DNA release into circulation. Utilization of three-dimensional (3D) spheroid cell cultures, models with characteristics closer to the in vivo...... cultures can serve as effective, simplified in vivo-simulating “closed-circuit” models since putative sources of cfDNA are limited to only the targeted cells. In addition, cfDNA can also serve as an alternative or auxiliary marker for tracking spheroid growth, development and culture stability. Biological...... significance 3D cell cultures can be used to translate “closed-circuit” in vitro model research into data that is relevant for in vivo studies and clinical applications. In turn, the utilization of cfDNA during 3D culture research can optimize sample collection without affecting the stability of the growth...

  7. Culture models of human mammary epithelial cell transformation

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R.; Yaswen, Paul

    2000-11-10

    Human pre-malignant breast diseases, particularly ductal carcinoma in situ (DCIS)3 already display several of the aberrant phenotypes found in primary breast cancers, including chromosomal abnormalities, telomerase activity, inactivation of the p53 gene and overexpression of some oncogenes. Efforts to model early breast carcinogenesis in human cell cultures have largely involved studies in vitro transformation of normal finite lifespan human mammary epithelial cells (HMEC) to immortality and malignancy. We present a model of HMEC immortal transformation consistent with the know in vivo data. This model includes a recently described, presumably epigenetic process, termed conversion, which occurs in cells that have overcome stringent replicative senescence and are thus able to maintain proliferation with critically short telomeres. The conversion process involves reactivation of telomerase activity, and acquisition of good uniform growth in the absence and presence of TFGB. We propose th at overcoming the proliferative constraints set by senescence, and undergoing conversion, represent key rate-limiting steps in human breast carcinogenesis, and occur during early stage breast cancer progression.

  8. Triple co-culture cell model as an in vitro model for oral particulate vaccine systems

    DEFF Research Database (Denmark)

    Nielsen, Line Hagner; De Rossi, C.; Lehr, C-M.

    ; this was not observed with ovalbumin and blank solution. An example of the results is shown in Figure 2 for IL-17A. An established co-culture of Caco-2, THP-1 and MUTZ-3 cells showed promise as an in vitro model for testing of oral vaccine formulations. Mobility of co-culture immune cells as well as cytokine production......A triple co-culture cell model of Caco-2 cells, dendritic cells and macrophages (Figure 1) has previously been developed for studying intestinal permeability in a state of inflammation [1],[2]. The aim of this study was to investigate the applicability of this cell model for testing...... the model antigen ovalbumin was spray dried to obtain a particulate vaccine model system for testing in the cell model. The precursors were shown to form cubosomes when dispersed in aqueous medium, and was therefore used as the vaccine formulation for testing on the co-cultures. After 11 days, the TEER...

  9. Stochastic modeling of oligodendrocyte generation in cell culture: model validation with time-lapse data

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    Noble Mark

    2006-05-01

    Full Text Available Abstract Background The purpose of this paper is two-fold. The first objective is to validate the assumptions behind a stochastic model developed earlier by these authors to describe oligodendrocyte generation in cell culture. The second is to generate time-lapse data that may help biomathematicians to build stochastic models of cell proliferation and differentiation under other experimental scenarios. Results Using time-lapse video recording it is possible to follow the individual evolutions of different cells within each clone. This experimental technique is very laborious and cannot replace model-based quantitative inference from clonal data. However, it is unrivalled in validating the structure of a stochastic model intended to describe cell proliferation and differentiation at the clonal level. In this paper, such data are reported and analyzed for oligodendrocyte precursor cells cultured in vitro. Conclusion The results strongly support the validity of the most basic assumptions underpinning the previously proposed model of oligodendrocyte development in cell culture. However, there are some discrepancies; the most important is that the contribution of progenitor cell death to cell kinetics in this experimental system has been underestimated.

  10. Good Cell Culture Practice for stem cells and stem-cell-derived models.

    Science.gov (United States)

    Pamies, David; Bal-Price, Anna; Simeonov, Anton; Tagle, Danilo; Allen, Dave; Gerhold, David; Yin, Dezhong; Pistollato, Francesca; Inutsuka, Takashi; Sullivan, Kristie; Stacey, Glyn; Salem, Harry; Leist, Marcel; Daneshian, Mardas; Vemuri, Mohan C; McFarland, Richard; Coecke, Sandra; Fitzpatrick, Suzanne C; Lakshmipathy, Uma; Mack, Amanda; Wang, Wen Bo; Yamazaki, Daiju; Sekino, Yuko; Kanda, Yasunari; Smirnova, Lena; Hartung, Thomas

    2017-01-01

    The first guidance on Good Cell Culture Practice (GCCP) dates back to 2005. This document expands this to include aspects of quality assurance for in vitro cell culture focusing on the increasingly diverse cell types and culture formats used in research, product development, testing and manufacture of biotechnology products and cell-based medicines. It provides a set of basic principles of best practice that can be used in training new personnel, reviewing and improving local procedures, and helping to assure standard practices and conditions for the comparison of data between laboratories and experimentation performed at different times. This includes recommendations for the documentation and reporting of culture conditions. It is intended as guidance to facilitate the generation of reliable data from cell culture systems, and is not intended to conflict with local or higher level legislation or regulatory requirements. It may not be possible to meet all recommendations in this guidance for practical, legal or other reasons. However, when it is necessary to divert from the principles of GCCP, the risk of decreasing the quality of work and the safety of laboratory staff should be addressed and any conclusions or alternative approaches justified. This workshop report is considered a first step toward a revised GCCP 2.0.

  11. Triple co-culture cell model as an in vitro model for oral particulate vaccine systems

    DEFF Research Database (Denmark)

    Nielsen, Line Hagner; De Rossi, C.; Lehr, C.-M.

    the immunostimulatory ability of particulate vaccine formulations designed for oral delivery. Levels of cytokine production in response to vaccine administration were measured following particulate vaccine administration, as an indication of dendritic cell and macrophage activation. Precursors of cubosomes containing......; this was not observed with ovalbumin and blank solution. An example of the results is shown in Figure 2 for IL-17A. An established co-culture of Caco-2, THP-1 and MUTZ-3 cells showed promise as an in vitro model for testing of oral vaccine formulations. Mobility of co-culture immune cells as well as cytokine production...... with particle formulations. This was not the case when incubating with ovalbumin solution or blank. The ELISA screening assay showed production of a wide range of cytokines following culture incubation with cubosomes (with and without ovalbumin) and LPS solutions, indicative of a stimulatory effect...

  12. The Human Glioblastoma Cell Culture Resource: Validated Cell Models Representing All Molecular Subtypes

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    Yuan Xie

    2015-10-01

    Full Text Available Glioblastoma (GBM is the most frequent and malignant form of primary brain tumor. GBM is essentially incurable and its resistance to therapy is attributed to a subpopulation of cells called glioma stem cells (GSCs. To meet the present shortage of relevant GBM cell (GC lines we developed a library of annotated and validated cell lines derived from surgical samples of GBM patients, maintained under conditions to preserve GSC characteristics. This collection, which we call the Human Glioblastoma Cell Culture (HGCC resource, consists of a biobank of 48 GC lines and an associated database containing high-resolution molecular data. We demonstrate that the HGCC lines are tumorigenic, harbor genomic lesions characteristic of GBMs, and represent all four transcriptional subtypes. The HGCC panel provides an open resource for in vitro and in vivo modeling of a large part of GBM diversity useful to both basic and translational GBM research.

  13. The Effect of Primary Cancer Cell Culture Models on the Results of Drug Chemosensitivity Assays: The Application of Perfusion Microbioreactor System as Cell Culture Vessel

    Science.gov (United States)

    Chen, Yi-Dao; Huang, Shiang-Fu; Wang, Hung-Ming

    2015-01-01

    To precisely and faithfully perform cell-based drug chemosensitivity assays, a well-defined and biologically relevant culture condition is required. For the former, a perfusion microbioreactor system capable of providing a stable culture condition was adopted. For the latter, however, little is known about the impact of culture models on the physiology and chemosensitivity assay results of primary oral cavity cancer cells. To address the issues, experiments were performed. Results showed that minor environmental pH change could significantly affect the metabolic activity of cells, demonstrating the importance of stable culture condition for such assays. Moreover, the culture models could also significantly influence the metabolic activity and proliferation of cells. Furthermore, the choice of culture models might lead to different outcomes of chemosensitivity assays. Compared with the similar test based on tumor-level assays, the spheroid model could overestimate the drug resistance of cells to cisplatin, whereas the 2D and 3D culture models might overestimate the chemosensitivity of cells to such anticancer drug. In this study, the 3D culture models with same cell density as that in tumor samples showed comparable chemosensitivity assay results as the tumor-level assays. Overall, this study has provided some fundamental information for establishing a precise and faithful drug chemosensitivity assay. PMID:25654105

  14. Establishment of mouse neuron and microglial cell co-cultured models and its action mechanism.

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    Zhang, Bo; Yang, Yunfeng; Tang, Jun; Tao, Yihao; Jiang, Bing; Chen, Zhi; Feng, Hua; Yang, Liming; Zhu, Gang

    2017-06-27

    The objective of this study is to establish a co-culture model of mouse neurons and microglial cells, and to analyze the mechanism of action of oxygen glucose deprivation (OGD) and transient oxygen glucose deprivation (tOGD) preconditioning cell models. Mouse primary neurons and BV2 microglial cells were successfully cultured, and the OGD and tOGD models were also established. In the co-culture of mouse primary neurons and microglial cells, the cell number of tOGD mouse neurons and microglial cells was larger than the OGD cell number, observed by a microscope. CCK-8 assay result showed that at 1h after treatment, the OD value in the control group is lower compared to all the other three groups (P control group compared to other three groups (P neurons cells were cultured. In the meantime mouse BV2 microglia cells were cultured. Two types of cells were co-cultured, and OGD and tOGD cell models were established. There were four groups in the experiment: control group (OGD), treatment group (tOGD+OGD), placebo group (tOGD+OGD+saline) and minocycline intervention group (tOGD+OGD+minocycline). CCK-8 kit was used to detect cell viability and flow cytometry was used to detect apoptosis. In this study, mouse primary neurons and microglial cells were co-cultured. The OGD and tOGD models were established successfully. tOGD was able to effectively protect neurons and microglial cells from damage, and inhibit the apoptosis caused by oxygen glucose deprivation.

  15. Computer modeling describes gravity-related adaptation in cell cultures.

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    Alexandrov, Ludmil B; Alexandrova, Stoyana; Usheva, Anny

    2009-12-16

    Questions about the changes of biological systems in response to hostile environmental factors are important but not easy to answer. Often, the traditional description with differential equations is difficult due to the overwhelming complexity of the living systems. Another way to describe complex systems is by simulating them with phenomenological models such as the well-known evolutionary agent-based model (EABM). Here we developed an EABM to simulate cell colonies as a multi-agent system that adapts to hyper-gravity in starvation conditions. In the model, the cell's heritable characteristics are generated and transferred randomly to offspring cells. After a qualitative validation of the model at normal gravity, we simulate cellular growth in hyper-gravity conditions. The obtained data are consistent with previously confirmed theoretical and experimental findings for bacterial behavior in environmental changes, including the experimental data from the microgravity Atlantis and the Hypergravity 3000 experiments. Our results demonstrate that it is possible to utilize an EABM with realistic qualitative description to examine the effects of hypergravity and starvation on complex cellular entities.

  16. Quantitative intracellular flux modeling and applications in biotherapeutic development and production using CHO cell cultures.

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    Huang, Zhuangrong; Lee, Dong-Yup; Yoon, Seongkyu

    2017-12-01

    Chinese hamster ovary (CHO) cells have been widely used for producing many recombinant therapeutic proteins. Constraint-based modeling, such as flux balance analysis (FBA) and metabolic flux analysis (MFA), has been developing rapidly for the quantification of intracellular metabolic flux distribution at a systematic level. Such methods would produce detailed maps of flows through metabolic networks, which contribute significantly to better understanding of metabolism in cells. Although these approaches have been extensively established in microbial systems, their application to mammalian cells is sparse. This review brings together the recent development of constraint-based models and their applications in CHO cells. The further development of constraint-based modeling approaches driven by multi-omics datasets is discussed, and a framework of potential modeling application in cell culture engineering is proposed. Improved cell culture system understanding will enable robust developments in cell line and bioprocess engineering thus accelerating consistent process quality control in biopharmaceutical manufacturing. © 2017 Wiley Periodicals, Inc.

  17. A carbon dioxide stripping model for mammalian cell culture in manufacturing scale bioreactors.

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    Xing, Zizhuo; Lewis, Amanda M; Borys, Michael C; Li, Zheng Jian

    2017-06-01

    Control of carbon dioxide within the optimum range is important in mammalian bioprocesses at the manufacturing scale in order to ensure robust cell growth, high protein yields, and consistent quality attributes. The majority of bioprocess development work is done in laboratory bioreactors, in which carbon dioxide levels are more easily controlled. Some challenges in carbon dioxide control can present themselves when cell culture processes are scaled up, because carbon dioxide accumulation is a common feature due to longer gas-residence time of mammalian cell culture in large scale bioreactors. A carbon dioxide stripping model can be used to better understand and optimize parameters that are critical to cell culture processes at the manufacturing scale. The prevailing carbon dioxide stripping models in literature depend on mass transfer coefficients and were applicable to cell culture processes with low cell density or at stationary/cell death phase. However, it was reported that gas bubbles are saturated with carbon dioxide before leaving the culture, which makes carbon dioxide stripping no longer depend on a mass transfer coefficient in the new generation cell culture processes characterized by longer exponential growth phase, higher peak viable cell densities, and higher specific production rate. Here, we present a new carbon dioxide stripping model for manufacturing scale bioreactors, which is independent of carbon dioxide mass transfer coefficient, but takes into account the gas-residence time and gas CO 2 saturation time. The model was verified by CHO cell culture processes with different peak viable cell densities (7 to 12 × 10 6  cells mL -1 ) for two products in 5,000-L and 25,000-L bioreactors. The model was also applied to a next generation cell culture process to optimize cell culture conditions and reduce carbon dioxide levels at manufacturing scale. The model provides a useful tool to understand and better control cell culture carbon dioxide

  18. Chromosomal instability and telomere shortening in long-term culture of hematopoietic stem cells: insights from a cell culture model of RPS14 haploinsufficiency.

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    Thomay, K; Schienke, A; Vajen, B; Modlich, U; Schambach, A; Hofmann, W; Schlegelberger, B; Göhring, G

    2014-01-01

    The fate of cultivated primary hematopoietic stem cells (HSCs) with respect to genetic instability and telomere attrition has not yet been described in great detail. Thus, knowledge of the genetic constitution of HSCs is important when interpreting results of HSCs in culture. While establishing a cell culture model for myelodysplastic syndrome with a deletion in 5q by performing RPS14 knockdown, we found surprising data that may be of importance for any CD34+ cell culture experiments. We performed cytogenetic analyses and telomere length measurement on transduced CD34+ cells and untransduced control cells to observe the effects of long-term culturing. Initially, CD34+ cells had a normal median telomere length of about 12 kb and showed no signs of chromosomal instability. During follow-up, the median telomere length seemed to decrease and, simultaneously, increased chromosomal instability could be observed - in modified and control cells. One culture showed a clonal monosomy 7 - independent of prior RPS14 knockdown. During further culturing, it seemed that the telomeres re-elongated, and chromosomes stabilized, while TERT expression was not elevated. In summary, irrespective of our results of RPS14 knockdown in the long-term culture of CD34+ cells, it becomes clear that cell culture artefacts inducing telomere shortening and chromosomal instability have to be taken into account and regular cytogenetic analyses should always be performed. © 2013 S. Karger AG, Basel.

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

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    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

  20. Stochastic cellular automata model of cell migration, proliferation and differentiation: validation with in vitro cultures of muscle satellite cells.

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    Garijo, N; Manzano, R; Osta, R; Perez, M A

    2012-12-07

    Cell migration and proliferation has been modelled in the literature as a process similar to diffusion. However, using diffusion models to simulate the proliferation and migration of cells tends to create a homogeneous distribution in the cell density that does not correlate to empirical observations. In fact, the mechanism of cell dispersal is not diffusion. Cells disperse by crawling or proliferation, or are transported in a moving fluid. The use of cellular automata, particle models or cell-based models can overcome this limitation. This paper presents a stochastic cellular automata model to simulate the proliferation, migration and differentiation of cells. These processes are considered as completely stochastic as well as discrete. The model developed was applied to predict the behaviour of in vitro cell cultures performed with adult muscle satellite cells. Moreover, non homogeneous distribution of cells has been observed inside the culture well and, using the above mentioned stochastic cellular automata model, we have been able to predict this heterogeneous cell distribution and compute accurate quantitative results. Differentiation was also incorporated into the computational simulation. The results predicted the myotube formation that typically occurs with adult muscle satellite cells. In conclusion, we have shown how a stochastic cellular automata model can be implemented and is capable of reproducing the in vitro behaviour of adult muscle satellite cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. A porcine astrocyte/endothelial cell co-culture model of the blood-brain barrier.

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    Jeliazkova-Mecheva, Valentina V; Bobilya, Dennis J

    2003-10-01

    A method for the isolation of porcine atrocytes as a simple extension of a previously described procedure for isolation of brain capillary endothelial cells from adolescent pigs [Methods Cell Sci. 17 (1995) 2] is described. The obtained astroglial culture purified through two passages and by the method of the selective detachment was validated by a phase contrast microscopy and through an immunofluorescent assay for the glial fibrillary acidic protein (GFAP). Porcine astrocytes were co-cultivated with porcine brain capillary endothelial cells (PBCEC) for the development of an in vitro blood-brain barrier (BBB) model. The model was visualized by an electron microscopy and showed elevated transendothellial electrical resistance and reduced inulin permeability. To our knowledge, this is the first report for the establishment of a porcine astrocyte/endothelial cell co-culture BBB model, which avoids interspecies and age differences between the two cell types, usually encountered in the other reported co-culture BBB models. Considering the availability of the porcine brain tissue and the close physiological and anatomical relation between the human and pig brain, the porcine astrocyte/endothelial cell co-culture system can serve as a reliable and easily reproducible model for different in vitro BBB studies.

  2. Establishment and characterization of a differentiated epithelial cell culture model derived from the porcine cervix uteri

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    Miessen Katrin

    2012-03-01

    Full Text Available Abstract Background Cervical uterine epithelial cells maintain a physiological and pathogen-free milieu in the female mammalian reproductive tract and are involved in sperm-epithelium interaction. Easily accessible, differentiated model systems of the cervical epithelium are not yet available to elucidate the underlying molecular mechanisms within these highly specialized cells. Therefore, the aim of the study was to establish a cell culture of the porcine cervical epithelium representing in vivo-like properties of the tissue. Results We tested different isolation methods and culture conditions and validated purity of the cultured cells by immunohistochemistry against keratins. We could reproducibly culture pure epithelial cells from cervical tissue explants. Based on a morphology score and the WST-1 Proliferation Assay, we optimized the growth medium composition. Primary porcine cervical cells performed best in conditioned Ham's F-12, containing 10% FCS, EGF and insulin. After cultivation in an air-liquid interface for three weeks, the cells showed a discontinuously multilayered phenotype. Finally, differentiation was validated via immunohistochemistry against beta catenin. Mucopolysaccharide production could be shown via alcian blue staining. Conclusions We provide the first suitable protocol to establish a differentiated porcine epithelial model of the cervix uteri, based on easily accessible cells using slaughterhouse material.

  3. Establishment and characterization of a differentiated epithelial cell culture model derived from the porcine cervix uteri.

    Science.gov (United States)

    Miessen, Katrin; Einspanier, Ralf; Schoen, Jennifer

    2012-03-19

    Cervical uterine epithelial cells maintain a physiological and pathogen-free milieu in the female mammalian reproductive tract and are involved in sperm-epithelium interaction. Easily accessible, differentiated model systems of the cervical epithelium are not yet available to elucidate the underlying molecular mechanisms within these highly specialized cells. Therefore, the aim of the study was to establish a cell culture of the porcine cervical epithelium representing in vivo-like properties of the tissue. We tested different isolation methods and culture conditions and validated purity of the cultured cells by immunohistochemistry against keratins. We could reproducibly culture pure epithelial cells from cervical tissue explants. Based on a morphology score and the WST-1 Proliferation Assay, we optimized the growth medium composition. Primary porcine cervical cells performed best in conditioned Ham's F-12, containing 10% FCS, EGF and insulin. After cultivation in an air-liquid interface for three weeks, the cells showed a discontinuously multilayered phenotype. Finally, differentiation was validated via immunohistochemistry against beta catenin. Mucopolysaccharide production could be shown via alcian blue staining. We provide the first suitable protocol to establish a differentiated porcine epithelial model of the cervix uteri, based on easily accessible cells using slaughterhouse material.

  4. Three-Dimensional Cell Culture Models for Infectious Disease and Drug Development

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    Nickerson, Cheryl A.; Honer zu Bentrup, Kerstin; Ott, C. Mark

    2005-01-01

    Three-dimensional (3-D) cell cultures hold enormous potential to advance our understanding of infectious disease and to effectively translate basic cellular research into clinical applications. Using novel NASA bioreactor technology, the rotating wall vessel (RWV), we have engineered physiologically relevant 3-D human tissue culture models for infectious disease studies. The design of the RWV is based on the understanding that organs and tissues function in a 3-D environment, and that this 3-D architecture is critical for the differentiated form and function of tissues in vivo. The RWV provides large numbers of cells which are amenable to a wide variety of experimental manipulations and provides an easy, reproducible, and cost-effective approach to enhance differentiated features of cell culture models.

  5. Continuous Release of Tumor-Derived Factors Improves the Modeling of Cachexia in Muscle Cell Culture

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    Robert W. Jackman

    2017-09-01

    Full Text Available Cachexia is strongly associated with a poor prognosis in cancer patients but the biological trigger is unknown and therefore no therapeutics exist. The loss of skeletal muscle is the most deleterious aspect of cachexia and it appears to depend on secretions from tumor cells. Models for studying wasting in cell culture consist of experiments where skeletal muscle cells are incubated with medium conditioned by tumor cells. This has led to candidates for cachectic factors but some of the features of cachexia in vivo are not yet well-modeled in cell culture experiments. Mouse myotube atrophy measured by myotube diameter in response to medium conditioned by mouse colon carcinoma cells (C26 is consistently less than what is seen in muscles of mice bearing C26 tumors with moderate to severe cachexia. One possible reason for this discrepancy is that in vivo the C26 tumor and skeletal muscle share a circulatory system exposing the muscle to tumor factors in a constant and increasing way. We have applied Transwell®-adapted cell culture conditions to more closely simulate conditions found in vivo where muscle is exposed to the ongoing kinetics of constant tumor secretion of active factors. C26 cells were incubated on a microporous membrane (a Transwell® insert that constitutes the upper compartment of wells containing plated myotubes. In this model, myotubes are exposed to a constant supply of cancer cell secretions in the medium but without direct contact with the cancer cells, analogous to a shared circulation of muscle and cancer cells in tumor-bearing animals. The results for myotube diameter support the idea that the use of Transwell® inserts serves as a more physiological model of the muscle wasting associated with cancer cachexia than the bolus addition of cancer cell conditioned medium. The Transwell® model supports the notion that the dose and kinetics of cachectic factor delivery to muscle play a significant role in the extent of pathology.

  6. A Simplified Model of Glycoprotein Production within Cell Culture

    OpenAIRE

    Lambert, A. B.; Smith, F. T.; Velayudhan, A.

    2017-01-01

    Complex biological products, such as those used to treat various forms of cancer, are typically produced by mammalian cells in bioreactors. The most important class of such biological medicines is proteins. These typically bind to sugars (glycans) in a process known as glycosylation, creating glycoproteins, which are more stable and effective medicines. The glycans are large polymers that are formed by a long sequence of enzyme catalysed reactions. This sequence is not always completed, thus ...

  7. Advanced Good Cell Culture Practice for human primary, stem cell-derived and organoid models as well as microphysiological systems.

    Science.gov (United States)

    Pamies, David; Bal-Price, Anna; Chesné, Christophe; Coecke, Sandra; Dinnyes, Andras; Eskes, Chantra; Grillari, Regina; Gstraunthaler, Gerhard; Hartung, Thomas; Jennings, Paul; Leist, Marcel; Martin, Ulrich; Passier, Robert; Schwamborn, Jens C; Stacey, Glyn N; Ellinger-Ziegelbauer, Heidrun; Daneshian, Mardas

    2018-04-13

    A major reason for the current reproducibility crisis in the life sciences is the poor implementation of quality control measures and reporting standards. Improvement is needed, especially regarding increasingly complex in vitro methods. Good Cell Culture Practice (GCCP) was an effort from 1996 to 2005 to develop such minimum quality standards also applicable in academia. This paper summarizes recent key developments in in vitro cell culture and addresses the issues resulting for GCCP, e.g. the development of induced pluripotent stem cells (iPSCs) and gene-edited cells. It further deals with human stem-cell-derived models and bioengineering of organo-typic cell cultures, including organoids, organ-on-chip and human-on-chip approaches. Commercial vendors and cell banks have made human primary cells more widely available over the last decade, increasing their use, but also requiring specific guidance as to GCCP. The characterization of cell culture systems including high-content imaging and high-throughput measurement technologies increasingly combined with more complex cell and tissue cultures represent a further challenge for GCCP. The increasing use of gene editing techniques to generate and modify in vitro culture models also requires discussion of its impact on GCCP. International (often varying) legislations and market forces originating from the commercialization of cell and tissue products and technologies are further impacting on the need for the use of GCCP. This report summarizes the recommendations of the second of two workshops, held in Germany in December 2015, aiming map the challenge and organize the process or developing a revised GCCP 2.0.

  8. Cell lineage identification and stem cell culture in a porcine model for the study of intestinal epithelial regeneration.

    Directory of Open Access Journals (Sweden)

    Liara M Gonzalez

    Full Text Available Significant advances in intestinal stem cell biology have been made in murine models; however, anatomical and physiological differences between mice and humans limit mice as a translational model for stem cell based research. The pig has been an effective translational model, and represents a candidate species to study intestinal epithelial stem cell (IESC driven regeneration. The lack of validated reagents and epithelial culture methods is an obstacle to investigating IESC driven regeneration in a pig model. In this study, antibodies against Epithelial Adhesion Molecule 1 (EpCAM and Villin marked cells of epithelial origin. Antibodies against Proliferative Cell Nuclear Antigen (PCNA, Minichromosome Maintenance Complex 2 (MCM2, Bromodeoxyuridine (BrdU and phosphorylated Histone H3 (pH3 distinguished proliferating cells at various stages of the cell cycle. SOX9, localized to the stem/progenitor cells zone, while HOPX was restricted to the +4/'reserve' stem cell zone. Immunostaining also identified major differentiated lineages. Goblet cells were identified by Mucin 2 (MUC2; enteroendocrine cells by Chromogranin A (CGA, Gastrin and Somatostatin; and absorptive enterocytes by carbonic anhydrase II (CAII and sucrase isomaltase (SIM. Transmission electron microscopy demonstrated morphologic and sub-cellular characteristics of stem cell and differentiated intestinal epithelial cell types. Quantitative PCR gene expression analysis enabled identification of stem/progenitor cells, post mitotic cell lineages, and important growth and differentiation pathways. Additionally, a method for long-term culture of porcine crypts was developed. Biomarker characterization and development of IESC culture in the porcine model represents a foundation for translational studies of IESC-driven regeneration of the intestinal epithelium in physiology and disease.

  9. A co-cultured skin model based on cell support membranes

    International Nuclear Information System (INIS)

    Dai, N.-T.; Yeh, M.-K.; Liu, Demeral David; Adams, E.F.; Chiang, C.-H.; Yen, C.-Y.; Shih, C.-M.; Sytwu, H.-K.; Chen, Tim-Mo; Wang, H.-J.; Williamson, M.R.; Coombes, A.G.A.

    2005-01-01

    Tissue engineering of skin based on collagen: PCL biocomposites using a designed co-culture system is reported. The collagen: PCL biocomposites having collagen: PCL (w/w) ratios of 1:4, 1:8, and 1:20 have been proven to be biocompatible materials to support both adult normal human epidermal Keratinocyte (NHEK) and mouse 3T3 fibroblast growth in cell culture, respectively, by Dai, Coombes, et al. in 2004. Films of collagen: PCL biocomposites were prepared using non-crosslinking method by impregnation of lyophilized collagen mats with PCL/dichloromethane solutions followed by solvent evaporation. To mimic the dermal/epidermal structure of skin, the 1:20 collagen: PCL biocomposites were selected for a feasibility study of a designed co-culture technique that would subsequently be used for preparing fibroblast/biocomposite/keratinocyte skin models. A 55.3% increase in cell number was measured in the designed co-culture system when fibroblasts were seeded on both sides of a biocomposite film compared with cell culture on one surface of the biocomposite in the feasibility study. The co-culture of human keratinocytes and 3T3 fibroblasts on each side of the membrane was therefore studied using the same co-culture system by growing keratinocytes on the top surface of membrane for 3 days and 3T3 fibroblasts underneath the membrane for 6 days. Scanning electron microscopy (SEM) and immunohistochemistry assay revealed good cell attachment and proliferation of both human keratinocytes and 3T3 fibroblasts with these two types of cells isolated well on each side of the membrane. Using a modified co-culture technique, a co-cultured skin model presenting a confluent epidermal sheet on one side of the biocomposite film and fibroblasts populated on the other side of the film was developed successfully in co-culture system for 28 days under investigations by SEM and immunohistochemistry assay. Thus, the design of a co-culture system based on 1:20 (w/w) collagen: PCL biocomposite

  10. Insect Cell Culture

    NARCIS (Netherlands)

    Oers, van M.M.; Lynn, D.E.

    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

  11. Interaction of lipid nanoparticles with human epidermis and an organotypic cell culture model

    DEFF Research Database (Denmark)

    Kuntsche, Judith; Bunjes, Heike; Fahr, Alfred

    2008-01-01

    Various lipid nanoparticle formulations were investigated with respect to (trans)dermal drug delivery with special regard to the mechanism of their effects on human and an organotypic cell culture epidermis. Potential alterations of stratum corneum lipid domains were studied using fluorescence...... assays with labeled liposomes and thermal analysis of isolated stratum corneum. Influences on the permeation of corticosterone were investigated and the occlusive properties of the nanoparticles were determined by measurements of the transepidermal water loss (TEWL). The penetration of a fluorescence dye...... studies and thermal analysis of human and cell culture epidermis indicate that surface lipids, which are not present to the same extent in the cell culture model than in human epidermis, seem to play an important role....

  12. Tumor cell culture on collagen–chitosan scaffolds as three-dimensional tumor model: A suitable model for tumor studies

    Directory of Open Access Journals (Sweden)

    Aziz Mahmoudzadeh

    2016-07-01

    Full Text Available Tumor cells naturally live in three-dimensional (3D microenvironments, while common laboratory tests and evaluations are done in two-dimensional (2D plates. This study examined the impact of cultured 4T1 cancer cells in a 3D collagen–chitosan scaffold compared with 2D plate cultures. Collagen–chitosan scaffolds were provided and passed confirmatory tests. 4T1 tumor cells were cultured on scaffolds and then tumor cells growth rate, resistance to X-ray radiation, and cyclophosphamide as a chemotherapy drug were analyzed. Furthermore, 4T1 cells were extracted from the scaffold model and were injected into the mice. Tumor growth rate, survival rate, and systemic immune responses were evaluated. Our results showed that 4T1 cells infiltrated the scaffolds pores and constructed a 3D microenvironment. Furthermore, 3D cultured tumor cells showed a slower proliferation rate, increased levels of survival to the X-ray irradiation, and enhanced resistance to chemotherapy drugs in comparison with 2D plate cultures. Transfer of extracted cells to the mice caused enhanced tumor volume and decreased life span. This study indicated that collagen–chitosan nanoscaffolds provide a suitable model of tumor that would be appropriate for tumor studies.

  13. Tumor cell culture on collagen-chitosan scaffolds as three-dimensional tumor model: A suitable model for tumor studies.

    Science.gov (United States)

    Mahmoudzadeh, Aziz; Mohammadpour, Hemn

    2016-07-01

    Tumor cells naturally live in three-dimensional (3D) microenvironments, while common laboratory tests and evaluations are done in two-dimensional (2D) plates. This study examined the impact of cultured 4T1 cancer cells in a 3D collagen-chitosan scaffold compared with 2D plate cultures. Collagen-chitosan scaffolds were provided and passed confirmatory tests. 4T1 tumor cells were cultured on scaffolds and then tumor cells growth rate, resistance to X-ray radiation, and cyclophosphamide as a chemotherapy drug were analyzed. Furthermore, 4T1 cells were extracted from the scaffold model and were injected into the mice. Tumor growth rate, survival rate, and systemic immune responses were evaluated. Our results showed that 4T1 cells infiltrated the scaffolds pores and constructed a 3D microenvironment. Furthermore, 3D cultured tumor cells showed a slower proliferation rate, increased levels of survival to the X-ray irradiation, and enhanced resistance to chemotherapy drugs in comparison with 2D plate cultures. Transfer of extracted cells to the mice caused enhanced tumor volume and decreased life span. This study indicated that collagen-chitosan nanoscaffolds provide a suitable model of tumor that would be appropriate for tumor studies. Copyright © 2016. Published by Elsevier B.V.

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

    Directory of Open Access Journals (Sweden)

    Stefan Przyborski

    2017-06-01

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

  15. Culture in embryonic kidney serum and xeno-free media as renal cell carcinoma and renal cell carcinoma cancer stem cells research model.

    Science.gov (United States)

    Krawczyk, Krzysztof M; Matak, Damian; Szymanski, Lukasz; Szczylik, Cezary; Porta, Camillo; Czarnecka, Anna M

    2018-04-01

    The use of fetal bovine serum hinders obtaining reproducible experimental results and should also be removed in hormone and growth factor studies. In particular hormones found in FBS act globally on cancer cell physiology and influence transcriptome and metabolome. The aim of our study was to develop a renal carcinoma serum free culture model optimized for (embryonal) renal cells in order to select the best study model for downstream auto-, para- or endocrine research. Secondary aim was to verify renal carcinoma stem cell culture for this application. In the study, we have cultured renal cell carcinoma primary tumour cell line (786-0) as well as human kidney cancer stem cells in standard 2D monolayer cultures in Roswell Park Memorial Institute Medium or Dulbecco's Modified Eagle's Medium and Complete Human Kidney Cancer Stem Cell Medium, respectively. Serum-free, animal-component free Human Embryonic Kidney 293 media were tested. Our results revealed that xeno-free embryonal renal cells optimized culture media provide a useful tool in RCC cancer biology research and at the same time enable effective growth of RCC. We propose bio-mimic RCC cell culture model with specific serum-free and xeno-free medium that promote RCC cell viability.

  16. Impaired development of cortico-striatal synaptic connectivity in a cell culture model of Huntington's disease.

    Science.gov (United States)

    Buren, Caodu; Parsons, Matthew P; Smith-Dijak, Amy; Raymond, Lynn A

    2016-03-01

    Huntington's disease (HD) is a genetically inherited neurodegenerative disease caused by a mutation in the gene encoding the huntingtin protein. This mutation results in progressive cell death that is particularly striking in the striatum. Recent evidence indicates that early HD is initially a disease of the synapse, in which subtle alterations in synaptic neurotransmission, particularly at the cortico-striatal (C-S) synapse, can be detected well in advance of cell death. Here, we used a cell culture model in which striatal neurons are co-cultured with cortical neurons, and monitored the development of C-S connectivity up to 21days in vitro (DIV) in cells cultured from either the YAC128 mouse model of HD or the background strain, FVB/N (wild-type; WT) mice. Our data demonstrate that while C-S connectivity in WT co-cultures develops rapidly and continuously from DIV 7 to 21, YAC128 C-S connectivity shows no significant growth from DIV 14 onward. Morphological and electrophysiological data suggest that a combination of pre- and postsynaptic mechanisms contribute to this effect, including a reduction in both the postsynaptic dendritic arborization and the size and replenishment rate of the presynaptic readily releasable pool of excitatory vesicles. Moreover, a chimeric culture strategy confirmed that the most robust impairment in C-S connectivity was only observed when mutant huntingtin was expressed both pre- and postsynaptically. In all, our data demonstrate a progressive HD synaptic phenotype in this co-culture system that may be exploited as a platform for identifying promising therapeutic strategies to prevent early HD-associated synaptopathy. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Nicotine permeability across the buccal TR146 cell culture model and porcine buccal mucosa in vitro

    DEFF Research Database (Denmark)

    Nielsen, Hanne Mørck; Rassing, Margrethe Rømer

    2002-01-01

    The present study was conducted to investigate and compare the effect of pH and drug concentration on nicotine permeability across the TR146 cell culture model and porcine buccal mucosa in vitro. As a further characterization of the TR146 cell culture model, it was explored whether the results were...... comparable for bi-directional and uni-directional transport in the presence of a transmembrane pH gradient. Nicotine concentrations between 10(-5) and 10(-2) M were applied to the apical side of the TR146 cell culture model or the mucosal side of porcine buccal mucosa. Buffers with pH values of 5.5, 7.......4 and 8.1 were used to obtain different fractions of non- and mono-ionized nicotine. The apparent permeability (P(app)) of nicotine across both models increased significantly with increasing pH, and the P(app) values obtained with the two models could be correlated in a linear manner. With increasing...

  18. Mammalian Cell Culture Process for Monoclonal Antibody Production: Nonlinear Modelling and Parameter Estimation

    Directory of Open Access Journals (Sweden)

    Dan Selişteanu

    2015-01-01

    Full Text Available Monoclonal antibodies (mAbs are at present one of the fastest growing products of pharmaceutical industry, with widespread applications in biochemistry, biology, and medicine. The operation of mAbs production processes is predominantly based on empirical knowledge, the improvements being achieved by using trial-and-error experiments and precedent practices. The nonlinearity of these processes and the absence of suitable instrumentation require an enhanced modelling effort and modern kinetic parameter estimation strategies. The present work is dedicated to nonlinear dynamic modelling and parameter estimation for a mammalian cell culture process used for mAb production. By using a dynamical model of such kind of processes, an optimization-based technique for estimation of kinetic parameters in the model of mammalian cell culture process is developed. The estimation is achieved as a result of minimizing an error function by a particle swarm optimization (PSO algorithm. The proposed estimation approach is analyzed in this work by using a particular model of mammalian cell culture, as a case study, but is generic for this class of bioprocesses. The presented case study shows that the proposed parameter estimation technique provides a more accurate simulation of the experimentally observed process behaviour than reported in previous studies.

  19. The importance of connexin hemichannels during chondroprogenitor cell differentiation in hydrogel versus microtissue culture models.

    Science.gov (United States)

    Schrobback, Karsten; Klein, Travis Jacob; Woodfield, Tim B F

    2015-06-01

    Appropriate selection of scaffold architecture is a key challenge in cartilage tissue engineering. Gap junction-mediated intercellular contacts play important roles in precartilage condensation of mesenchymal cells. However, scaffold architecture could potentially restrict cell-cell communication and differentiation. This is particularly important when choosing the appropriate culture platform as well as scaffold-based strategy for clinical translation, that is, hydrogel or microtissues, for investigating differentiation of chondroprogenitor cells in cartilage tissue engineering. We, therefore, studied the influence of gap junction-mediated cell-cell communication on chondrogenesis of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and articular chondrocytes. Expanded human chondrocytes and BM-MSCs were either (re-) differentiated in micromass cell pellets or encapsulated as isolated cells in alginate hydrogels. Samples were treated with and without the gap junction inhibitor 18-α glycyrrhetinic acid (18αGCA). DNA and glycosaminoglycan (GAG) content and gene expression levels (collagen I/II/X, aggrecan, and connexin 43) were quantified at various time points. Protein localization was determined using immunofluorescence, and adenosine-5'-triphosphate (ATP) was measured in conditioned media. While GAG/DNA was higher in alginate compared with pellets for chondrocytes, there were no differences in chondrogenic gene expression between culture models. Gap junction blocking reduced collagen II and extracellular ATP in all chondrocyte cultures and in BM-MSC hydrogels. However, differentiation capacity was not abolished completely by 18αGCA. Connexin 43 levels were high throughout chondrocyte cultures and peaked only later during BM-MSC differentiation, consistent with the delayed response of BM-MSCs to 18αGCA. Alginate hydrogels and microtissues are equally suited culture platforms for the chondrogenic (re-)differentiation of expanded human articular

  20. Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model.

    Science.gov (United States)

    Pilgrim, Matthew G; Lengyel, Imre; Lanzirotti, Antonio; Newville, Matt; Fearn, Sarah; Emri, Eszter; Knowles, Jonathan C; Messinger, Jeffrey D; Read, Russell W; Guidry, Clyde; Curcio, Christine A

    2017-02-01

    Extracellular deposits containing hydroxyapatite, lipids, proteins, and trace metals that form between the basal lamina of the RPE and the inner collagenous layer of Bruch's membrane are hallmarks of early AMD. We examined whether cultured RPE cells could produce extracellular deposits containing all of these molecular components. Retinal pigment epithelium cells isolated from freshly enucleated porcine eyes were cultured on Transwell membranes for up to 6 months. Deposit composition and structure were characterized using light, fluorescence, and electron microscopy; synchrotron x-ray diffraction and x-ray fluorescence; secondary ion mass spectroscopy; and immunohistochemistry. Apparently functional primary RPE cells, when cultured on 10-μm-thick inserts with 0.4-μm-diameter pores, can produce sub-RPE deposits that contain hydroxyapatite, lipids, proteins, and trace elements, without outer segment supplementation, by 12 weeks. The data suggest that sub-RPE deposit formation is initiated, and probably regulated, by the RPE, as well as the loss of permeability of the Bruch's membrane and choriocapillaris complex associated with age and early AMD. This cell culture model of early AMD lesions provides a novel system for testing new therapeutic interventions against sub-RPE deposit formation, an event occurring well in advance of the onset of vision loss.

  1. A population balance equation model of aggregation dynamics in Taxus suspension cell cultures.

    Science.gov (United States)

    Kolewe, Martin E; Roberts, Susan C; Henson, Michael A

    2012-02-01

    The nature of plant cells to grow as multicellular aggregates in suspension culture has profound effects on bioprocess performance. Recent advances in the measurement of plant cell aggregate size allow for routine process monitoring of this property. We have exploited this capability to develop a conceptual model to describe changes in the aggregate size distribution that are observed over the course of a Taxus cell suspension batch culture. We utilized the population balance equation framework to describe plant cell aggregates as a particulate system, accounting for the relevant phenomenological processes underlying aggregation, such as growth and breakage. We compared model predictions to experimental data to select appropriate kernel functions, and found that larger aggregates had a higher breakage rate, biomass was partitioned asymmetrically following a breakage event, and aggregates grew exponentially. Our model was then validated against several datasets with different initial aggregate size distributions and was able to quantitatively predict changes in total biomass and mean aggregate size, as well as actual size distributions. We proposed a breakage mechanism where a fraction of biomass was lost upon each breakage event, and demonstrated that even though smaller aggregates have been shown to produce more paclitaxel, an optimum breakage rate was predicted for maximum paclitaxel accumulation. We believe this is the first model to use a segregated, corpuscular approach to describe changes in the size distribution of plant cell aggregates, and represents an important first step in the design of rational strategies to control aggregation and optimize process performance. Copyright © 2011 Wiley Periodicals, Inc.

  2. Brain Aggregates: An Effective In Vitro Cell Culture System Modeling Neurodegenerative Diseases.

    Science.gov (United States)

    Ahn, Misol; Kalume, Franck; Pitstick, Rose; Oehler, Abby; Carlson, George; DeArmond, Stephen J

    2016-03-01

    Drug discovery for neurodegenerative diseases is particularly challenging because of the discrepancies in drug effects between in vitro and in vivo studies. These discrepancies occur in part because current cell culture systems used for drug screening have many limitations. First, few cell culture systems accurately model human aging or neurodegenerative diseases. Second, drug efficacy may differ between dividing and stationary cells, the latter resembling nondividing neurons in the CNS. Brain aggregates (BrnAggs) derived from embryonic day 15 gestation mouse embryos may represent neuropathogenic processes in prion disease and reflect in vivo drug efficacy. Here, we report a new method for the production of BrnAggs suitable for drug screening and suggest that BrnAggs can model additional neurological diseases such as tauopathies. We also report a functional assay with BrnAggs by measuring electrophysiological activities. Our data suggest that BrnAggs could serve as an effective in vitro cell culture system for drug discovery for neurodegenerative diseases. © 2016 American Association of Neuropathologists, Inc. All rights reserved.

  3. The potential of chitosan in enhancing peptide and protein absorption across the TR146 cell culture model-an in vitro model of the buccal epithelium

    DEFF Research Database (Denmark)

    Portero, Ana; Remuñán-López, Carmen; Nielsen, Hanne Mørck

    2002-01-01

    To investigate the potential of chitosan (CS) to enhance buccal peptide and protein absorption, the TR146 cell culture model, a model of the buccal epithelium, was used.......To investigate the potential of chitosan (CS) to enhance buccal peptide and protein absorption, the TR146 cell culture model, a model of the buccal epithelium, was used....

  4. A 3D human neural cell culture system for modeling Alzheimer’s disease

    Science.gov (United States)

    Kim, Young Hye; Choi, Se Hoon; D’Avanzo, Carla; Hebisch, Matthias; Sliwinski, Christopher; Bylykbashi, Enjana; Washicosky, Kevin J.; Klee, Justin B.; Brüstle, Oliver; Tanzi, Rudolph E.; Kim, Doo Yeon

    2015-01-01

    Stem cell technologies have facilitated the development of human cellular disease models that can be used to study pathogenesis and test therapeutic candidates. These models hold promise for complex neurological diseases such as Alzheimer’s disease (AD) because existing animal models have been unable to fully recapitulate all aspects of pathology. We recently reported the characterization of a novel three-dimensional (3D) culture system that exhibits key events in AD pathogenesis, including extracellular aggregation of β-amyloid and accumulation of hyperphosphorylated tau. Here we provide instructions for the generation and analysis of 3D human neural cell cultures, including the production of genetically modified human neural progenitor cells (hNPCs) with familial AD mutations, the differentiation of the hNPCs in a 3D matrix, and the analysis of AD pathogenesis. The 3D culture generation takes 1–2 days. The aggregation of β-amyloid is observed after 6-weeks of differentiation followed by robust tau pathology after 10–14 weeks. PMID:26068894

  5. Basics elements for modelling the dynamics of cell migration in cell culture

    International Nuclear Information System (INIS)

    FarIas, Ro; Vidal, Cs; Rapacioli, M; Flores, V

    2007-01-01

    This paper introduces some basic elements for modelling the dynamics of cell migration activity over a bi-dimensional substratum. A square matrix, representing the substratum, is implemented in order to generate virtual cells with an initial random uniform distribution, with the ability to freely move within the matrix and to interact with each others by mean of adhesive forces. Two different conditions were examined: A) cells can freely move and after contacting with another cell they both completely inhibit their migration; B) cells that come into contact have the ability to rotate respect to each other without losing their contacts and retaining the ability to move together but at a slower rate, being the decrease in the rate of movement proportional to the number of contacting cells. The dynamics of the migration process in these two conditions was evaluated by recording the evolution of several parameters as a function of time. Minor modifications in some parameters (mobility, intensity of cell-cell and cell-substratum adhesiveness) significantly change the dynamics and the final result of the virtual migrating cells

  6. Cell Culture Made Easy.

    Science.gov (United States)

    Dye, Frank J.

    1985-01-01

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

  7. Macroscopic Dynamic Modeling of Sequential Batch Cultures of Hybridoma Cells: An Experimental Validation

    Directory of Open Access Journals (Sweden)

    Laurent Dewasme

    2017-02-01

    Full Text Available Hybridoma cells are commonly grown for the production of monoclonal antibodies (MAb. For monitoring and control purposes of the bioreactors, dynamic models of the cultures are required. However these models are difficult to infer from the usually limited amount of available experimental data and do not focus on target protein production optimization. This paper explores an experimental case study where hybridoma cells are grown in a sequential batch reactor. The simplest macroscopic reaction scheme translating the data is first derived using a maximum likelihood principal component analysis. Subsequently, nonlinear least-squares estimation is used to determine the kinetic laws. The resulting dynamic model reproduces quite satisfactorily the experimental data, as evidenced in direct and cross-validation tests. Furthermore, model predictions can also be used to predict optimal medium renewal time and composition.

  8. Efficient generation of patient-matched malignant and normal primary cell cultures from clear cell renal cell carcinoma patients: clinically relevant models for research and personalized medicine

    International Nuclear Information System (INIS)

    Lobo, Nazleen C.; Gedye, Craig; Apostoli, Anthony J.; Brown, Kevin R.; Paterson, Joshua; Stickle, Natalie; Robinette, Michael; Fleshner, Neil; Hamilton, Robert J.; Kulkarni, Girish; Zlotta, Alexandre; Evans, Andrew; Finelli, Antonio; Moffat, Jason; Jewett, Michael A. S.; Ailles, Laurie

    2016-01-01

    Patients with clear cell renal cell carcinoma (ccRCC) have few therapeutic options, as ccRCC is unresponsive to chemotherapy and is highly resistant to radiation. Recently targeted therapies have extended progression-free survival, but responses are variable and no significant overall survival benefit has been achieved. Commercial ccRCC cell lines are often used as model systems to develop novel therapeutic approaches, but these do not accurately recapitulate primary ccRCC tumors at the genomic and transcriptional levels. Furthermore, ccRCC exhibits significant intertumor genetic heterogeneity, and the limited cell lines available fail to represent this aspect of ccRCC. Our objective was to generate accurate preclinical in vitro models of ccRCC using tumor tissues from ccRCC patients. ccRCC primary single cell suspensions were cultured in fetal bovine serum (FBS)-containing media or defined serum-free media. Established cultures were characterized by genomic verification of mutations present in the primary tumors, expression of renal epithelial markers, and transcriptional profiling. The apparent efficiency of primary cell culture establishment was high in both culture conditions, but genotyping revealed that the majority of cultures contained normal, not cancer cells. ccRCC characteristically shows biallelic loss of the von Hippel Lindau (VHL) gene, leading to accumulation of hypoxia-inducible factor (HIF) and expression of HIF target genes. Purification of cells based on expression of carbonic anhydrase IX (CA9), a cell surface HIF target, followed by culture in FBS enabled establishment of ccRCC cell cultures with an efficiency of >80 %. Culture in serum-free conditions selected for growth of normal renal proximal tubule epithelial cells. Transcriptional profiling of ccRCC and matched normal cell cultures identified up- and down-regulated networks in ccRCC and comparison to The Cancer Genome Atlas confirmed the clinical validity of our cell cultures. The ability

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

    International Nuclear Information System (INIS)

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

    2000-01-01

    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 copies of a transgene comprised of the ovine β-lactoglobulin milk protein gene promoter, driving expression of a temperature-sensitive variant of simian virus-40 (SV40) large T antigen (T-Ag). KIM-2 cells have a characteristic luminal epithelial cell morphology and a stable, nontransformed phenotype at the semipermissive temperature of 37°C. In contrast, at the permissive temperature of 33°C the cells have an elongated spindle-like morphology and become transformed after prolonged culture. Differentiation of KIM-2 cells at 37°C, in response to lactogenic hormones, results in the formation of polarized dome-like structures with tight junctions. This is accompanied by expression of the milk protein genes that encode β-casein and whey acidic protein (WAP), and activation of the prolactin signalling molecule, signal transducer and activator of transcription (STAT)5. Fully differentiated KIM-2 cultures at 37°C become dependent on lactogenic hormones for survival and undergo extensive apoptosis upon hormone withdrawal, as indicated by nuclear morphology and flow cytometric analysis. KIM-2 cells can be genetically modified by stable transfection and clonal lines isolated that retain the characteristics of untransfected cells. KIM-2 cells are a valuable addition, therefore, to currently available lines of mammary epithelial cells. Their capacity for extensive differentiation in the absence of exogenously added basement membrane, and ability to undergo apoptosis in response to physiological signals will provide an invaluable model system for the study of signal transduction pathways and transcriptional regulatory mechanisms that control differentiation and involution in the mammary gland

  10. Modelling glioblastoma tumour-host cell interactions using adult brain organotypic slice co-culture

    Directory of Open Access Journals (Sweden)

    Maria Angeles Marques-Torrejon

    2018-02-01

    Full Text Available Glioblastoma multiforme (GBM is an aggressive incurable brain cancer. The cells that fuel the growth of tumours resemble neural stem cells found in the developing and adult mammalian forebrain. These are referred to as glioma stem cells (GSCs. Similar to neural stem cells, GSCs exhibit a variety of phenotypic states: dormant, quiescent, proliferative and differentiating. How environmental cues within the brain influence these distinct states is not well understood. Laboratory models of GBM can be generated using either genetically engineered mouse models, or via intracranial transplantation of cultured tumour initiating cells (mouse or human. Unfortunately, these approaches are expensive, time-consuming, low-throughput and ill-suited for monitoring live cell behaviours. Here, we explored whole adult brain coronal organotypic slices as an alternative model. Mouse adult brain slices remain viable in a serum-free basal medium for several weeks. GSCs can be easily microinjected into specific anatomical sites ex vivo, and we demonstrate distinct responses of engrafted GSCs to diverse microenvironments in the brain tissue. Within the subependymal zone – one of the adult neural stem cell niches – injected tumour cells could effectively engraft and respond to endothelial niche signals. Tumour-transplanted slices were treated with the antimitotic drug temozolomide as proof of principle of the utility in modelling responses to existing treatments. Engraftment of mouse or human GSCs onto whole brain coronal organotypic brain slices therefore provides a simplified, yet flexible, experimental model. This will help to increase the precision and throughput of modelling GSC-host brain interactions and complements ongoing in vivo studies. This article has an associated First Person interview with the first author of the paper.

  11. An in vitro co-culture model of esophageal cells identifies ascorbic acid as a modulator of cell competition

    Directory of Open Access Journals (Sweden)

    Gardiner Kristin L

    2011-10-01

    Full Text Available Abstract Background The evolutionary dynamics between interacting heterogeneous cell types are fundamental properties of neoplastic progression but can be difficult to measure and quantify. Cancers are heterogeneous mixtures of mutant clones but the direct effect of interactions between these clones is rarely documented. The implicit goal of most preventive interventions is to bias competition in favor of normal cells over neoplastic cells. However, this is rarely explicitly tested. Here we have developed a cell culture competition model to allow for direct observation of the effect of chemopreventive or therapeutic agents on two interacting cell types. We have examined competition between normal and Barrett's esophagus cell lines, in the hopes of identifying a system that could screen for potential chemopreventive agents. Methods One fluorescently-labeled normal squamous esophageal cell line (EPC2-hTERT was grown in competition with one of four Barrett's esophagus cell lines (CP-A, CP-B, CP-C, CP-D under varying conditions and the outcome of competition measured over 14 days by flow cytometry. Results We demonstrate that ascorbic acid (vitamin C can help squamous cells outcompete Barrett's cells in this system. We are also able to show that ascorbic acid's boost to the relative fitness of squamous cells was increased in most cases by mimicking the pH conditions of gastrointestinal reflux in the lower esophagus. Conclusions This model is able to integrate differential fitness effects on various cell types, allowing us to simultaneously capture effects on interacting cell types without having to perform separate experiments. This model system may be used to screen for new classes of cancer prevention agents designed to modulate the competition between normal and neoplastic cells.

  12. A new cell culture model to genetically dissect the complete human papillomavirus life cycle.

    Science.gov (United States)

    Bienkowska-Haba, Malgorzata; Luszczek, Wioleta; Myers, Julia E; Keiffer, Timothy R; DiGiuseppe, Stephen; Polk, Paula; Bodily, Jason M; Scott, Rona S; Sapp, Martin

    2018-03-01

    Herein, we describe a novel infection model that achieves highly efficient infection of primary keratinocytes with human papillomavirus type 16 (HPV16). This cell culture model does not depend on immortalization and is amenable to extensive genetic analyses. In monolayer cell culture, the early but not late promoter was active and yielded a spliced viral transcript pattern similar to HPV16-immortalized keratinocytes. However, relative levels of the E8^E2 transcript increased over time post infection suggesting the expression of this viral repressor is regulated independently of other early proteins and that it may be important for the shift from the establishment to the maintenance phase of the viral life cycle. Both the early and the late promoter were strongly activated when infected cells were subjected to differentiation by growth in methylcellulose. When grown as organotypic raft cultures, HPV16-infected cells expressed late E1^E4 and L1 proteins and replication foci were detected, suggesting that they supported the completion of the viral life cycle. As a proof of principle that the infection system may be used for genetic dissection of viral factors, we analyzed E1, E6 and E7 translation termination linker mutant virus for establishment of infection and genome maintenance. E1 but not E6 and E7 was essential to establish infection. Furthermore, E6 but not E7 was required for episomal genome maintenance. Primary keratinocytes infected with wild type HPV16 immortalized, whereas keratinocytes infected with E6 and E7 knockout virus began to senesce 25 to 35 days post infection. The novel infection model provides a powerful genetic tool to study the role of viral proteins throughout the viral life cycle but especially for immediate early events and enables us to compare low- and high-risk HPV types in the context of infection.

  13. A new cell culture model to genetically dissect the complete human papillomavirus life cycle.

    Directory of Open Access Journals (Sweden)

    Malgorzata Bienkowska-Haba

    2018-03-01

    Full Text Available Herein, we describe a novel infection model that achieves highly efficient infection of primary keratinocytes with human papillomavirus type 16 (HPV16. This cell culture model does not depend on immortalization and is amenable to extensive genetic analyses. In monolayer cell culture, the early but not late promoter was active and yielded a spliced viral transcript pattern similar to HPV16-immortalized keratinocytes. However, relative levels of the E8^E2 transcript increased over time post infection suggesting the expression of this viral repressor is regulated independently of other early proteins and that it may be important for the shift from the establishment to the maintenance phase of the viral life cycle. Both the early and the late promoter were strongly activated when infected cells were subjected to differentiation by growth in methylcellulose. When grown as organotypic raft cultures, HPV16-infected cells expressed late E1^E4 and L1 proteins and replication foci were detected, suggesting that they supported the completion of the viral life cycle. As a proof of principle that the infection system may be used for genetic dissection of viral factors, we analyzed E1, E6 and E7 translation termination linker mutant virus for establishment of infection and genome maintenance. E1 but not E6 and E7 was essential to establish infection. Furthermore, E6 but not E7 was required for episomal genome maintenance. Primary keratinocytes infected with wild type HPV16 immortalized, whereas keratinocytes infected with E6 and E7 knockout virus began to senesce 25 to 35 days post infection. The novel infection model provides a powerful genetic tool to study the role of viral proteins throughout the viral life cycle but especially for immediate early events and enables us to compare low- and high-risk HPV types in the context of infection.

  14. Universal Capacitance Model for Real-Time Biomass in Cell Culture

    Directory of Open Access Journals (Sweden)

    Viktor Konakovsky

    2015-09-01

    Full Text Available Capacitance probes have the potential to revolutionize bioprocess control due to their safe and robust use and ability to detect even the smallest capacitors in the form of biological cells. Several techniques have evolved to model biomass statistically, however, there are problems with model transfer between cell lines and process conditions. Errors of transferred models in the declining phase of the culture range for linear models around +100% or worse, causing unnecessary delays with test runs during bioprocess development. The goal of this work was to develop one single universal model which can be adapted by considering a potentially mechanistic factor to estimate biomass in yet untested clones and scales. The novelty of this work is a methodology to select sensitive frequencies to build a statistical model which can be shared among fermentations with an error between 9% and 38% (mean error around 20% for the whole process, including the declining phase. A simple linear factor was found to be responsible for the transferability of biomass models between cell lines, indicating a link to their phenotype or physiology.

  15. First complete and productive cell culture model for members of the genus Iridovirus.

    Science.gov (United States)

    D'Costa, Susan M; Vigerust, David J; Perales-Hull, Marsha R; Lodhi, Sundus A; Viravathana, Polrit; Bilimoria, Shän L

    2012-11-01

    Chilo iridescent virus (CIV; the type strain of the genus Iridovirus) replicates productively in larvae of the boll weevil, Anthonomus grandis. This study focuses on characterizing productive infections of a boll weevil cell line, BRL-AG-3A (AG3A), starting with CIV reared in the waxworm, Galleria mellonella. We show that CIV can be continually and productively passaged to high titer in AG3A cells. The replication of larval-derived CIV in AG3A was analyzed by observing viral DNA replication and restriction endonuclease digestion profiles, morphogenesis, and infectivity using TCID(50) assays with AG3A as an indicator cell line. The data showed that virus passaged in the AG3A host is stable. AG3A cells are more efficient than previously utilized CF-124T cells from Choristoneura fumiferana. This system constitutes a superior model for cellular and molecular studies on CIV; it represents the first complete, productive cell culture model for the replication of CIV or any member of the genus Iridovirus.

  16. Cellular track model of biological damage to mammalian cell cultures from galactic cosmic rays

    Science.gov (United States)

    Cucinotta, Francis A.; Katz, Robert; Wilson, John W.; Townsend, Lawrence W.; Nealy, John E.; Shinn, Judy L.

    1991-01-01

    The assessment of biological damage from the galactic cosmic rays (GCR) is a current interest for exploratory class space missions where the highly ionizing, high-energy, high-charge ions (HZE) particles are the major concern. The relative biological effectiveness (RBE) values determined by ground-based experiments with HZE particles are well described by a parametric track theory of cell inactivation. Using the track model and a deterministic GCR transport code, the biological damage to mammalian cell cultures is considered for 1 year in free space at solar minimum for typical spacecraft shielding. Included are the effects of projectile and target fragmentation. The RBE values for the GCR spectrum which are fluence-dependent in the track model are found to be more severe than the quality factors identified by the International Commission on Radiological Protection publication 26 and seem to obey a simple scaling law with the duration period in free space.

  17. Cellular track model of biological damage to mammalian cell cultures from galactic cosmic rays

    International Nuclear Information System (INIS)

    Cucinotta, F.A.; Katz, R.; Wilson, J.W.; Townsend, L.W.; Nealy, J.E.; Shinn, J.L.

    1991-02-01

    The assessment of biological damage from the galactic cosmic rays (GCR) is a current interest for exploratory class space missions where the highly ionizing, high-energy, high-charge ions (HZE) particles are the major concern. The relative biological effectiveness (RBE) values determined by ground-based experiments with HZE particles are well described by a parametric track theory of cell inactivation. Using the track model and a deterministic GCR transport code, the biological damage to mammalian cell cultures is considered for 1 year in free space at solar minimum for typical spacecraft shielding. Included are the effects of projectile and target fragmentation. The RBE values for the GCR spectrum which are fluence-dependent in the track model are found to be more severe than the quality factors identified by the International Commission on Radiological Protection publication 26 and seem to obey a simple scaling law with the duration period in free space

  18. Mesenchymal stem cells support neuronal fiber growth in an organotypic brain slice co-culture model.

    Science.gov (United States)

    Sygnecka, Katja; Heider, Andreas; Scherf, Nico; Alt, Rüdiger; Franke, Heike; Heine, Claudia

    2015-04-01

    Mesenchymal stem cells (MSCs) have been identified as promising candidates for neuroregenerative cell therapies. However, the impact of different isolation procedures on the functional and regenerative characteristics of MSC populations has not been studied thoroughly. To quantify these differences, we directly compared classically isolated bulk bone marrow-derived MSCs (bulk BM-MSCs) to the subpopulation Sca-1(+)Lin(-)CD45(-)-derived MSCs(-) (SL45-MSCs), isolated by fluorescence-activated cell sorting from bulk BM-cell suspensions. Both populations were analyzed with respect to functional readouts, that are, frequency of fibroblast colony forming units (CFU-f), general morphology, and expression of stem cell markers. The SL45-MSC population is characterized by greater morphological homogeneity, higher CFU-f frequency, and significantly increased nestin expression compared with bulk BM-MSCs. We further quantified the potential of both cell populations to enhance neuronal fiber growth, using an ex vivo model of organotypic brain slice co-cultures of the mesocortical dopaminergic projection system. The MSC populations were cultivated underneath the slice co-cultures without direct contact using a transwell system. After cultivation, the fiber density in the border region between the two brain slices was quantified. While both populations significantly enhanced fiber outgrowth as compared with controls, purified SL45-MSCs stimulated fiber growth to a larger degree. Subsequently, we analyzed the expression of different growth factors in both cell populations. The results show a significantly higher expression of brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor in the SL45-MSCs population. Altogether, we conclude that MSC preparations enriched for primary MSCs promote neuronal regeneration and axonal regrowth, more effectively than bulk BM-MSCs, an effect that may be mediated by a higher BDNF secretion.

  19. Development of an in situ evaluation system for neural cells using extracellular matrix-modeled gel culture.

    Science.gov (United States)

    Nagai, Takayuki; Ikegami, Yasuhiro; Mizumachi, Hideyuki; Shirakigawa, Nana; Ijima, Hiroyuki

    2017-10-01

    Two-dimensional monolayer culture is the most popular cell culture method. However, the cells may not respond as they do in vivo because the culture conditions are different from in vivo conditions. However, hydrogel-embedding culture, which cultures cells in a biocompatible culture substrate, can produce in vivo-like cell responses, but in situ evaluation of cells in a gel is difficult. In this study, we realized an in vivo-like environment in vitro to produce cell responses similar to those in vivo and established an in situ evaluation system for hydrogel-embedded cell responses. The extracellular matrix (ECM)-modeled gel consisted of collagen and heparin (Hep-col) to mimic an in vivo-like environment. The Hep-col gel could immobilize growth factors, which is important for ECM functions. Neural stem/progenitor cells cultured in the Hep-col gel grew and differentiated more actively than in collagen, indicating an in vivo-like environment in the Hep-col gel. Second, a thin-layered gel culture system was developed to realize in situ evaluation of the gel-embedded cells. Cells in a 200-μm-thick gel could be evaluated clearly by a phase-contrast microscope and immunofluorescence staining through reduced optical and diffusional effects. Finally, we found that the neural cells cultured in this system had synaptic connections and neuronal action potentials by immunofluorescence staining and Ca 2+ imaging. In conclusion, this culture method may be a valuable evaluation system for neurotoxicity testing. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  20. Mammalian Cell Culture Simplified.

    Science.gov (United States)

    Moss, Robert; Solomon, Sondra

    1991-01-01

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

  1. Modulating and modeling aggregation of cell-seeded microcarriers in stirred culture system for macrotissue engineering.

    Science.gov (United States)

    Mei, Yang; Luo, Houyong; Tang, Qiang; Ye, Zhaoyang; Zhou, Yan; Tan, Wen-Song

    2010-11-01

    A recently developed protocol, "microtissue assembly" holds great promise to address the issue of limited mass transfer within engineered large tissue replacements (macrotissues), wherein small "building blocks" (microtissues) are prepared and then assembled into macrotissues. Previous studies suggested that aggregation behavior of microcarrier-based microtissues were very important for macrotissue engineering. However, a systematic study on the aggregation behavior of microtissues is still missing. In this study, to examine the aggregation behavior of microtissues, effects of key operation parameters in dynamic culture including cell seeding density, microcarrier concentration, L-ascorbic acid 2-phosphate (V(c)) and agitating speed were investigated. The aggregation process could be divided into three phases (i.e., lag, growth and stable). Aggregation efficiency (S) was found to be modulated by cell seeding density, microcarrier concentration, addition of V(c) and agitating speed. A mathematical model correlating the operation parameters with S at different phases of aggregation was developed and experimentally proved to be able to predict S with varied operation parameters. In the end, a cylindrical macrotissue (diameter × height: 2.0 cm × 0.8 cm) with fairly good integrity and cellularity and uniform cell distribution was successfully engineered through perfusion assembling microtissues with controlled S under selected culture conditions. Our study showed that aggregation of microtissues could be precisely modulated, which would definitely facilitate engineering macrotissues with high quality. Copyright © 2010 Elsevier B.V. All rights reserved.

  2. Bacterial cell culture

    OpenAIRE

    sprotocols

    2014-01-01

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

  3. A critical review of cell culture strategies for modelling intracortical brain implant material reactions.

    Science.gov (United States)

    Gilmour, A D; Woolley, A J; Poole-Warren, L A; Thomson, C E; Green, R A

    2016-06-01

    The capacity to predict in vivo responses to medical devices in humans currently relies greatly on implantation in animal models. Researchers have been striving to develop in vitro techniques that can overcome the limitations associated with in vivo approaches. This review focuses on a critical analysis of the major in vitro strategies being utilized in laboratories around the world to improve understanding of the biological performance of intracortical, brain-implanted microdevices. Of particular interest to the current review are in vitro models for studying cell responses to penetrating intracortical devices and their materials, such as electrode arrays used for brain computer interface (BCI) and deep brain stimulation electrode probes implanted through the cortex. A background on the neural interface challenge is presented, followed by discussion of relevant in vitro culture strategies and their advantages and disadvantages. Future development of 2D culture models that exhibit developmental changes capable of mimicking normal, postnatal development will form the basis for more complex accurate predictive models in the future. Although not within the scope of this review, innovations in 3D scaffold technologies and microfluidic constructs will further improve the utility of in vitro approaches. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. A 3D Culture Model to Study How Fluid Pressure and Flow Affect the Behavior of Aggregates of Epithelial Cells.

    Science.gov (United States)

    Piotrowski-Daspit, Alexandra S; Simi, Allison K; Pang, Mei-Fong; Tien, Joe; Nelson, Celeste M

    2017-01-01

    Cells are surrounded by mechanical stimuli in their microenvironment. It is important to determine how cells respond to the mechanical information that surrounds them in order to understand both development and disease progression, as well as to be able to predict cell behavior in response to physical stimuli. Here we describe a protocol to determine the effects of interstitial fluid flow on the migratory behavior of an aggregate of epithelial cells in a three-dimensional (3D) culture model. This protocol includes detailed methods for the fabrication of a 3D cell culture chamber with hydrostatic pressure control, the culture of epithelial cells as an aggregate in a collagen gel, and the analysis of collective cell behavior in response to pressure-driven flow.

  5. Cytocompatibility of calcium silicate-based sealers in a three-dimensional cell culture model.

    Science.gov (United States)

    da Silva, Emmanuel João Nogueira Leal; Zaia, Alexandre A; Peters, Ove A

    2017-06-01

    The aim of the present study was to evaluate cytotoxic effects and cytokine production of calcium silicate-based sealers (EndoSeal, EndoSequence BC Sealer, and MTA Fillapex) using an in vitro root canal filling model and three-dimensional (3D) cell culture. AH Plus as a reference was compared to contemporary calcium silicate cements regarding cell viability and cytokine production. Root canals of 30 human maxillary incisors were prepared using a single-file reciprocating technique. The samples were randomly distributed and canals filled with either AH Plus, EndoSeal, EndoSequence BC Sealer, and MTA Fillapex (n = 6). In the negative control group, the root canal remained unfilled. Sealers were placed into the canals along with a gutta-percha cone placed to working length. Balb/c 3T3 fibroblasts, cultured in a type I collagen 3D scaffold, were exposed to filling material and the respective root apex for 24 h. Cytocompatibility of the materials was evaluated using the methyl-thiazoldiphenyl-tetrazolium (MTT) assay. The production of IL-1β, IL-6, and IL-8 was analyzed using enzyme-linked immunosorbent assay (ELISA). One-way analysis of variance was performed, and when the F-ratios were significant, data were compared by Duncan's multiple-range test. The alpha-type error was set at 0.05. EndoSeal, Endosequence BC Sealer and AH Plus showed cell viability that was similar to the negative control group (P > 0.05), while MTA Fillapex sealer was cytotoxic (P culture, AH Plus, EndoSeal, and EndoSequence BC Sealer were cytocompatible. These results may suggest that AH Plus, EndoSeal and EndoSequence BC Sealer may achieve better biological response when compared to MTA Fillapex.

  6. Human pericyte-endothelial cell interactions in co-culture models mimicking the diabetic retinal microvascular environment.

    Science.gov (United States)

    Tarallo, Sonia; Beltramo, Elena; Berrone, Elena; Porta, Massimo

    2012-12-01

    Pericytes regulate vascular tone, perfusion pressure and endothelial cell (EC) proliferation in capillaries. Thiamine and benfotiamine counteract high glucose-induced damage in vascular cells. We standardized two human retinal pericyte (HRP)/EC co-culture models to mimic the diabetic retinal microvascular environment. We aimed at evaluating the interactions between co-cultured HRP and EC in terms of proliferation/apoptosis and the possible protective role of thiamine and benfotiamine against high glucose-induced damage. EC and HRP were co-cultured in physiological glucose and stable or intermittent high glucose, with or without thiamine/benfotiamine. No-contact model: EC were plated on a porous membrane suspended into the medium and HRP on the bottom of the same well. Cell-to-cell contact model: EC and HRP were plated on the opposite sides of the same membrane. Proliferation (cell counts and DNA synthesis), apoptosis and tubule formation in Matrigel were assessed. In the no-contact model, stable high glucose reduced proliferation of co-cultured EC/HRP and EC alone and increased co-cultured EC/HRP apoptosis. In the contact model, both stable and intermittent high glucose reduced co-cultured EC/HRP proliferation and increased apoptosis. Stable high glucose had no effects on HRP in separate cultures. Both EC and HRP proliferated better when co-cultured. Thiamine and benfotiamine reversed high glucose-induced damage in all cases. HRP are sensitive to soluble factors released by EC when cultured in high glucose conditions, as suggested by conditioned media assays. In the Matrigel models, addition of thiamine and benfotiamine re-established the high glucose-damaged interactions between EC/HRP and stabilized microtubules.

  7. Development of microfluidic cell culture devices towards an in vitro human intestinal barrier model

    DEFF Research Database (Denmark)

    Tan, Hsih-Yin

    to enable real-time detection of cell responses, adjustment of cellular stimulation etc. leading to establishment of conditional experiments. In this project, microfluidic systems engineering was leveraged to develop an eight chamber multi-layer microchip for intestinal barrier studies. Sandwiched between...... the layers was a modified Teflon porous membrane for cell culture. The novelty lies in modifying the surface of the porous Teflon support membrane using thiol-ene ‘click’ chemistry, thus allowing the modified Teflon membrane to be bonded between the chip layers to form an enclosed microchip. Successful...... application of the multi-layer microchip was demonstrated by integrating the microchip to an existing cell culture fluidic system to culture the human intestinal epithelial cells, Caco-2, for long term studies. Under the continuous low flow conditions, the cells differentiated into columnar cells displaying...

  8. BioSig3D: High Content Screening of Three-Dimensional Cell Culture Models.

    Directory of Open Access Journals (Sweden)

    Cemal Cagatay Bilgin

    Full Text Available BioSig3D is a computational platform for high-content screening of three-dimensional (3D cell culture models that are imaged in full 3D volume. It provides an end-to-end solution for designing high content screening assays, based on colony organization that is derived from segmentation of nuclei in each colony. BioSig3D also enables visualization of raw and processed 3D volumetric data for quality control, and integrates advanced bioinformatics analysis. The system consists of multiple computational and annotation modules that are coupled together with a strong use of controlled vocabularies to reduce ambiguities between different users. It is a web-based system that allows users to: design an experiment by defining experimental variables, upload a large set of volumetric images into the system, analyze and visualize the dataset, and either display computed indices as a heatmap, or phenotypic subtypes for heterogeneity analysis, or download computed indices for statistical analysis or integrative biology. BioSig3D has been used to profile baseline colony formations with two experiments: (i morphogenesis of a panel of human mammary epithelial cell lines (HMEC, and (ii heterogeneity in colony formation using an immortalized non-transformed cell line. These experiments reveal intrinsic growth properties of well-characterized cell lines that are routinely used for biological studies. BioSig3D is being released with seed datasets and video-based documentation.

  9. Liver Cell Culture Devices

    NARCIS (Netherlands)

    Andria, B.; Bracco, A.; Cirino, G.; Chamuleau, R. A. F. M.

    2010-01-01

    In the last 15 years many different liver cell culture devices, consisting of functional liver cells and artificial materials, have been developed. They have been devised for numerous different applications, such as temporary organ replacement (a bridge to liver transplantation or native liver

  10. Quality by control: Towards model predictive control of mammalian cell culture bioprocesses.

    Science.gov (United States)

    Sommeregger, Wolfgang; Sissolak, Bernhard; Kandra, Kulwant; von Stosch, Moritz; Mayer, Martin; Striedner, Gerald

    2017-07-01

    The industrial production of complex biopharmaceuticals using recombinant mammalian cell lines is still mainly built on a quality by testing approach, which is represented by fixed process conditions and extensive testing of the end-product. In 2004 the FDA launched the process analytical technology initiative, aiming to guide the industry towards advanced process monitoring and better understanding of how critical process parameters affect the critical quality attributes. Implementation of process analytical technology into the bio-production process enables moving from the quality by testing to a more flexible quality by design approach. The application of advanced sensor systems in combination with mathematical modelling techniques offers enhanced process understanding, allows on-line prediction of critical quality attributes and subsequently real-time product quality control. In this review opportunities and unsolved issues on the road to a successful quality by design and dynamic control implementation are discussed. A major focus is directed on the preconditions for the application of model predictive control for mammalian cell culture bioprocesses. Design of experiments providing information about the process dynamics upon parameter change, dynamic process models, on-line process state predictions and powerful software environments seem to be a prerequisite for quality by control realization. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. A phasor approach analysis of multiphoton FLIM measurements of three-dimensional cell culture models

    Science.gov (United States)

    Lakner, P. H.; Möller, Y.; Olayioye, M. A.; Brucker, S. Y.; Schenke-Layland, K.; Monaghan, M. G.

    2016-03-01

    Fluorescence lifetime imaging microscopy (FLIM) is a useful approach to obtain information regarding the endogenous fluorophores present in biological samples. The concise evaluation of FLIM data requires the use of robust mathematical algorithms. In this study, we developed a user-friendly phasor approach for analyzing FLIM data and applied this method on three-dimensional (3D) Caco-2 models of polarized epithelial luminal cysts in a supporting extracellular matrix environment. These Caco-2 based models were treated with epidermal growth factor (EGF), to stimulate proliferation in order to determine if FLIM could detect such a change in cell behavior. Autofluorescence from nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) in luminal Caco-2 cysts was stimulated by 2-photon laser excitation. Using a phasor approach, the lifetimes of involved fluorophores and their contribution were calculated with fewer initial assumptions when compared to multiexponential decay fitting. The phasor approach simplified FLIM data analysis, making it an interesting tool for non-experts in numerical data analysis. We observed that an increased proliferation stimulated by EGF led to a significant shift in fluorescence lifetime and a significant alteration of the phasor data shape. Our data demonstrates that multiphoton FLIM analysis with the phasor approach is a suitable method for the non-invasive analysis of 3D in vitro cell culture models qualifying this method for monitoring basic cellular features and the effect of external factors.

  12. Radiation damage, repopulation and cell recovery analysis of in vitro tumour cell megacolony culture data using a non-Poissonian cell repopulation TCP model

    International Nuclear Information System (INIS)

    Stavrev, P; Weldon, M; Warkentin, B; Stavreva, N; Fallone, B G

    2005-01-01

    The effects of radiation damage, tumour repopulation and cell sublethal damage repair and the possibility of extracting information about the model parameters describing them are investigated in this work. Previously published data on two different cultured cell lines were analysed with the help of a tumour control probability (TCP) model that describes tumour cell dynamics properly. Different versions of a TCP model representing the cases of full or partial cell recovery between fractions of radiation, accompanied by repopulation or no repopulation were used to fit the data and were ranked according to statistical criteria. The data analysis shows the importance of the linear-quadratic mechanism of cell damage for the description of the in vitro cell dynamics. In a previous work where in vivo data were analysed, the employment of the single hit model of cell kill and cell repopulation produced the best fit, while ignoring the quadratic term of cell damage in the current analysis leads to poor fits. It is also concluded that more experiments using different fractionation regimes producing diverse data are needed to help model analysis and better ranking of the models

  13. Human airway epithelial cell cultures for modeling respiratory syncytial virus infection.

    Science.gov (United States)

    Pickles, Raymond J

    2013-01-01

    Respiratory syncytial virus (RSV) is an important human respiratory pathogen with narrow species tropism. Limited availability of human pathologic specimens during early RSV-induced lung disease and ethical restrictions for RSV challenge studies in the lower airways of human volunteers has slowed our understanding of how RSV causes airway disease and greatly limited the development of therapeutic strategies for reducing RSV disease burden. Our current knowledge of RSV infection and pathology is largely based on in vitro studies using nonpolarized epithelial cell-lines grown on plastic or in vivo studies using animal models semipermissive for RSV infection. Although these models have revealed important aspects of RSV infection, replication, and associated inflammatory responses, these models do not broadly recapitulate the early interactions and potential consequences of RSV infection of the human columnar airway epithelium in vivo. In this chapter, the pro et contra of in vitro models of human columnar airway epithelium and their usefulness in respiratory virus pathogenesis and vaccine development studies will be discussed. The use of such culture models to predict characteristics of RSV infection and the correlation of these findings to the human in vivo situation will likely accelerate our understanding of RSV pathogenesis potentially identifying novel strategies for limiting the severity of RSV-associated airway disease.

  14. Stochastic modelling of Listeria monocytogenes single cell growth in cottage cheese with mesophilic lactic acid bacteria from aroma producing cultures

    DEFF Research Database (Denmark)

    Østergaard, Nina Bjerre; Christiansen, Lasse Engbo; Dalgaard, Paw

    2015-01-01

    . 2014. Modelling the effect of lactic acid bacteria from starter- and aroma culture on growth of Listeria monocytogenes in cottage cheese. International Journal of Food Microbiology. 188, 15-25]. Growth of L. monocytogenes single cells, using lag time distributions corresponding to three different......A stochastic model was developed for simultaneous growth of low numbers of Listeria monocytogenes and populations of lactic acid bacteria from the aroma producing cultures applied in cottage cheese. During more than two years, different batches of cottage cheese with aroma culture were analysed...

  15. Changes of heterogeneous cell populations in the Ishikawa cell line during long-term culture: Proposal for an in vitro clonal evolution model of tumor cells.

    Science.gov (United States)

    Kasai, Fumio; Hirayama, Noriko; Ozawa, Midori; Iemura, Masashi; Kohara, Arihiro

    2016-06-01

    Genomic changes in tumor cell lines can occur during culture, leading to differences between cell lines carrying the same name. In this study, genome profiles between low and high passages were investigated in the Ishikawa 3-H-12 cell line (JCRB1505). Cells contained between 43 and 46 chromosomes and the modal number changed from 46 to 45 during culture. Cytogenetic analysis revealed that a translocation t(9;14), observed in all metaphases, is a robust marker for this cell line. Single-nucleotide polymorphism microarrays showed a heterogeneous copy number in the early passages and distinct profiles at late passages. These results demonstrate that cell culture can lead to elimination of ancestral clones by sequential selection, resulting in extensive replacement with a novel clone. Our observations on Ishikawa cells in vitro are different from the in vivo heterogeneity in which ancestral clones are often retained during tumor evolution and suggest a model for in vitro clonal evolution. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Cell Culturing of Cytoskeleton

    Science.gov (United States)

    2004-01-01

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

  17. Differential susceptibility of primary cultured human skin cells to hypericin PDT in an in vitro model.

    Science.gov (United States)

    Popovic, A; Wiggins, T; Davids, L M

    2015-08-01

    Skin cancer is the most common cancer worldwide, and its incidence rate in South Africa is increasing. Photodynamic therapy (PDT) has been shown to be an effective treatment modality, through topical administration, for treatment of non-melanoma skin cancers. Our group investigates hypericin-induced PDT (HYP-PDT) for the treatment of both non-melanoma and melanoma skin cancers. However, a prerequisite for effective cancer treatments is efficient and selective targeting of the tumoral cells with minimal collateral damage to the surrounding normal cells, as it is well established that cancer therapies have bystander effects on normal cells in the body, often causing undesirable side effects. The aim of this study was to investigate the cellular and molecular effects of HYP-PDT on normal primary human keratinocytes (Kc), melanocytes (Mc) and fibroblasts (Fb) in an in vitro tissue culture model which represented both the epidermal and dermal cellular compartments of human skin. Cell viability analysis revealed a differential cytotoxic response to a range of HYP-PDT doses in all the human skin cell types, showing that Fb (LD50=1.75μM) were the most susceptible to HYP-PDT, followed by Mc (LD50=3.5μM) and Kc (LD50>4μM HYP-PDT) These results correlated with the morphological analysis which displayed distinct morphological changes in Fb and Mc, 24h post treatment with non-lethal (1μM) and lethal (3μM) doses of HYP-PDT, but the highest HYP-PDT doses had no effect on Kc morphology. Fluorescent microscopy displayed cytoplasmic localization of HYP in all the 3 skin cell types and additionally, HYP was excluded from the nuclei in all the cell types. Intracellular ROS levels measured in Fb at 3μM HYP-PDT, displayed a significant 3.8 fold (phuman skin cells thus highlighting the efficacy and indeed, the potential bystander effect of if administered in vivo. This study contributes toward our knowledge of the cellular response of the epidermis to photodynamic therapies and

  18. Oscillating Cell Culture Bioreactor

    Science.gov (United States)

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

    2010-01-01

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

  19. A Cell Culture Model of Latent and Lytic Herpes Simplex Virus Type 1 Infection in Spiral Ganglion.

    Science.gov (United States)

    Liu, Yuehong; Li, Shufeng

    2015-01-01

    Reactivation of latent herpes simplex virus type 1 (HSV-1) in spiral ganglion neurons (SGNs) is supposed to be one of the causes of idiopathic sudden sensorineural hearing loss. This study aims to establish a cell culture model of latent and lytic HSV-1 infection in spiral ganglia. In the presence of acyclovir, primary cultures of SGNs were latently infected with HSV-1 expressing green fluorescent protein. Four days later, these cells were treated with trichostatin A (TSA), a known chemical reactivator of HSV-1. TCID50 was used to measure the titers of virus in cultures on Vero cells. RNA from cultures was detected for the presence of transcripts of ICP27 and latency-associated transcript (LAT) using reverse transcription polymerase chain reaction. There is no detectable infectious HSV-1 in latently infected cultures, whereas they could be observed in both lytically infected and latently infected/TSA-treated cultures. LAT was the only detectable transcript during latent infection, whereas lytic ICP27 transcript was detected in lytically infected and latently infected/TSA-treated cultures. Cultured SGNs can be both latently and lytically infected with HSV-1. Furthermore, latently infected SGNs can be reactivated using TSA, yielding infectious virus.

  20. Modeling industrial centrifugation of mammalian cell culture using a capillary based scale-down system.

    Science.gov (United States)

    Westoby, Matthew; Rogers, Jameson K; Haverstock, Ryan; Romero, Jonathan; Pieracci, John

    2011-05-01

    Continuous-flow centrifugation is widely utilized as the primary clarification step in the recovery of biopharmaceuticals from cell culture. However, it is a challenging operation to develop and characterize due to the lack of easy to use, small-scale, systems that can be used to model industrial processes. As a result, pilot-scale continuous centrifugation is typically employed to model large-scale systems requiring a significant amount of resources. In an effort to reduce resource requirements and create a system which is easy to construct and utilize, a capillary shear device, capable of producing energy dissipation rates equivalent to those present in the feed zones of industrial disk stack centrifuges, was developed and evaluated. When coupled to a bench-top, batch centrifuge, the capillary device reduced centrate turbidity prediction error from 37% to 4% compared to using a bench-top centrifuge alone. Laboratory-scale parameters that are analogous to those routinely varied during industrial-scale continuous centrifugation were identified and evaluated for their utility in emulating disk stack centrifuge performance. The resulting relationships enable bench-scale process modeling of continuous disk stack centrifuges using an easily constructed, scalable, capillary shear device coupled to a typical bench-top centrifuge. Copyright © 2010 Wiley Periodicals, Inc.

  1. Personality and Cultural Modeling for Agent-Based Representation of a Terrorist Cell, Phase 1

    National Research Council Canada - National Science Library

    Hogan, C. M; Van Houten, Robert A; La, Nini

    2003-01-01

    This report describes the research into the use of personality, cultural and socio-political modeling in order to provide a robust asymmetric opponent for Military Operation in Urban Terrain training...

  2. Plant cell culture initiation

    NARCIS (Netherlands)

    Hall, R.D.

    2000-01-01

    The use of cultured plant cells in either organized or unorganized form has increased vey considerably in the last 10-15 yr. Many new technologies have been developed and applications in both fundamental and applied research have led to the development of some powerful tools for improving our

  3. Synchronized mammalian cell culture: part II--population ensemble modeling and analysis for development of reproducible processes.

    Science.gov (United States)

    Jandt, Uwe; Barradas, Oscar Platas; Pörtner, Ralf; Zeng, An-Ping

    2015-01-01

    The consideration of inherent population inhomogeneities of mammalian cell cultures becomes increasingly important for systems biology study and for developing more stable and efficient processes. However, variations of cellular properties belonging to different sub-populations and their potential effects on cellular physiology and kinetics of culture productivity under bioproduction conditions have not yet been much in the focus of research. Culture heterogeneity is strongly determined by the advance of the cell cycle. The assignment of cell-cycle specific cellular variations to large-scale process conditions can be optimally determined based on the combination of (partially) synchronized cultivation under otherwise physiological conditions and subsequent population-resolved model adaptation. The first step has been achieved using the physical selection method of countercurrent flow centrifugal elutriation, recently established in our group for different mammalian cell lines which is presented in Part I of this paper series. In this second part, we demonstrate the successful adaptation and application of a cell-cycle dependent population balance ensemble model to describe and understand synchronized bioreactor cultivations performed with two model mammalian cell lines, AGE1.HNAAT and CHO-K1. Numerical adaptation of the model to experimental data allows for detection of phase-specific parameters and for determination of significant variations between different phases and different cell lines. It shows that special care must be taken with regard to the sampling frequency in such oscillation cultures to minimize phase shift (jitter) artifacts. Based on predictions of long-term oscillation behavior of a culture depending on its start conditions, optimal elutriation setup trade-offs between high cell yields and high synchronization efficiency are proposed. © 2014 American Institute of Chemical Engineers.

  4. Short-term spheroid culture of primary colorectal cancer cells as an in vitro model for personalizing cancer medicine

    DEFF Research Database (Denmark)

    Jeppesen, Maria; Hagel, Grith; Glenthoj, Anders

    2017-01-01

    Chemotherapy treatment of cancer remains a challenge due to the molecular and functional heterogeneity displayed by tumours originating from the same cell type. The pronounced heterogeneity makes it difficult for oncologists to devise an effective therapeutic strategy for the patient. One approac...... and combinations most commonly used for treatment of colorectal cancer. In summary, short-term spheroid culture of primary colorectal adenocarcinoma cells represents a promising in vitro model for use in personalized medicine....... for increasing treatment efficacy is to test the chemosensitivity of cancer cells obtained from the patient's tumour. 3D culture represents a promising method for modelling patient tumours in vitro. The aim of this study was therefore to evaluate how closely short-term spheroid cultures of primary colorectal...... cancer cells resemble the original tumour. Colorectal cancer cells were isolated from human tumour tissue and cultured as spheroids. Spheroid cultures were established with a high success rate and remained viable for at least 10 days. The spheroids exhibited significant growth over a period of 7 days...

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

    Science.gov (United States)

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

    2015-01-01

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

  6. Mutation in cultured mammalian cells

    International Nuclear Information System (INIS)

    Nakamura, N.; Okada, S.

    1982-01-01

    Mammalian cell cultures were exposed to gamma-rays at various dose rates. Dose-rate effects were observed in cultured somatic cells of the mouse for cell killing and mutations resistant to 6-thioguanine (TGsup(r)) and to methotrexate (MTXsup(r)). Linear quadratic model may be applied to cell killing and TGsup(r) mutations in some cases but can not explain the whole data. Results at low doses with far low dose-rate were not predictable from data at high doses with acute or chronic irradiation. Radioprotective effects of dimethyl sulfoxide were seen only after acute exposure but not after chronic one, suggesting that damages by indirect action of radiations may be potentially reparable by cells. TGsup(r) mutations seem to contain gross structural changes whereas MTXsup(r) ones may have smaller alterations. (Namekawa, K.)

  7. An imbalance in progenitor cell populations reflects tumour progression in breast cancer primary culture models

    LENUS (Irish Health Repository)

    Donatello, Simona

    2011-04-26

    Abstract Background Many factors influence breast cancer progression, including the ability of progenitor cells to sustain or increase net tumour cell numbers. Our aim was to define whether alterations in putative progenitor populations could predict clinicopathological factors of prognostic importance for cancer progression. Methods Primary cultures were established from human breast tumour and adjacent non-tumour tissue. Putative progenitor cell populations were isolated based on co-expression or concomitant absence of the epithelial and myoepithelial markers EPCAM and CALLA respectively. Results Significant reductions in cellular senescence were observed in tumour versus non-tumour cultures, accompanied by a stepwise increase in proliferation:senescence ratios. A novel correlation between tumour aggressiveness and an imbalance of putative progenitor subpopulations was also observed. Specifically, an increased double-negative (DN) to double-positive (DP) ratio distinguished aggressive tumours of high grade, estrogen receptor-negativity or HER2-positivity. The DN:DP ratio was also higher in malignant MDA-MB-231 cells relative to non-tumourogenic MCF-10A cells. Ultrastructural analysis of the DN subpopulation in an invasive tumour culture revealed enrichment in lipofuscin bodies, markers of ageing or senescent cells. Conclusions Our results suggest that an imbalance in tumour progenitor subpopulations imbalances the functional relationship between proliferation and senescence, creating a microenvironment favouring tumour progression.

  8. Effects of external radiation in a co-culture model of endothelial cells and adipose-derived stem cells

    International Nuclear Information System (INIS)

    Haubner, Frank; Leyh, Michaela; Ohmann, Elisabeth; Pohl, Fabian; Prantl, Lukas; Gassner, Holger G

    2013-01-01

    The inflammatory response clinically observed after radiation has been described to correlate with elevated expression of cytokines and adhesion molecules by endothelial cells. Therapeutic compensation for this microvascular compromise could be an important approach in the treatment of irradiated wounds. Clinical reports describe the potential of adipose-derived stem cells to enhance wound healing, but the underlying cellular mechanisms remain largely unclear. Human dermal microvascular endothelial cells (HDMEC) and human adipose-derived stem cells (ASC) were cultured in a co-culture setting and irradiated with sequential doses of 2 to 12 Gy. Cell count was determined 48 h after radiation using a semi-automated cell counting system. Levels of interleukin-6 (IL-6), basic fibroblast growth factor (FGF), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were determined in the supernatants using enzyme-linked immunosorbent assay (ELISA). Irradiated HDMEC and ASC as well as non-irradiated co-cultures, HDMEC or ASC respectively were used as controls. Cell count was significantly reduced in irradiated co-cultures of HDMEC and ASC compared to non-irradiated controls. Levels of IL-6, FGF, ICAM-1 and VCAM-1 in the supernatants of the co-cultures were significantly less affected by external radiation in comparison to HDMEC. The increased expression of cytokines and adhesion molecules by HDMEC after external radiation is mitigated in the co-culture setting with ASC. These in vitro changes seem to support the clinical observation that ASC may have a stabilizing effect when injected into irradiated wounds

  9. Epithelial Cell Cultures

    Directory of Open Access Journals (Sweden)

    Imran S. Chaudhry

    2011-01-01

    Full Text Available The biological effects of only a finite number of tobacco toxins have been studied. Here, we describe exposure of cultures of human bronchial epithelial cells to low concentrations of tobacco carcinogens: nickel sulphate, benzo(bfluoranthene, N-nitrosodiethylamine, and 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK. After a 24-hour exposure, EGFR was expressed in cell membrane and cytoplasm, BCL-2 was expressed only in the irregular nuclei of large atypical cells, MKI67 was expressed in nuclei with no staining in larger cells, cytoplasmic BIRC5 with stronger nuclear staining was seen in large atypical cells, and nuclear TP53 was strongly expressed in all cells. After only a 24-hour exposure, cells exhibited atypical nuclear and cytoplasmic features. After a 48-hour exposure, EGFR staining was localized to the nucleus, BCL-2 was slightly decreased in intensity, BIRC5 was localized to the cytoplasm, and TP53 staining was increased in small and large cells. BCL2L1 was expressed in both the cytoplasm and nuclei of cells at 24- and 48-hour exposures. We illustrate that short-termexposure of a bronchial epithelial cell line to smoking-equivalent concentrations of tobacco carcinogens alters the expression of key proliferation regulatory genes, EGFR, BCL-2, BCL2L1, BIRC5, TP53, and MKI67, similar to that reported in biopsy specimens of pulmonary epithelium described to be preneoplastic lesions.

  10. Comparison of Biocompatibility and Adsorption Properties of Different Plastics for Advanced Microfluidic Cell and Tissue Culture Models

    NARCIS (Netherlands)

    van Midwoud, Paul M.; Janse, Arnout; Merema, M.T.; Groothuis, Geny M. M.; Verpoorte, Elisabeth

    2012-01-01

    Microfluidic technology is providing new routes toward advanced cell and tissue culture models to better understand human biology and disease. Many advanced devices have been made from poly(dimethylsiloxane) (PDMS) to enable experiments, for example, to study drug metabolism by use of precision cut

  11. Cell culture techniques in honey bee research

    Science.gov (United States)

    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...

  12. Perfusion based cell culture chips

    DEFF Research Database (Denmark)

    Heiskanen, Arto; Emnéus, Jenny; Dufva, Martin

    2010-01-01

    Performing cell culture in miniaturized perfusion chambers gives possibilities to experiment with cells under near in vivo like conditions. In contrast to traditional batch cultures, miniaturized perfusion systems provide precise control of medium composition, long term unattended cultures...... and tissue like structuring of the cultures. However, as this chapter illustrates, many issues remain to be identified regarding perfusion cell culture such as design, material choice and how to use these systems before they will be widespread amongst biomedical researchers....

  13. Cell culture for three-dimensional modeling in rotating-wall vessels: an application of simulated microgravity

    Science.gov (United States)

    Schwarz, R. P.; Goodwin, T. J.; Wolf, D. A.

    1992-01-01

    High-density, three-dimensional cell cultures are difficult to grow in vitro. The rotating-wall vessel (RWV) described here has cultured BHK-21 cells to a density of 1.1 X 10(7) cells/ml. Cells on microcarriers were observed to grow with enhanced bridging in this batch culture system. The RWV is a horizontally rotated tissue culture vessel with silicon membrane oxygenation. This design results in a low-turbulence, low-shear cell culture environment with abundant oxygenation. The RWV has the potential to culture a wide variety of normal and neoplastic cells.

  14. Microfluidic Cell Culture Device

    Science.gov (United States)

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

    2014-01-01

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

  15. Elevated hydrostatic pressures induce apoptosis and oxidative stress through mitochondrial membrane depolarization in PC12 neuronal cells: A cell culture model of glaucoma.

    Science.gov (United States)

    Tök, Levent; Nazıroğlu, Mustafa; Uğuz, Abdülhadi Cihangir; Tök, Ozlem

    2014-10-01

    Despite the importance of oxidative stress and apoptosis through mitochondrial depolarization in neurodegenerative diseases, their roles in etiology of glaucoma are poorly understood. We aimed to investigate whether oxidative stress and apoptosis formation are altered in rat pheochromocytoma-derived cell line-12 (PC12) neuronal cell cultures exposed to elevated different hydrostatic pressures as a cell culture model of glaucoma. Cultured PC12 cells were subjected to 0, 15 and 70 mmHg hydrostatic pressure for 1 and 24 h. Then, the following values were analyzed: (a) cell viability; (b) lipid peroxidation and intracellular reactive oxygen species production; (c) mitochondrial membrane depolarization; (d) cell apoptosis; (e) caspase-3 and caspase-9 activities; (f) reduced glutathione (GSH) and glutathione peroxidase (GSH-Px). The hydrostatic pressures (15 and 70 mmHg) increased oxidative cell damage through a decrease of GSH and GSH-Px values, and increasing mitochondrial membrane potential. Additionally, 70 mmHg hydrostatic pressure for 24 h indicated highest apoptotic effects, as demonstrated by plate reader analyses of apoptosis, caspase-3 and -9 values. The present data indicated oxidative stress, apoptosis and mitochondrial changes in PC12 cell line during different hydrostatic pressure as a cell culture model of glaucoma. This findings support the view that mitochondrial oxidative injury contributes early to glaucomatous optic neuropathy.

  16. An improved in vitro blood-brain barrier model: rat brain endothelial cells co-cultured with astrocytes.

    Science.gov (United States)

    Abbott, N Joan; Dolman, Diana E M; Drndarski, Svetlana; Fredriksson, Sarah M

    2012-01-01

    In vitro blood-brain barrier (BBB) models using primary cultured brain endothelial cells are important for establishing cellular and molecular mechanisms of BBB function. Co-culturing with BBB-associated cells especially astrocytes to mimic more closely the in vivo condition leads to upregulation of the BBB phenotype in the brain endothelial cells. Rat brain endothelial cells (RBECs) are a valuable tool allowing ready comparison with in vivo studies in rodents; however, it has been difficult to obtain pure brain endothelial cells, and few models achieve a transendothelial electrical resistance (TEER, measure of tight junction efficacy) of >200 Ω cm(2), i.e. the models are still relatively leaky. Here, we describe methods for preparing high purity RBECs and neonatal rat astrocytes, and a co-culture method that generates a robust, stable BBB model that can achieve TEER >600 Ω cm(2). The method is based on >20 years experience with RBEC culture, together with recent improvements to kill contaminating cells and encourage BBB differentiation.Astrocytes are isolated by mechanical dissection and cell straining and are frozen for later co-culture. RBECs are isolated from 3-month-old rat cortices. The brains are cleaned of meninges and white matter and enzymatically and mechanically dissociated. Thereafter, the tissue homogenate is centrifuged in bovine serum albumin to separate vessel fragments from other cells that stick to the myelin plug. The vessel fragments undergo a second enzyme digestion to separate pericytes from vessels and break down vessels into shorter segments, after which a Percoll gradient is used to separate capillaries from venules, arterioles, and single cells. To kill remaining contaminating cells such as pericytes, the capillary fragments are plated in puromycin-containing medium and RBECs grown to 50-60% confluence. They are then passaged onto filters for co-culture with astrocytes grown in the bottom of the wells. The whole procedure takes ∼2

  17. AlgiMatrix™ based 3D cell culture system as an in-vitro tumor model for anticancer studies.

    Directory of Open Access Journals (Sweden)

    Chandraiah Godugu

    Full Text Available Three-dimensional (3D in-vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in-vivo conditions. Taking the advantages of 3D culture, we have developed the in-vitro tumor model for anticancer drug screening.Cancer cells grown in 6 and 96 well AlgiMatrix™ scaffolds resulted in the formation of multicellular spheroids in the size range of 100-300 µm. Spheroids were grown in two weeks in cultures without compromising the growth characteristics. Different marketed anticancer drugs were screened by incubating them for 24 h at 7, 9 and 11 days in 3D cultures and cytotoxicity was measured by AlamarBlue® assay. Effectiveness of anticancer drug treatments were measured based on spheroid number and size distribution. Evaluation of apoptotic and anti-apoptotic markers was done by immunohistochemistry and RT-PCR. The 3D results were compared with the conventional 2D monolayer cultures. Cellular uptake studies for drug (Doxorubicin and nanoparticle (NLC were done using spheroids.IC(50 values for anticancer drugs were significantly higher in AlgiMatrix™ systems compared to 2D culture models. The cleaved caspase-3 expression was significantly decreased (2.09 and 2.47 folds respectively for 5-Fluorouracil and Camptothecin in H460 spheroid cultures compared to 2D culture system. The cytotoxicity, spheroid size distribution, immunohistochemistry, RT-PCR and nanoparticle penetration data suggested that in vitro tumor models show higher resistance to anticancer drugs and supporting the fact that 3D culture is a better model for the cytotoxic evaluation of anticancer drugs in vitro.The results from our studies are useful to develop a high throughput in vitro tumor model to study the effect of various anticancer agents and various molecular pathways affected by the anticancer drugs and formulations.

  18. Model-based strategy for cell culture seed train layout verified at lab scale.

    Science.gov (United States)

    Kern, Simon; Platas-Barradas, Oscar; Pörtner, Ralf; Frahm, Björn

    2016-08-01

    Cell culture seed trains-the generation of a sufficient viable cell number for the inoculation of the production scale bioreactor, starting from incubator scale-are time- and cost-intensive. Accordingly, a seed train offers potential for optimization regarding its layout and the corresponding proceedings. A tool has been developed to determine the optimal points in time for cell passaging from one scale into the next and it has been applied to two different cell lines at lab scale, AGE1.HN AAT and CHO-K1. For evaluation, experimental seed train realization has been evaluated in comparison to its layout. In case of the AGE1.HN AAT cell line, the results have also been compared to the formerly manually designed seed train. The tool provides the same seed train layout based on the data of only two batches.

  19. Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption.

    Science.gov (United States)

    Sagrillo-Fagundes, Lucas; Clabault, Hélène; Laurent, Laetitia; Hudon-Thibeault, Andrée-Anne; Salustiano, Eugênia Maria Assunção; Fortier, Marlène; Bienvenue-Pariseault, Josianne; Wong Yen, Philippe; Sanderson, J Thomas; Vaillancourt, Cathy

    2016-07-30

    This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.

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

    OpenAIRE

    Przyborski, Stefan

    2017-01-01

    Human tissues are mostly composed of different cell types, that are often highly organised in relation to each other. Often cells are arranged in distinct layers that enable signalling and cell-to-cell interactions. Here we describe the application of scaffold-based technology, that can be used to create advanced organotypic 3D models of various tissue types that more closely resemble in vivo-like conditions (Knight et al., 2011). The scaffold comprises a highly porous polystyrene material, e...

  1. Critical analysis of 3-D organoid in vitro cell culture models for high-throughput drug candidate toxicity assessments.

    Science.gov (United States)

    Astashkina, Anna; Grainger, David W

    2014-04-01

    Drug failure due to toxicity indicators remains among the primary reasons for staggering drug attrition rates during clinical studies and post-marketing surveillance. Broader validation and use of next-generation 3-D improved cell culture models are expected to improve predictive power and effectiveness of drug toxicological predictions. However, after decades of promising research significant gaps remain in our collective ability to extract quality human toxicity information from in vitro data using 3-D cell and tissue models. Issues, challenges and future directions for the field to improve drug assay predictive power and reliability of 3-D models are reviewed. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Exposing primary rat retina cell cultures to γ-rays: An in vitro model for evaluating radiation responses.

    Science.gov (United States)

    Gaddini, Lucia; Balduzzi, Maria; Campa, Alessandro; Esposito, Giuseppe; Malchiodi-Albedi, Fiorella; Patrono, Clarice; Matteucci, Andrea

    2018-01-01

    Retinal tissue can receive incidental γ-rays exposure during radiotherapy either of tumors of the eye and optic nerve or of head-and-neck tumors, and during medical diagnostic procedures. Healthy retina is therefore at risk of suffering radiation-related side effects and the knowledge of pathophysiological response of retinal cells to ionizing radiations could be useful to design possible strategies of prevention and management of radiotoxicity. In this study, we have exploited an in vitro model (primary rat retinal cell culture) to study an array of biological effects induced on retinal neurons by γ-rays. Most of the different cell types present in retinal tissue - either of the neuronal or glial lineages - are preserved in primary rat retinal cultures. Similar to the retina in situ, neuronal cells undergo in vitro a maturational development shown by the formation of polarized neuritic trees and operating synapses. Since 2 Gy is the incidental dose received by the healthy retina per fraction when the standard treatment is delivered to the brain, retina cell cultures have been exposed to 1 or 2 Gy of γ-rays at different level of neuronal differentiation in vitro: days in vitro (DIV)2 or DIV8. At DIV9, retinal cultures were analyzed in terms of viability, apoptosis and characterized by immunocytochemistry to identify alterations in neuronal differentiation. After irradiation at DIV2, MTT assay revealed an evident loss of cell viability and βIII-tubulin immunostaining highlighted a marked neuritic damage, indicating that survived neurons showed an impaired differentiation. Differentiated cultures (DIV8) appeared to be more resistant with respect to undifferentiated, DIV2 cultures, both in terms of cell viability and differentiation. Apoptosis evaluated with TUNEL assay showed that irradiation at both DIV2 and DIV8 induced a significant increase in the apoptotic rate. To further investigate the effects of γ-rays on retinal neurons, we evaluated the

  3. Modeling long-term host cell-Giardia lamblia interactions in an in vitro co-culture system.

    Directory of Open Access Journals (Sweden)

    Bridget S Fisher

    Full Text Available Globally, there are greater than 700,000 deaths per year associated with diarrheal disease. The flagellated intestinal parasite, Giardia lamblia, is one of the most common intestinal pathogens in both humans and animals throughout the world. While attached to the gastrointestinal epithelium, Giardia induces epithelial cell apoptosis, disrupts tight junctions, and increases intestinal permeability. The underlying cellular and molecular mechanisms of giardiasis, including the role lamina propria immune cells, such as macrophages, play in parasite control or clearance are poorly understood. Thus far, one of the major obstacles in ascertaining the mechanisms of Giardia pathology is the lack of a functionally relevant model for the long-term study of the parasite in vitro. Here we report on the development of an in vitro co-culture model which maintains the basolateral-apical architecture of the small intestine and allows for long-term survival of the parasite. Using transwell inserts, Caco-2 intestinal epithelial cells and IC-21 macrophages are co-cultured in the presence of Giardia trophozoites. Using the developed model, we show that Giardia trophozoites survive over 21 days and proliferate in a combination media of Caco-2 cell and Giardia medium. Giardia induces apoptosis of epithelial cells through caspase-3 activation and macrophages do not abrogate this response. Additionally, macrophages induce Caco-2 cells to secrete the pro-inflammatory cytokines, GRO and IL-8, a response abolished by Giardia indicating parasite induced suppression of the host immune response. The co-culture model provides additional complexity and information when compared to a single-cell model. This model will be a valuable tool for answering long-standing questions on host-parasite biology that may lead to discovery of new therapeutic interventions.

  4. Importance of a diffusion-dominant small volume to activate cell-secreted soluble factor signaling in embryonic stem cell culture in microbioreactors: a mathematical model based study.

    Science.gov (United States)

    Chowdhury, Mohammad Mahfuz; Fujii, Teruo; Sakai, Yasuyuki

    2013-07-01

    In our previous studies, we observed that cell-secreted BMP4 had a prominent influence on mouse embryonic stem cell (mESC) behaviors in a membrane-based two-chambered microbioreactor (MB), but not in a macro-scale culture (6-well plate/6WP). In this study, we investigated how the physical aspects of these cultures regulated BMP4 signaling by developing mathematical models of the cultures. The models estimated signaling activity in the cultures by considering size of the undifferentiated mESC colonies and their growth, diffusion of BMP4, and BMP4 trafficking process in the colonies. The models successfully depicted measured profile of BMP4 concentration in the culture medium which was two times higher in the MB than that in the 6WP during 5-day culture. The models estimated that, owing to the small volume and the membrane, cells were exposed to a higher BMP4 concentration in the top chamber of the MB than that in the 6WP culture. The higher concentration of BMP4 induced a higher concentration of BMP4-bound receptor in the colony in the MB than in the 6WP, thereby leading to the higher activation of BMP4 signaling in the MB. The models also predicted that the size of the MB, but not that of the 6WP, was suitable for maximizing BMP4 accumulation and upregulating its signaling. This study will be helpful in analyzing culture systems, designing microfluidic devices for controlling ESC or other cell behavior. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  5. Induction of calcification by serum depletion in cell culture: a model for focal calcification in aortas related to atherosclerosis

    Directory of Open Access Journals (Sweden)

    Villar Maria T

    2008-01-01

    Full Text Available Abstract Background Since aortic calcification has been shown to initiate in the lower zone of well-thickened plaques (LZP adjacent to the aortic media of rabbits fed supplemental cholesterol diets, a restricted supply of serum to vascular cells could play a role in vascular calcification. This study was designed to use a cell culture model to support this hypothesis. Results Rabbit aortic smooth muscle cells were grown to confluence in a culture media containing 10 % fetal bovine serum (FBS. The confluent cells were then exposed to the media for 2 hrs with or without serum at a Ca × P ion product range of 4.5–9.4 mM2. In contrast to the cells cultured in the presence of FBS, confluent cells in its absence displayed marked mineral-positive alizarin red staining and infrared absorption of mineral phosphate. A kinetic parameter C1/2 was used to designate the concentration of serum or its protein constituents needed to reduce the deposition of Ca and P by half. The C1/2 for FBS and rabbit serum was 0.04–0.07 % The C1/2 value for rabbit serum proteins was 13.5 μg/ml corresponding to the protein concentration in 0.06 % of serum. This C1/2 was markedly smaller than 86.2 μg/ml for bovine serum albumin present in 0.37 % serum (p Conclusion The aortic smooth muscle cell culture model suggests that serum depletion may play a role in the initiation of aortic calcification. The serum exhibits remarkable ability to inhibit cell-mediated calcification.

  6. An innovative intermittent hypoxia model for cell cultures allowing fast Po2 oscillations with minimal gas consumption.

    Science.gov (United States)

    Minoves, Mélanie; Morand, Jessica; Perriot, Frédéric; Chatard, Morgane; Gonthier, Brigitte; Lemarié, Emeline; Menut, Jean-Baptiste; Polak, Jan; Pépin, Jean-Louis; Godin-Ribuot, Diane; Briançon-Marjollet, Anne

    2017-10-01

    Performing hypoxia-reoxygenation cycles in cell culture with a cycle duration accurately reflecting what occurs in obstructive sleep apnea (OSA) patients is a difficult but crucial technical challenge. Our goal was to develop a novel device to expose multiple cell culture dishes to intermittent hypoxia (IH) cycles relevant to OSA with limited gas consumption. With gas flows as low as 200 ml/min, our combination of plate holders with gas-permeable cultureware generates rapid normoxia-hypoxia cycles. Cycles alternating 1 min at 20% O 2 followed by 1 min at 2% O 2 resulted in Po 2 values ranging from 124 to 44 mmHg. Extending hypoxic and normoxic phases to 10 min allowed Po 2 variations from 120 to 25 mmHg. The volume of culture medium or the presence of cells only modestly affected the Po 2 variations. In contrast, the nadir of the hypoxia phase increased when measured at different heights above the membrane. We validated the physiological relevance of this model by showing that hypoxia inducible factor-1α expression was significantly increased by IH exposure in human aortic endothelial cells, murine breast carcinoma (4T1) cells as well as in a blood-brain barrier model (2.5-, 1.5-, and 6-fold increases, respectively). In conclusion, we have established a new device to perform rapid intermittent hypoxia cycles in cell cultures, with minimal gas consumption and the possibility to expose several culture dishes simultaneously. This device will allow functional studies of the consequences of IH and deciphering of the molecular biology of IH at the cellular level using oxygen cycles that are clinically relevant to OSA. Copyright © 2017 the American Physiological Society.

  7. Comparative SAXS and DSC study on stratum corneum structural organization in an epidermal cell culture model (ROC)

    DEFF Research Database (Denmark)

    Kuntsche, Judith; Herre, Angela; Fahr, Alfred

    2013-01-01

    barrier similar to that of human stratum corneum is, however, a prerequisite. In this study, the stratum corneum lipid organization in an epidermal cell culture model based on rat epidermal keratinocytes (REK organotypic culture, ROC) was investigated by small-angle X-ray scattering (SAXS) in dependence......Cell cultured skin equivalents present an alternative for dermatological in vitro evaluations of drugs and excipients as they provide the advantage of availability, lower variability and higher assay robustness compared to native skin. For penetration/permeation studies, an adequate stratum corneum...... and SC lipid organization. Cultivation for 21days resulted in further minor changes in the structural organization of ROC SC. The SAXS patterns of ROC SC had overall large similarities with that of human SC and point to the presence of a long periodicity phase with a repeat distance of about 122Å, e...

  8. A Triple Culture Model of the Blood-Brain Barrier Using Porcine Brain Endothelial cells, Astrocytes and Pericytes.

    Science.gov (United States)

    Thomsen, Louiza Bohn; Burkhart, Annette; Moos, Torben

    2015-01-01

    In vitro blood-brain barrier (BBB) models based on primary brain endothelial cells (BECs) cultured as monoculture or in co-culture with primary astrocytes and pericytes are useful for studying many properties of the BBB. The BECs retain their expression of tight junction proteins and efflux transporters leading to high trans-endothelial electric resistance (TEER) and low passive paracellular permeability. The BECs, astrocytes and pericytes are often isolated from small rodents. Larger species as cows and pigs however, reveal a higher yield, are readily available and have a closer resemblance to humans, which make them favorable high-throughput sources for cellular isolation. The aim of the present study has been to determine if the preferable combination of purely porcine cells isolated from the 6 months old domestic pigs, i.e. porcine brain endothelial cells (PBECs) in co-culture with porcine astrocytes and pericytes, would compare with PBECs co-cultured with astrocytes and pericytes isolated from newborn rats with respect to TEER value and low passive permeability. The astrocytes and pericytes were grown both as contact and non-contact co-cultures as well as in triple culture to examine their effects on the PBECs for barrier formation as revealed by TEER, passive permeability, and expression patterns of tight junction proteins, efflux transporters and the transferrin receptor. This syngenic porcine in vitro BBB model is comparable to triple cultures using PBECs, rat astrocytes and rat pericytes with respect to TEER formation, low passive permeability, and expression of hallmark proteins signifying the brain endothelium (tight junction proteins claudin 5 and occludin, the efflux transporters P-glycoprotein (PgP) and breast cancer related protein (BCRP), and the transferrin receptor).

  9. A Triple Culture Model of the Blood-Brain Barrier Using Porcine Brain Endothelial cells, Astrocytes and Pericytes.

    Directory of Open Access Journals (Sweden)

    Louiza Bohn Thomsen

    Full Text Available In vitro blood-brain barrier (BBB models based on primary brain endothelial cells (BECs cultured as monoculture or in co-culture with primary astrocytes and pericytes are useful for studying many properties of the BBB. The BECs retain their expression of tight junction proteins and efflux transporters leading to high trans-endothelial electric resistance (TEER and low passive paracellular permeability. The BECs, astrocytes and pericytes are often isolated from small rodents. Larger species as cows and pigs however, reveal a higher yield, are readily available and have a closer resemblance to humans, which make them favorable high-throughput sources for cellular isolation. The aim of the present study has been to determine if the preferable combination of purely porcine cells isolated from the 6 months old domestic pigs, i.e. porcine brain endothelial cells (PBECs in co-culture with porcine astrocytes and pericytes, would compare with PBECs co-cultured with astrocytes and pericytes isolated from newborn rats with respect to TEER value and low passive permeability. The astrocytes and pericytes were grown both as contact and non-contact co-cultures as well as in triple culture to examine their effects on the PBECs for barrier formation as revealed by TEER, passive permeability, and expression patterns of tight junction proteins, efflux transporters and the transferrin receptor. This syngenic porcine in vitro BBB model is comparable to triple cultures using PBECs, rat astrocytes and rat pericytes with respect to TEER formation, low passive permeability, and expression of hallmark proteins signifying the brain endothelium (tight junction proteins claudin 5 and occludin, the efflux transporters P-glycoprotein (PgP and breast cancer related protein (BCRP, and the transferrin receptor.

  10. Protein Biomarkers Associated With Growth And Synaptogenesis In a cell culture model of neuronal development

    Science.gov (United States)

    Cerebellar granule cells (CGC) provide a homogenous population of cells which can be used as an in vitro model for studying the cellular processes involved in the normal development of the CNS. They may also be useful for hazard identification as in vitro screens fo...

  11. Conserved host response to highly pathogenic avian influenza virus infection in human cell culture, mouse and macaque model systems

    Directory of Open Access Journals (Sweden)

    McDermott Jason E

    2011-11-01

    Full Text Available Abstract Background Understanding host response to influenza virus infection will facilitate development of better diagnoses and therapeutic interventions. Several different experimental models have been used as a proxy for human infection, including cell cultures derived from human cells, mice, and non-human primates. Each of these systems has been studied extensively in isolation, but little effort has been directed toward systematically characterizing the conservation of host response on a global level beyond known immune signaling cascades. Results In the present study, we employed a multivariate modeling approach to characterize and compare the transcriptional regulatory networks between these three model systems after infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Using this approach we identified functions and pathways that display similar behavior and/or regulation including the well-studied impact on the interferon response and the inflammasome. Our results also suggest a primary response role for airway epithelial cells in initiating hypercytokinemia, which is thought to contribute to the pathogenesis of H5N1 viruses. We further demonstrate that we can use a transcriptional regulatory model from the human cell culture data to make highly accurate predictions about the behavior of important components of the innate immune system in tissues from whole organisms. Conclusions This is the first demonstration of a global regulatory network modeling conserved host response between in vitro and in vivo models.

  12. Microfluidic cell culture systems for drug research.

    Science.gov (United States)

    Wu, Min-Hsien; Huang, Song-Bin; Lee, Gwo-Bin

    2010-04-21

    In pharmaceutical research, an adequate cell-based assay scheme to efficiently screen and to validate potential drug candidates in the initial stage of drug discovery is crucial. In order to better predict the clinical response to drug compounds, a cell culture model that is faithful to in vivo behavior is required. With the recent advances in microfluidic technology, the utilization of a microfluidic-based cell culture has several advantages, making it a promising alternative to the conventional cell culture methods. This review starts with a comprehensive discussion on the general process for drug discovery and development, the role of cell culture in drug research, and the characteristics of the cell culture formats commonly used in current microfluidic-based, cell-culture practices. Due to the significant differences in several physical phenomena between microscale and macroscale devices, microfluidic technology provides unique functionality, which is not previously possible by using traditional techniques. In a subsequent section, the niches for using microfluidic-based cell culture systems for drug research are discussed. Moreover, some critical issues such as cell immobilization, medium pumping or gradient generation in microfluidic-based, cell-culture systems are also reviewed. Finally, some practical applications of microfluidic-based, cell-culture systems in drug research particularly those pertaining to drug toxicity testing and those with a high-throughput capability are highlighted.

  13. Cluster–cluster aggregation with particle replication and chemotaxy: a simple model for the growth of animal cells in culture

    International Nuclear Information System (INIS)

    Alves, S G; Martins, M L

    2010-01-01

    Aggregation of animal cells in culture comprises a series of motility, collision and adhesion processes of basic relevance for tissue engineering, bioseparations, oncology research and in vitro drug testing. In the present paper, a cluster–cluster aggregation model with stochastic particle replication and chemotactically driven motility is investigated as a model for the growth of animal cells in culture. The focus is on the scaling laws governing the aggregation kinetics. Our simulations reveal that in the absence of chemotaxy the mean cluster size and the total number of clusters scale in time as stretched exponentials dependent on the particle replication rate. Also, the dynamical cluster size distribution functions are represented by a scaling relation in which the scaling function involves a stretched exponential of the time. The introduction of chemoattraction among the particles leads to distribution functions decaying as power laws with exponents that decrease in time. The fractal dimensions and size distributions of the simulated clusters are qualitatively discussed in terms of those determined experimentally for several normal and tumoral cell lines growing in culture. It is shown that particle replication and chemotaxy account for the simplest cluster size distributions of cellular aggregates observed in culture

  14. More Novel Hantaviruses and Diversifying Reservoir Hosts — Time for Development of Reservoir-Derived Cell Culture Models?

    Directory of Open Access Journals (Sweden)

    Isabella Eckerle

    2014-02-01

    Full Text Available Due to novel, improved and high-throughput detection methods, there is a plethora of newly identified viruses within the genus Hantavirus. Furthermore, reservoir host species are increasingly recognized besides representatives of the order Rodentia, now including members of the mammalian orders Soricomorpha/Eulipotyphla and Chiroptera. Despite the great interest created by emerging zoonotic viruses, there is still a gross lack of in vitro models, which reflect the exclusive host adaptation of most zoonotic viruses. The usually narrow host range and genetic diversity of hantaviruses make them an exciting candidate for studying virus-host interactions on a cellular level. To do so, well-characterized reservoir cell lines covering a wide range of bat, insectivore and rodent species are essential. Most currently available cell culture models display a heterologous virus-host relationship and are therefore only of limited value. Here, we review the recently established approaches to generate reservoir-derived cell culture models for the in vitro study of virus-host interactions. These successfully used model systems almost exclusively originate from bats and bat-borne viruses other than hantaviruses. Therefore we propose a parallel approach for research on rodent- and insectivore-borne hantaviruses, taking the generation of novel rodent and insectivore cell lines from wildlife species into account. These cell lines would be also valuable for studies on further rodent-borne viruses, such as orthopox- and arenaviruses.

  15. HCV Genotype 6a Escape From and Resistance to Velpatasvir, Pibrentasvir, and Sofosbuvir in Robust Infectious Cell Culture Models

    DEFF Research Database (Denmark)

    Pham, Long V; Ramirez, Santseharay; Gottwein, Judith M

    2018-01-01

    V was able to propagate and escape in the presence of pibrentasvir with emergence of NS5A-L28S, conferring a high level of resistance to this inhibitor. CONCLUSIONS: Strains of HCV genotype 6a isolated from patients can be adapted to propagate in cultured cells, permitting studies of the complete...... infectious cell culture models of HCV genotype 6a infection to study the effects of these inhibitors and the development of resistance. METHODS: The consensus sequences of prototype strains HK2 (MG717925) and HK6a (MG717928), originating from serum of patients with chronic HCV infection, were determined...... by Sanger sequencing of genomes amplified by reverse-transcription polymerase chain reaction. In vitro noninfectious full-length clones of these 6a strains were subsequently adapted in Huh7.5 cells, primarily by using substitutions identified in JFH1-based Core-NS5A and Core-NS5B genotype 6a recombinants...

  16. Serum-free keloid fibroblast cell culture: an in vitro model for the study of aberrant wound healing.

    Science.gov (United States)

    Koch, R J; Goode, R L; Simpson, G T

    1997-04-01

    The purpose of this study was to develop an in vitro serum-free keloid fibroblast model. Keloid formation remains a problem for every surgeon. Prior evaluations of fibroblast characteristics in vitro, especially those of growth factor measurement, have been confounded by the presence of serum-containing tissue culture media. The serum itself contains growth factors, yet has been a "necessary evil" to sustain cell growth. The design of this study is laboratory-based and uses keloid fibroblasts obtained from five patients undergoing facial (ear lobule) keloid removal in a university-affiliated clinic. Keloid fibroblasts were established in primary cell culture and then propagated in a serum-free environment. The main outcome measures included sustained keloid fibroblast growth and viability, which was comparable to serum-based models. The keloid fibroblast cell cultures exhibited logarithmic growth, sustained a high cellular viability, maintained a monolayer, and displayed contact inhibition. Demonstrating model consistency, there was no statistically significant difference between the mean cell counts of the five keloid fibroblast cell lines at each experimental time point. The in vitro growth of keloid fibroblasts in a serum-free model has not been done previous to this study. The results of this study indicate that the proliferative characteristics described are comparable to those of serum-based models. The described model will facilitate the evaluation of potential wound healing modulators, and cellular effects and collagen modifications of laser resurfacing techniques, and may serve as a harvest source for contaminant-free fibroblast autoimplants. Perhaps its greatest utility will be in the evaluation of endogenous and exogenous growth factors.

  17. In vitro models of the blood–brain barrier: An overview of commonly used brain endothelial cell culture models and guidelines for their use

    OpenAIRE

    Helms, Hans C; Abbott, N Joan; Burek, Malgorzata; Cecchelli, Romeo; Couraud, Pierre-Olivier; Deli, Maria A; Förster, Carola; Galla, Hans J; Romero, Ignacio A; Shusta, Eric V; Stebbins, Matthew J; Vandenhaute, Elodie; Weksler, Babette; Brodin, Birger

    2016-01-01

    The endothelial cells lining the brain capillaries separate the blood from the brain parenchyma. The endothelial monolayer of the brain capillaries serves both as a crucial interface for exchange of nutrients, gases, and metabolites between blood and brain, and as a barrier for neurotoxic components of plasma and xenobiotics. This “blood-brain barrier” function is a major hindrance for drug uptake into the brain parenchyma. Cell culture models, based on either primary cells or immortalized br...

  18. Three-Dimensional Organotypic Co-Culture Model of Intestinal Epithelial Cells and Macrophages to Study "Salmonella Enterica" Colonization Patterns

    Science.gov (United States)

    Ott, Mark; Yang, J; Barilla, J.; Crabbe, A.; Sarker, S. F.; Liu, Y.

    2017-01-01

    Three-dimensional/3-D organotypic models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by 2-D monolayers and respond to Salmonella in ways that reflect in vivo infections. To further enhance the physiological relevance of 3-D models to more closely approximate in vivo intestinal microenvironments during infection, we developed and validated a novel 3-D intestinal co-culture model containing multiple epithelial cell types and phagocytic macrophages, and applied to study enteric infection by different Salmonella pathovars.

  19. Organizational culture modeling

    OpenAIRE

    Valentina Mihaela GHINEA; Constantin BRĂTIANU

    2012-01-01

    The purpose of this paper is to present a conceptual analysis of organizational culture modeling in the framework of system dynamics. Tom Peters and Robert Waterman demonstrated through their seminal research that organizational culture constitutes one of the most important key success factors in any company trying to achieve excellence in its business. Organizational culture is a strong nonlinear integrator of the organizational intellectual capital acting especially on the emotional knowled...

  20. Cultural Resource Predictive Modeling

    Science.gov (United States)

    2017-10-01

    CR cultural resource CRM cultural resource management CRPM Cultural Resource Predictive Modeling DoD Department of Defense ESTCP Environmental...resource management ( CRM ) legal obligations under NEPA and the NHPA, military installations need to demonstrate that CRM decisions are based on objective...maxim “one size does not fit all,” and demonstrate that DoD installations have many different CRM needs that can and should be met through a variety

  1. A system identification approach for developing model predictive controllers of antibody quality attributes in cell culture processes.

    Science.gov (United States)

    Downey, Brandon; Schmitt, John; Beller, Justin; Russell, Brian; Quach, Anthony; Hermann, Elizabeth; Lyon, David; Breit, Jeffrey

    2017-11-01

    As the biopharmaceutical industry evolves to include more diverse protein formats and processes, more robust control of Critical Quality Attributes (CQAs) is needed to maintain processing flexibility without compromising quality. Active control of CQAs has been demonstrated using model predictive control techniques, which allow development of processes which are robust against disturbances associated with raw material variability and other potentially flexible operating conditions. Wide adoption of model predictive control in biopharmaceutical cell culture processes has been hampered, however, in part due to the large amount of data and expertise required to make a predictive model of controlled CQAs, a requirement for model predictive control. Here we developed a highly automated, perfusion apparatus to systematically and efficiently generate predictive models using application of system identification approaches. We successfully created a predictive model of %galactosylation using data obtained by manipulating galactose concentration in the perfusion apparatus in serialized step change experiments. We then demonstrated the use of the model in a model predictive controller in a simulated control scenario to successfully achieve a %galactosylation set point in a simulated fed-batch culture. The automated model identification approach demonstrated here can potentially be generalized to many CQAs, and could be a more efficient, faster, and highly automated alternative to batch experiments for developing predictive models in cell culture processes, and allow the wider adoption of model predictive control in biopharmaceutical processes. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 33:1647-1661, 2017. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers.

  2. Blue-light filtering alters angiogenic signaling in human retinal pigmented epithelial cells culture model.

    Science.gov (United States)

    Vila, Natalia; Siblini, Aya; Esposito, Evangelina; Bravo-Filho, Vasco; Zoroquiain, Pablo; Aldrees, Sultan; Logan, Patrick; Arias, Lluis; Burnier, Miguel N

    2017-11-02

    Light exposure and more specifically the spectrum of blue light contribute to the oxidative stress in Age-related macular degeneration (AMD). The purpose of the study was to establish whether blue light filtering could modify proangiogenic signaling produced by retinal pigmented epithelial (RPE) cells under different conditions simulating risk factors for AMD. Three experiments were carried out in order to expose ARPE-19 cells to white light for 48 h with and without blue light-blocking filters (BLF) in different conditions. In each experiment one group was exposed to light with no BLF protection, a second group was exposed to light with BLF protection, and a control group was not exposed to light. The ARPE-19 cells used in each experiment prior to light exposure were cultured for 24 h as follows: Experiment 1) Normoxia, Experiment 2) Hypoxia, and Experiment 3) Lutein supplemented media in normoxia. The media of all groups was harvested after light exposure for sandwich ELISA-based assays to quantify 10 pro-angiogenic cytokines. A significant decrease in angiogenin secretion levels and a significant increase in bFGF were observed following light exposure, compared to dark conditions, in both normoxia and hypoxia conditions. With the addition of a blue light-blocking filter in normoxia, a significant increase in angiogenin levels was observed. Although statistical significance was not achieved, blue light filters reduce light-induced secretion of bFGF and VEGF to near normal levels. This trend is also observed when ARPE-19 cells are grown under hypoxic conditions and when pre-treated with lutein prior to exposure to experimental conditions. Following light exposure, there is a decrease in angiogenin secretion by ARPE-19 cells, which was abrogated with a blue light - blocking filter. Our findings support the position that blue light filtering affects the secretion of angiogenic factors by retinal pigmented epithelial cells under normoxic, hypoxic, and lutein

  3. Modeling human disease using organotypic cultures

    DEFF Research Database (Denmark)

    Schweiger, Pawel J; Jensen, Kim B

    2016-01-01

    animal models and in vitro cell culture systems. However, it has been exceedingly difficult to model disease at the tissue level. Since recently, the gap between cell line studies and in vivo modeling has been narrowing thanks to progress in biomaterials and stem cell research. Development of reliable 3D...... culture systems has enabled a rapid expansion of sophisticated in vitro models. Here we focus on some of the latest advances and future perspectives in 3D organoids for human disease modeling....

  4. Acrolein stimulates the synthesis of IL-6 and C-reactive protein (CRP) in thrombosis model mice and cultured cells.

    Science.gov (United States)

    Saiki, Ryotaro; Hayashi, Daisuke; Ikuo, Yukiko; Nishimura, Kazuhiro; Ishii, Itsuko; Kobayashi, Kaoru; Chiba, Kan; Toida, Toshihiko; Kashiwagi, Keiko; Igarashi, Kazuei

    2013-12-01

    Measurements of protein-conjugated acrolein (PC-Acro), IL-6, and C-reactive protein (CRP) in plasma were useful for identifying silent brain infarction with high sensitivity and specificity. The aim of this study was to determine whether acrolein causes increased production of IL-6 and CRP in thrombosis model mice and cultured cells. In mice with photochemically induced thrombosis, acrolein produced at the locus of infarction increased the level of IL-6 and then CRP in plasma. This was confirmed in cell culture systems - acrolein stimulated the production of IL-6 in mouse neuroblastoma Neuro-2a cells, mouse macrophage-like J774.1 cells, and human umbilical vein endothelial cells (HUVEC), and IL-6 in turn stimulated the production of CRP in human hepatocarcinoma cells. The level of IL-6 mRNA was increased by acrolein through an increase in phosphorylation of the transcription factors, c-Jun, and NF-κB p65. Furthermore, CRP stimulated IL-6 production in mouse macrophage-like J774.1 cells and HUVEC. IL-6 functioned as a protective factor against acrolein toxicity in Neuro-2a cells and HUVEC. These results show that acrolein stimulates the synthesis of IL-6 and CRP, which function as protecting factors against acrolein toxicity, and that the combined measurement of PC-Acro, IL-6, and CRP is effective for identification of silent brain infarction. The combined measurements of protein-conjugated acrolein (PC-Acro), IL-6, and C-reactive protein (CRP) in plasma were useful for identifying silent brain infarction. The aim of this study was to determine whether acrolein causes increased production of IL-6 and CRP, and indeed acrolein increased IL-6 synthesis and IL-6 in turn increased CRP synthesis. Furthermore, IL-6 decreased acrolein toxicity in several cell lines. © 2013 International Society for Neurochemistry.

  5. Pediatric and Adult High-Grade Glioma Stem Cell Culture Models Are Permissive to Lytic Infection with Parvovirus H-1.

    Science.gov (United States)

    Josupeit, Rafael; Bender, Sebastian; Kern, Sonja; Leuchs, Barbara; Hielscher, Thomas; Herold-Mende, Christel; Schlehofer, Jörg R; Dinsart, Christiane; Witt, Olaf; Rommelaere, Jean; Lacroix, Jeannine

    2016-05-19

    Combining virus-induced cytotoxic and immunotherapeutic effects, oncolytic virotherapy represents a promising therapeutic approach for high-grade glioma (HGG). A clinical trial has recently provided evidence for the clinical safety of the oncolytic parvovirus H-1 (H-1PV) in adult glioblastoma relapse patients. The present study assesses the efficacy of H-1PV in eliminating HGG initiating cells. H-1PV was able to enter and to transduce all HGG neurosphere culture models (n = 6), including cultures derived from adult glioblastoma, pediatric glioblastoma, and diffuse intrinsic pontine glioma. Cytotoxic effects induced by the virus have been observed in all HGG neurospheres at half maximal inhibitory concentration (IC50) doses of input virus between 1 and 10 plaque forming units per cell. H-1PV infection at this dose range was able to prevent tumorigenicity of NCH421k glioblastoma multiforme (GBM) "stem-like" cells in NOD/SCID mice. Interestingly NCH421R, an isogenic subclone with equal capacity of xenograft formation, but resistant to H-1PV infection could be isolated from the parental NCH421k culture. To reveal changes in gene expression associated with H-1PV resistance we performed a comparative gene expression analysis in these subclones. Several dysregulated genes encoding receptor proteins, endocytosis factors or regulators innate antiviral responses were identified and represent intriguing candidates for to further study molecular mechanisms of H-1PV resistance.

  6. Bactericidal Activity of Ceragenin CSA-13 in Cell Culture and in an Animal Model of Peritoneal Infection.

    Science.gov (United States)

    Bucki, Robert; Niemirowicz, Katarzyna; Wnorowska, Urszula; Byfield, Fitzroy J; Piktel, Ewelina; Wątek, Marzena; Janmey, Paul A; Savage, Paul B

    2015-10-01

    Ceragenins constitute a novel family of cationic antibiotics characterized by a broad spectrum of antimicrobial activities, which have mostly been assessed in vitro. Using a polarized human lung epithelial cell culture system, we evaluated the antibacterial activities of the ceragenin CSA-13 against two strains of Pseudomonas aeruginosa (PAO1 and Xen5). Additionally, the biodistribution and bactericidal activity of a CSA-13-IRDye 800CW derivate were assessed using an animal model of peritoneal infection after PAO1 challenge. In cell culture, CSA-13 bactericidal activities against PAO1 and Xen5 were higher than the activities of the human cathelicidin peptide LL-37. Increased CSA-13 activity was observed in polarized human lung epithelial cell cultures subjected to butyric acid treatment, which is known to increase endogenous LL-37 production. Eight hours after intravenous or intraperitoneal injection, the greatest CSA-13-IRDye 800CW accumulation was observed in mouse liver and kidneys. CSA-13-IRDye 800CW administration resulted in decreased bacterial outgrowth from abdominal fluid collected from animals subjected to intraperitoneal PAO1 infection. These observations indicate that CSA-13 may synergistically interact with antibacterial factors that are naturally present at mucosal surfaces and it maintains its antibacterial activity in the infected abdominal cavity. Cationic lipids such as CSA-13 represent excellent candidates for the development of new antibacterial compounds. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. AlgiMatrix™-Based 3D Cell Culture System as an In Vitro Tumor Model: An Important Tool in Cancer Research.

    Science.gov (United States)

    Godugu, Chandraiah; Singh, Mandip

    2016-01-01

    Routinely used two-dimensional cell culture-based models often fail while translating the observations into in vivo models. This setback is more common in cancer research, due to several reasons. The extracellular matrix and cell-to-cell interactions are not present in two-dimensional (2D) cell culture models. Diffusion of drug molecules into cancer cells is hindered by barriers of extracellular components in in vivo conditions, these barriers are absent in 2D cell culture models. To better mimic or simulate the in vivo conditions present in tumors, the current study used the alginate based three-dimensional cell culture (AlgiMatrix™) model, which resembles close to the in vivo tumor models. The current study explains the detailed protocols involved in AlgiMatrix™ based in vitro non-small-cell lung cancer (NSCLC) models. The suitability of this model was studied by evaluating, cytotoxicity, apoptosis, and penetration of nanoparticles into the in vitro tumor spheroids. This study also demonstrated the effect of EphA2 receptor targeted docetaxel-loaded nanoparticles on MDA-MB-468 TNBC cell lines. The methods section is subdivided into three subsections such as (1) preparation of AlgiMatrix™-based 3D in vitro tumor models and cytotoxicity assays, (2) free drug and nanoparticle uptake into spheroid studies, and (3) western blot, IHC, and RT-PCR studies.

  8. Anaerobic co-culture of mesenchymal stem cells and anaerobic pathogens - a new in vitro model system.

    Directory of Open Access Journals (Sweden)

    Katja Kriebel

    Full Text Available BACKGROUND: Human mesenchymal stem cells (hMSCs are multipotent by nature and are originally isolated from bone marrow. In light of a future application of hMSCs in the oral cavity, a body compartment with varying oxygen partial pressures and an omnipresence of different bacterial species i.e. periodontitis pathogens, we performed this study to gain information about the behavior of hMSC in an anaerobic system and the response in interaction with oral bacterial pathogens. METHODOLOGY/PRINCIPAL FINDINGS: We established a model system with oral pathogenic bacterial species and eukaryotic cells cultured in anaerobic conditions. The facultative anaerobe bacteria Fusobacterium nucleatum, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were studied. Their effects on hMSCs and primary as well as permanent gingival epithelial cells (Ca9-22, HGPEC were comparatively analyzed. We show that hMSCs cope with anoxic conditions, since 40% vital cells remain after 72 h of anaerobic culture. The Ca9-22 and HGPEC cells are significantly more sensitive to lack of oxygen. All bacterial species reveal a comparatively low adherence to and internalization into hMSCs (0.2% and 0.01% of the initial inoculum, respectively. In comparison, the Ca9-22 and HGPEC cells present better targets for bacterial adherence and internalization. The production of the pro-inflammatory chemokine IL-8 is higher in both gingival epithelial cell lines compared to hMSCs and Fusobacterium nucleatum induce a time-dependent cytokine secretion in both cell lines. Porphyromonas gingivalis is less effective in stimulating secretion of IL-8 in the co-cultivation experiments. CONCLUSIONS/SIGNIFICANCE: HMSCs are suitable for use in anoxic regions of the oral cavity. The interaction with local pathogenic bacteria does not result in massive pro-inflammatory cytokine responses. The test system established in this study allowed further investigation of parameters prior to set up of

  9. Tracheobronchial air-liquid interface cell culture: a model for innate mucosal defense of the upper airways?

    Science.gov (United States)

    Kesimer, Mehmet; Kirkham, Sara; Pickles, Raymond J.; Henderson, Ashley G.; Alexis, Neil E.; DeMaria, Genevieve; Knight, David; Thornton, David J.; Sheehan, John K.

    2009-01-01

    Human tracheobronchial epithelial cells grown in air-liquid interface culture have emerged as a powerful tool for the study of airway biology. In this study, we have investigated whether this culture system produces “mucus” with a protein composition similar to that of in vivo, induced airway secretions. Previous compositional studies of mucous secretions have greatly underrepresented the contribution of mucins, which are major structural components of normal mucus. To overcome this limitation, we have used a mass spectrometry-based approach centered on prior separation of the mucins from the majority of the other proteins. Using this approach, we have compared the protein composition of apical secretions (AS) from well-differentiated primary human tracheobronchial cells grown at air-liquid interface and human tracheobronchial normal induced sputum (IS). A total of 186 proteins were identified, 134 from AS and 136 from IS; 84 proteins were common to both secretions, with host defense proteins being predominant. The epithelial mucins MUC1, MUC4, and MUC16 and the gel-forming mucins MUC5B and MUC5AC were identified in both secretions. Refractometry showed that the gel-forming mucins were the major contributors by mass to both secretions. When the composition of the IS was corrected for proteins that were most likely derived from saliva, serum, and migratory cells, there was considerable similarity between the two secretions, in particular, in the category of host defense proteins, which includes the mucins. This shows that the primary cell culture system is an important model for study of aspects of innate defense of the upper airways related specifically to mucus consisting solely of airway cell products. PMID:18931053

  10. Dynamized Preparations in Cell Culture

    Directory of Open Access Journals (Sweden)

    Ellanzhiyil Surendran Sunila

    2009-01-01

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

  11. Agent-Based Computational Modeling of Cell Culture: Understanding Dosimetry In Vitro as Part of In Vitro to In Vivo Extrapolation

    Science.gov (United States)

    Quantitative characterization of cellular dose in vitro is needed for alignment of doses in vitro and in vivo. We used the agent-based software, CompuCell3D (CC3D), to provide a stochastic description of cell growth in culture. The model was configured so that isolated cells assu...

  12. In vitro models of the blood–brain barrier: An overview of commonly used brain endothelial cell culture models and guidelines for their use

    Science.gov (United States)

    Helms, Hans C; Abbott, N Joan; Burek, Malgorzata; Cecchelli, Romeo; Couraud, Pierre-Olivier; Deli, Maria A; Förster, Carola; Galla, Hans J; Romero, Ignacio A; Shusta, Eric V; Stebbins, Matthew J; Vandenhaute, Elodie; Weksler, Babette

    2016-01-01

    The endothelial cells lining the brain capillaries separate the blood from the brain parenchyma. The endothelial monolayer of the brain capillaries serves both as a crucial interface for exchange of nutrients, gases, and metabolites between blood and brain, and as a barrier for neurotoxic components of plasma and xenobiotics. This “blood-brain barrier” function is a major hindrance for drug uptake into the brain parenchyma. Cell culture models, based on either primary cells or immortalized brain endothelial cell lines, have been developed, in order to facilitate in vitro studies of drug transport to the brain and studies of endothelial cell biology and pathophysiology. In this review, we aim to give an overview of established in vitro blood–brain barrier models with a focus on their validation regarding a set of well-established blood–brain barrier characteristics. As an ideal cell culture model of the blood–brain barrier is yet to be developed, we also aim to give an overview of the advantages and drawbacks of the different models described. PMID:26868179

  13. Principles of cancer cell culture.

    Science.gov (United States)

    Cree, Ian A

    2011-01-01

    The basics of cell culture are now relatively common, though it was not always so. The pioneers of cell culture would envy our simple access to manufactured plastics, media and equipment for such studies. The prerequisites for cell culture are a well lit and suitably ventilated laboratory with a laminar flow hood (Class II), CO(2) incubator, benchtop centrifuge, microscope, plasticware (flasks and plates) and a supply of media with or without serum supplements. Not only can all of this be ordered easily over the internet, but large numbers of well-characterised cell lines are available from libraries maintained to a very high standard allowing the researcher to commence experiments rapidly and economically. Attention to safety and disposal is important, and maintenance of equipment remains essential. This chapter should enable researchers with little prior knowledge to set up a suitable laboratory to do basic cell culture, but there is still no substitute for experience within an existing well-run laboratory.

  14. Evaluation of silicon nitride as a substrate for culture of PC12 cells: an interfacial model for functional studies in neurons.

    Directory of Open Access Journals (Sweden)

    Johan Jaime Medina Benavente

    Full Text Available Silicon nitride is a biocompatible material that is currently used as an interfacial surface between cells and large-scale integration devices incorporating ion-sensitive field-effect transistor technology. Here, we investigated whether a poly-L-lysine coated silicon nitride surface is suitable for the culture of PC12 cells, which are widely used as a model for neural differentiation, and we characterized their interaction based on cell behavior when seeded on the tested material. The coated surface was first examined in terms of wettability and topography using contact angle measurements and atomic force microscopy and then, conditioned silicon nitride surface was used as the substrate for the study of PC12 cell culture properties. We found that coating silicon nitride with poly-L-lysine increased surface hydrophilicity and that exposing this coated surface to an extracellular aqueous environment gradually decreased its roughness. When PC12 cells were cultured on a coated silicon nitride surface, adhesion and spreading were facilitated, and the cells showed enhanced morphological differentiation compared to those cultured on a plastic culture dish. A bromodeoxyuridine assay demonstrated that, on the coated silicon nitride surface, higher proportions of cells left the cell cycle, remained in a quiescent state and had longer survival times. Therefore, our study of the interaction of the silicon nitride surface with PC12 cells provides important information for the production of devices that need to have optimal cell culture-supporting properties in order to be used in the study of neuronal functions.

  15. HCV Genotype 6a Escape from and Resistance to Velpatasvir, Pibrentasvir, and Sofosbuvir in Robust Infectious Cell Culture Models

    DEFF Research Database (Denmark)

    Pham, Long; Ramirez Almeida, Santseharay; Gottwein, Judith Margarete

    was able to propagate and escape in the presence of pibrentasvir with emergence of NS5A-L28S, conferring a high level of resistance to this inhibitor. CONCLUSIONS: Strains of HCV genotype 6a isolated from patients can be adapted to propagate in cultured cells, permitting studies of the complete life cycle...... infectious cell culture models of HCV genotype 6a infection to study the effects of these inhibitors and the development of resistance. METHODS: The consensus sequences of prototype strains HK2 (MG717925) and HK6a (MG717928), originating from serum of patients with chronic HCV infection, were determined...... by Sanger sequencing of genomes amplified by reverse transcription-PCR. In vitro non-infectious full-length clones of these 6a strains were subsequently adapted in Huh7.5 cells, primarily by using substitutions identified in JFH1-based core-NS5A and core-NS5B genotype 6a recombinants. We studied...

  16. Optimization of high grade glioma cell culture from surgical specimens for use in clinically relevant animal models and 3D immunochemistry.

    Science.gov (United States)

    Hasselbach, Laura A; Irtenkauf, Susan M; Lemke, Nancy W; Nelson, Kevin K; Berezovsky, Artem D; Carlton, Enoch T; Transou, Andrea D; Mikkelsen, Tom; deCarvalho, Ana C

    2014-01-07

    Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor. Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

  17. The anti-epileptic drug substance vigabatrin inhibits taurine transport in intestinal and renal cell culture models

    DEFF Research Database (Denmark)

    Plum, Jakob Munk; Nøhr, Martha Kampp; Hansen, Steen H

    2014-01-01

    , such evidence does not preclude the involvement of other transporters. The aim of the present study was, therefore, to investigate if vigabatrin interacts with taurine transport. The uptake of taurine was measured in intestinal human Caco-2 and canine MDCK cell monolayers in the absence or presence of amino...... acids such as GABA and vigabatrin. Vigabatrin inhibits the uptake of taurine in Caco-2 and MDCK cells to 34±3 and 53±2%, respectively, at a concentration of 30mM. In Caco-2 cells the uptake of vigabatrin under neutral pH conditions is concentration-dependent and saturable with a Km-value of 27mM (log......Km is 1.43±0.09). In conclusion, the present study shows that vigabatrin was able to inhibit the uptake of taurine in intestinal and renal cell culture models. Furthermore, uptake of vigabatrin in Caco-2 cells under neutral pH conditions was concentration-dependent and saturable and suggesting...

  18. Neuroprotective effect of the natural iron chelator, phytic acid in a cell culture model of Parkinson's disease

    International Nuclear Information System (INIS)

    Xu Qi; Kanthasamy, Anumantha G.; Reddy, Manju B.

    2008-01-01

    Disrupted iron metabolism and excess iron accumulation has been reported in the brains of Parkinson's disease (PD) patients. Because excessive iron can induce oxidative stress subsequently causing degradation of nigral dopaminergic neurons in PD, we determined the protective effect of a naturally occurring iron chelator, phytic acid (IP6), on 1-methyl-4-phenylpyridinium (MPP + )-induced cell death in immortalized rat mesencephalic/dopaminergic cells. Cell death was induced with MPP + in normal and iron-excess conditions and cytotoxicity was measured by thiazolyl blue tetrazolium bromide (MTT assay) and trypan blue staining. Apoptotic cell death was also measured with caspase-3 activity, DNA fragmentation, and Hoechst nuclear staining. Compared to MPP + treatment, IP6 (30 μmol/L) increased cell viability by 19% (P + treatment was decreased by 55% (P < 0.01) and 52% (P < 0.05), respectively with IP6. Cell survival was increased by 18% (P < 0.05) and 42% (P < 0.001) with 30 and 100 μmol/L of IP6, respectively in iron-excess conditions. A 40% and 52% (P < 0.001) protection was observed in caspase-3 activity with 30 and 100 μmol/L IP6, respectively in iron-excess condition. Similarly, a 45% reduction (P < 0.001) in DNA fragmentation was found with 100 μmol/L IP6. In addition, Hoechst nuclear staining results confirmed the protective effect of IP6 against apoptosis. Similar protection was also observed with the differentiated cells. Collectively, our results demonstrate a significant neuroprotective effect of phytate in a cell culture model of PD

  19. LDLR expression and localization are altered in mouse and human cell culture models of Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Jose F Abisambra

    Full Text Available BACKGROUND: Alzheimer's disease (AD is a chronic neurodegenerative disorder and the most common form of dementia. The major molecular risk factor for late-onset AD is expression of the epsilon-4 allele of apolipoprotein E (apoE, the major cholesterol transporter in the brain. The low-density lipoprotein receptor (LDLR has the highest affinity for apoE and plays an important role in brain cholesterol metabolism. METHODOLOGY/PRINCIPAL FINDINGS: Using RT-PCR and western blotting techniques we found that over-expression of APP caused increases in both LDLR mRNA and protein levels in APP transfected H4 neuroglioma cells compared to H4 controls. Furthermore, immunohistochemical experiments showed aberrant localization of LDLR in H4-APP neuroglioma cells, Abeta-treated primary neurons, and in the PSAPP transgenic mouse model of AD. Finally, immunofluorescent staining of LDLR and of gamma- and alpha-tubulin showed a change in LDLR localization preferentially away from the plasma membrane that was paralleled by and likely the result of a disruption of the microtubule-organizing center and associated microtubule network. CONCLUSIONS/SIGNIFICANCE: These data suggest that increased APP expression and Abeta exposure alters microtubule function, leading to reduced transport of LDLR to the plasma membrane. Consequent deleterious effects on apoE uptake and function will have implications for AD pathogenesis and/or progression.

  20. Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture

    Directory of Open Access Journals (Sweden)

    Olga Schmal

    2016-01-01

    Full Text Available Efficient ex vivo expansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34+ hematopoietic stem and progenitor cells (HSPCs with bone marrow-derived mesenchymal stromal cells (MSCs. When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard.

  1. Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture.

    Science.gov (United States)

    Schmal, Olga; Seifert, Jan; Schäffer, Tilman E; Walter, Christina B; Aicher, Wilhelm K; Klein, Gerd

    2016-01-01

    Efficient ex vivo expansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D) cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34(+) hematopoietic stem and progenitor cells (HSPCs) with bone marrow-derived mesenchymal stromal cells (MSCs). When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer) shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard.

  2. Embryonic Cell Grafts in a Culture Model of Spinal Cord Lesion: Neuronal Relay Formation is Essential for Functional Regeneration

    Directory of Open Access Journals (Sweden)

    Anne Tscherter

    2016-09-01

    Full Text Available Presently there exists no cure for spinal cord injury. However, transplantation of embryonic tissue into spinal cord lesions resulted in axon outgrowth across the lesion site and some functional recovery, fostering hope for future stem cell therapies. Although in vivo evidence for functional recovery is given, the exact cellular mechanism of the graft support remains elusive: either the grafted cells provide a permissive environment for the host tissue to regenerate itself or the grafts actually integrate functionally into the host neuronal network reconnecting the separated spinal cord circuits. We tested the two hypotheses in an in vitro spinal cord lesion model that is based on propagation of activity between two rat organotypic spinal cord slices in culture. Transplantation of dissociated cells from E14 rat spinal cord or forebrain re-established the relay of activity over the lesion site and, thus, provoked functional regeneration. Combining patch-clamp recordings from transplanted cells with network activity measurements from the host tissue on multi-electrode arrays we here show that neurons differentiate from the grafted cells and integrate into the host circuits. Optogenetic silencing of neurons developed from transplanted embryonic mouse forebrain cells provides clear evidence that they replace the lost neuronal connections to relay and synchronize activity between the separated spinal cord circuits. In contrast, transplantation of neurospheres induced neither the differentiation of mature neurons from the grafts nor an improvement of functional regeneration. Together these findings suggest, that the formation of neuronal relays from grafted embryonic cells is essential to re-connect segregated spinal cord circuits.

  3. TRPV4 Regulates Tight Junctions and Affects Differentiation in a Cell Culture Model of the Corneal Epithelium.

    Science.gov (United States)

    Martínez-Rendón, Jacqueline; Sánchez-Guzmán, Erika; Rueda, Angélica; González, James; Gulias-Cañizo, Rosario; Aquino-Jarquín, Guillermo; Castro-Muñozledo, Federico; García-Villegas, Refugio

    2017-07-01

    TRPV4 (transient receptor potential vanilloid 4) is a cation channel activated by hypotonicity, moderate heat, or shear stress. We describe the expression of TRPV4 during the differentiation of a corneal epithelial cell model, RCE1(5T5) cells. TRPV4 is a late differentiation feature that is concentrated in the apical membrane of the outmost cell layer of the stratified epithelia. Ca 2+ imaging experiments showed that TRPV4 activation with GSK1016790A produced an influx of calcium that was blunted by the specific TRPV4 blocker RN-1734. We analyzed the involvement of TRPV4 in RCE1(5T5) epithelial differentiation by measuring the development of transepithelial electrical resistance (TER) as an indicator of the tight junction (TJ) assembly. We showed that TRPV4 activity was necessary to establish the TJ. In differentiated epithelia, activation of TRPV4 increases the TER and the accumulation of claudin-4 in cell-cell contacts. Epidermal Growth Factor (EGF) up-regulates the TER of corneal epithelial cultures, and we show here that TRPV4 activation mimicked this EGF effect. Conversely, TRPV4 inhibition or knock down by specific shRNA prevented the increase in TER. Moreover, TRPP2, an EGF-activated channel that forms heteromeric complexes with TRPV4, is also concentrated in the outmost cell layer of differentiated RCE1(5T5) sheets. This suggests that the EGF regulation of the TJ may involve a heterotetrameric TRPV4-TRPP2 channel. These results demonstrated TRPV4 activity was necessary for the correct establishment of TJ in corneal epithelia and as well as the regulation of both the barrier function of TJ and its ability to respond to EGF. J. Cell. Physiol. 232: 1794-1807, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  4. In-depth evaluation of Gly-Sar transport parameters as a function of culture time in the Caco-2 cell model

    DEFF Research Database (Denmark)

    Bravo, Silvina A.; Nielsen, Carsten Uhd; Amstrup, Jan

    2004-01-01

    The aim of the present study was to investigate the influence of culture time on hPEPT1-mediated transport in Caco-2 cell monolayers. Peptide transport activity in Caco-2 cells grown in standard media and in a "rapid" 4-day model was first compared. The rapid 4-day Caco-2 cell model, cultured using...... a cocktail of growth factors and agonists, displayed lower peptide uptake capacity than Caco-2 cells grown for 4 days in conventional media, and was judged to be unsuitable for peptide transport studies. Peptide transport activity as well as monolayer integrity and tissue morphology were evaluated...... in the standard >21 days model as a function of the culture time. Peptide transport activity was studied using [14C]-glycylsarcosine ([ 14C]-Gly-Sar). Monolayer integrity was evaluated by transepithelial electrical resistance (TEER) measurements and [3H]-mannitol permeabilities. Tissue morphology and hPEPT1...

  5. Evaluation of metallothionein formation as a proxy for zinc absorption in an in vitro digestion/caco-2 cell culture model

    Science.gov (United States)

    Caco-2 cell metallothionein (MT) formation was studied to determine if MT could be used as a proxy for zinc (Zn) absorption in a cell culture model. MT intracellular concentration was determined by using a cadmium/hemoglobin affinity assay. Cellular Zn uptake was determined in acid digests (5% HNO3)...

  6. Geometry optimization of a fibrous scaffold based on mathematical modelling and CFD simulation of a dynamic cell culture

    DEFF Research Database (Denmark)

    Tajsoleiman, Tannaz; J. Abdekhodaie, Mohammad; Gernaey, Krist

    2016-01-01

    simulation of cartilage cell culture under a perfusion flow, which allows not only to characterize the supply of nutrients and metabolic products inside a fibrous scaffold, but also to assess the overall culture condition and predict the cell growth rate. Afterwards, the simulation results supported finding...... an optimized design of the scaffold within a new mathematical optimization algorithm that is proposed. The main concept of this optimization routine isto maintain a large effective surface while simultaneously keeping the shear stress levelin an operating range that is expected to be supporting growth....... Therewith, it should bepossible to gradually reach improved culture efficiency as defined in the objective function....

  7. An imbalance in progenitor cell populations reflects tumour progression in breast cancer primary culture models.

    LENUS (Irish Health Repository)

    Donatello, Simona

    2011-01-01

    Many factors influence breast cancer progression, including the ability of progenitor cells to sustain or increase net tumour cell numbers. Our aim was to define whether alterations in putative progenitor populations could predict clinicopathological factors of prognostic importance for cancer progression.

  8. Immortalization protocols used in cell culture models of human breast morphogenesis

    DEFF Research Database (Denmark)

    Gudjonsson, T; Villadsen, R; Rønnov-Jessen, L

    2004-01-01

    of the tissue of origin. In recent years, we have sought to establish immortalized primary breast cells, which retain crucial characteristics of their original in situ tissue pattern. This review discusses various approaches to immortalization of breast-derived epithelial and stromal cells and the application...

  9. Zika Virus Exhibits Lineage-Specific Phenotypes in Cell Culture, in Aedes aegypti Mosquitoes, and in an Embryo Model

    Directory of Open Access Journals (Sweden)

    Katherine A. Willard

    2017-12-01

    Full Text Available Zika virus (ZIKV has quietly circulated in Africa and Southeast Asia for the past 65 years. However, the recent ZIKV epidemic in the Americas propelled this mosquito-borne virus to the forefront of flavivirus research. Based on historical evidence, ZIKV infections in Africa were sporadic and caused mild symptoms such as fever, skin rash, and general malaise. In contrast, recent Asian-lineage ZIKV infections in the Pacific Islands and the Americas are linked to birth defects and neurological disorders. The aim of this study is to compare replication, pathogenicity, and transmission efficiency of two historic and two contemporary ZIKV isolates in cell culture, the mosquito host, and an embryo model to determine if genetic variation between the African and Asian lineages results in phenotypic differences. While all tested isolates replicated at similar rates in Vero cells, the African isolates displayed more rapid viral replication in the mosquito C6/36 cell line, yet they exhibited poor infection rates in Aedes aegypti mosquitoes compared to the contemporary Asian-lineage isolates. All isolates could infect chicken embryos; however, infection with African isolates resulted in higher embryo mortality than infection with Asian-lineage isolates. These results suggest that genetic variation between ZIKV isolates can significantly alter experimental outcomes.

  10. Small airway epithelial cells exposure to printer-emitted engineered nanoparticles induces cellular effects on human microvascular endothelial cells in an alveolar-capillary co-culture model.

    Science.gov (United States)

    Sisler, Jennifer D; Pirela, Sandra V; Friend, Sherri; Farcas, Mariana; Schwegler-Berry, Diane; Shvedova, Anna; Castranova, Vincent; Demokritou, Philip; Qian, Yong

    2015-01-01

    The printer is one of the most common office equipment. Recently, it was reported that toner formulations for printing equipment constitute nano-enabled products (NEPs) and contain engineered nanomaterials (ENMs) that become airborne during printing. To date, insufficient research has been performed to understand the potential toxicological properties of printer-emitted particles (PEPs) with several studies using bulk toner particles as test particles. These studies demonstrated the ability of toner particles to cause chronic inflammation and fibrosis in animal models. However, the toxicological implications of inhalation exposures to ENMs emitted from laser printing equipment remain largely unknown. The present study investigates the toxicological effects of PEPs using an in vitro alveolar-capillary co-culture model with Human Small Airway Epithelial Cells (SAEC) and Human Microvascular Endothelial Cells (HMVEC). Our data demonstrate that direct exposure of SAEC to low concentrations of PEPs (0.5 and 1.0 µg/mL) caused morphological changes of actin remodeling and gap formations within the endothelial monolayer. Furthermore, increased production of reactive oxygen species (ROS) and angiogenesis were observed in the HMVEC. Analysis of cytokine and chemokine levels demonstrates that interleukin (IL)-6 and MCP-1 may play a major role in the cellular communication observed between SAEC and HMVEC and the resultant responses in HMVEC. These data indicate that PEPs at low, non-cytotoxic exposure levels are bioactive and affect cellular responses in an alveolar-capillary co-culture model, which raises concerns for potential adverse health effects.

  11. Organotypic three-dimensional culture model of mesenchymal and epithelial cells to examine tissue fusion events.

    Science.gov (United States)

    Tissue fusion during early mammalian development requires coordination of multiple cell types, the extracellular matrix, and complex signaling pathways. Fusion events during processes including heart development, neural tube closure, and palatal fusion are dependent on signaling ...

  12. EGCG Protects against 6-OHDA-Induced Neurotoxicity in a Cell Culture Model

    OpenAIRE

    Chen, Dan; Kanthasamy, Anumantha G.; Reddy, Manju B.

    2015-01-01

    Background. Parkinson's disease (PD) is a progressive neurodegenerative disease that causes severe brain dopamine depletion. Disruption of iron metabolism may be involved in the PD progression. Objective. To test the protective effect of (?)-epigallocatechin-3-gallate (EGCG) against 6-hydroxydopamine- (6-OHDA-) induced neurotoxicity by regulating iron metabolism in N27 cells. Methods. Protection by EGCG in N27 cells was assessed by SYTOX green assay, MTT, and caspase-3 activity. Iron regulato...

  13. Cell culture compositions

    Science.gov (United States)

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

    2014-03-18

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

  14. Vitamin E-Mediated Modulation of Glutamate Receptor Expression in an Oxidative Stress Model of Neural Cells Derived from Embryonic Stem Cell Cultures

    Directory of Open Access Journals (Sweden)

    Afifah Abd Jalil

    2017-01-01

    Full Text Available Glutamate is the primary excitatory neurotransmitter in the central nervous system. Excessive concentrations of glutamate in the brain can be excitotoxic and cause oxidative stress, which is associated with Alzheimer’s disease. In the present study, the effects of vitamin E in the form of tocotrienol-rich fraction (TRF and alpha-tocopherol (α-TCP in modulating the glutamate receptor and neuron injury markers in an in vitro model of oxidative stress in neural-derived embryonic stem (ES cell cultures were elucidated. A transgenic mouse ES cell line (46C was differentiated into a neural lineage in vitro via induction with retinoic acid. These cells were then subjected to oxidative stress with a significantly high concentration of glutamate. Measurement of reactive oxygen species (ROS was performed after inducing glutamate excitotoxicity, and recovery from this toxicity in response to vitamin E was determined. The gene expression levels of glutamate receptors and neuron-specific enolase were elucidated using real-time PCR. The results reveal that neural cells derived from 46C cells and subjected to oxidative stress exhibit downregulation of NMDA, kainate receptor, and NSE after posttreatment with different concentrations of TRF and α-TCP, a sign of neurorecovery. Treatment of either TRF or α-TCP reduced the levels of ROS in neural cells subjected to glutamate-induced oxidative stress; these results indicated that vitamin E is a potent antioxidant.

  15. EGCG Protects against 6-OHDA-Induced Neurotoxicity in a Cell Culture Model

    Directory of Open Access Journals (Sweden)

    Dan Chen

    2015-01-01

    Full Text Available Background. Parkinson’s disease (PD is a progressive neurodegenerative disease that causes severe brain dopamine depletion. Disruption of iron metabolism may be involved in the PD progression. Objective. To test the protective effect of (−-epigallocatechin-3-gallate (EGCG against 6-hydroxydopamine- (6-OHDA- induced neurotoxicity by regulating iron metabolism in N27 cells. Methods. Protection by EGCG in N27 cells was assessed by SYTOX green assay, MTT, and caspase-3 activity. Iron regulatory gene and protein expression were measured by RT-PCR and Western blotting. Intracellular iron uptake was measured using 55Fe. The EGCG protection was further tested in primary mesencephalic dopaminergic neurons by immunocytochemistry. Results. EGCG protected against 6-OHDA-induced cell toxicity. 6-OHDA treatment significantly (p<0.05 increased divalent metal transporter-1 (DMT1 and hepcidin and decreased ferroportin 1 (Fpn1 level, whereas pretreatment with EGCG counteracted the effects. The increased 55Fe (by 96%, p<0.01 cell uptake confirmed the iron burden by 6-OHDA and was reduced by EGCG by 27% (p<0.05, supporting the DMT1 results. Pretreatment with EGCG and 6-OHDA significantly increased (p<0.0001 TH+ cell count (~3-fold and neurite length (~12-fold compared to 6-OHDA alone in primary mesencephalic neurons. Conclusions. Pretreatment with EGCG protected against 6-OHDA-induced neurotoxicity by regulating genes and proteins involved in brain iron homeostasis, especially modulating hepcidin levels.

  16. Predictivity of dog co-culture model, primary human hepatocytes and HepG2 cells for the detection of hepatotoxic drugs in humans

    International Nuclear Information System (INIS)

    Atienzar, Franck A.; Novik, Eric I.; Gerets, Helga H.; Parekh, Amit; Delatour, Claude; Cardenas, Alvaro; MacDonald, James; Yarmush, Martin L.; Dhalluin, Stéphane

    2014-01-01

    Drug Induced Liver Injury (DILI) is a major cause of attrition during early and late stage drug development. Consequently, there is a need to develop better in vitro primary hepatocyte models from different species for predicting hepatotoxicity in both animals and humans early in drug development. Dog is often chosen as the non-rodent species for toxicology studies. Unfortunately, dog in vitro models allowing long term cultures are not available. The objective of the present manuscript is to describe the development of a co-culture dog model for predicting hepatotoxic drugs in humans and to compare the predictivity of the canine model along with primary human hepatocytes and HepG2 cells. After rigorous optimization, the dog co-culture model displayed metabolic capacities that were maintained up to 2 weeks which indicates that such model could be also used for long term metabolism studies. Most of the human hepatotoxic drugs were detected with a sensitivity of approximately 80% (n = 40) for the three cellular models. Nevertheless, the specificity was low approximately 40% for the HepG2 cells and hepatocytes compared to 72.7% for the canine model (n = 11). Furthermore, the dog co-culture model showed a higher superiority for the classification of 5 pairs of close structural analogs with different DILI concerns in comparison to both human cellular models. Finally, the reproducibility of the canine system was also satisfactory with a coefficient of correlation of 75.2% (n = 14). Overall, the present manuscript indicates that the dog co-culture model may represent a relevant tool to perform chronic hepatotoxicity and metabolism studies. - Highlights: • Importance of species differences in drug development. • Relevance of dog co-culture model for metabolism and toxicology studies. • Hepatotoxicity: higher predictivity of dog co-culture vs HepG2 and human hepatocytes

  17. Predictivity of dog co-culture model, primary human hepatocytes and HepG2 cells for the detection of hepatotoxic drugs in humans

    Energy Technology Data Exchange (ETDEWEB)

    Atienzar, Franck A., E-mail: franck.atienzar@ucb.com [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium); Novik, Eric I. [H mu rel Corporation, 675 U.S. Highway 1, North Brunswick, NJ 08902 (United States); Gerets, Helga H. [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium); Parekh, Amit [H mu rel Corporation, 675 U.S. Highway 1, North Brunswick, NJ 08902 (United States); Delatour, Claude; Cardenas, Alvaro [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium); MacDonald, James [Chrysalis Pharma Consulting, LLC, 385 Route 24, Suite 1G, Chester, NJ 07930 (United States); Yarmush, Martin L. [Department of Biomedical Engineering, Rutgers University, Piscataway, NJ 08854 (United States); Dhalluin, Stéphane [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium)

    2014-02-15

    Drug Induced Liver Injury (DILI) is a major cause of attrition during early and late stage drug development. Consequently, there is a need to develop better in vitro primary hepatocyte models from different species for predicting hepatotoxicity in both animals and humans early in drug development. Dog is often chosen as the non-rodent species for toxicology studies. Unfortunately, dog in vitro models allowing long term cultures are not available. The objective of the present manuscript is to describe the development of a co-culture dog model for predicting hepatotoxic drugs in humans and to compare the predictivity of the canine model along with primary human hepatocytes and HepG2 cells. After rigorous optimization, the dog co-culture model displayed metabolic capacities that were maintained up to 2 weeks which indicates that such model could be also used for long term metabolism studies. Most of the human hepatotoxic drugs were detected with a sensitivity of approximately 80% (n = 40) for the three cellular models. Nevertheless, the specificity was low approximately 40% for the HepG2 cells and hepatocytes compared to 72.7% for the canine model (n = 11). Furthermore, the dog co-culture model showed a higher superiority for the classification of 5 pairs of close structural analogs with different DILI concerns in comparison to both human cellular models. Finally, the reproducibility of the canine system was also satisfactory with a coefficient of correlation of 75.2% (n = 14). Overall, the present manuscript indicates that the dog co-culture model may represent a relevant tool to perform chronic hepatotoxicity and metabolism studies. - Highlights: • Importance of species differences in drug development. • Relevance of dog co-culture model for metabolism and toxicology studies. • Hepatotoxicity: higher predictivity of dog co-culture vs HepG2 and human hepatocytes.

  18. New Strategy in Spinal Cord Repair with Cryofrozen Primary Cell Cultures in Model In Vivo

    Directory of Open Access Journals (Sweden)

    Rosa Margarita Gomez Bello

    2015-02-01

    The results as show evidence that when evaluating the locomotor recuperation, after a medullar section in rats through the implantation of OECs of cryopreserved cells combined with aFGF + FG. In addition, the conclusion it is potencial use in reparative phenomena in the injured medullar tissue but making further studies necessary before considering their use.

  19. Track structure model for damage to mammalian cell cultures during solar proton events

    Science.gov (United States)

    Cucinotta, F. A.; Wilson, J. W.; Townsend, L. W.; Shinn, J. L.; Katz, R.

    1992-01-01

    Solar proton events (SPEs) occur infrequently and unpredictably, thus representing a potential hazard to interplanetary space missions. Biological damage from SPEs will be produced principally through secondary electron production in tissue, including important contributions due to delta rays from nuclear reaction products. We review methods for estimating the biological effectiveness of SPEs using a high energy proton model and the parametric cellular track model. Results of the model are presented for several of the historically largest flares using typical levels and body shielding.

  20. In vitro culture and characterization of human lung cancer circulating tumor cells isolated by size exclusion from an orthotopic nude-mouse model expressing fluorescent protein.

    Science.gov (United States)

    Kolostova, Katarina; Zhang, Yong; Hoffman, Robert M; Bobek, Vladimir

    2014-09-01

    In the present study, we demonstrate an animal model and recently introduced size-based exclusion method for circulating tumor cells (CTCs) isolation. The methodology enables subsequent in vitro CTC-culture and characterization. Human lung cancer cell line H460, expressing red fluorescent protein (H460-RFP), was orthotopically implanted in nude mice. CTCs were isolated by a size-based filtration method and successfully cultured in vitro on the separating membrane (MetaCell®), analyzed by means of time-lapse imaging. The cultured CTCs were heterogeneous in size and morphology even though they originated from a single tumor. The outer CTC-membranes were blebbing in general. Abnormal mitosis resulting in three daughter cells was frequently observed. The expression of RFP ensured that the CTCs originated from lung tumor. These readily isolatable, identifiable and cultivable CTCs can be used to characterize individual patient cancers and for screening of more effective treatment.

  1. Hepcidin Plays a Key Role in 6-OHDA Induced Iron Overload and Apoptotic Cell Death in a Cell Culture Model of Parkinson’s Disease

    Directory of Open Access Journals (Sweden)

    Qi Xu

    2016-01-01

    Full Text Available Background. Elevated brain iron levels have been implicated in the pathogenesis of Parkinson’s disease (PD. However, the precise mechanism underlying abnormal iron accumulation in PD is not clear. Hepcidin, a hormone primarily produced by hepatocytes, acts as a key regulator in both systemic and cellular iron homeostasis. Objective. We investigated the role of hepcidin in 6-hydroxydopamine (6-OHDA induced apoptosis in a cell culture model of PD. Methods. We downregulated hepcidin using siRNA interference in N27 dopaminergic neuronal cells and made a comparison with control siRNA transfected cells to investigate the role of hepcidin in 6-OHDA induced neurodegeneration. Results. Hepcidin knockdown (32.3%, P<0.0001 upregulated ferroportin 1 expression and significantly (P<0.05 decreased intracellular iron by 25%. Hepcidin knockdown also reduced 6-OHDA induced caspase-3 activity by 42% (P<0.05 and DNA fragmentation by 29% (P=0.086 and increased cell viability by 22% (P<0.05. In addition, hepcidin knockdown significantly attenuated 6-OHDA induced protein carbonyls by 52% (P<0.05 and intracellular iron by 28% (P<0.01, indicating the role of hepcidin in oxidative stress. Conclusions. Our results demonstrate that hepcidin knockdown protected N27 cells from 6-OHDA induced apoptosis and that hepcidin plays a major role in reducing cellular iron burden and oxidative damage by possibly regulating cellular iron export mediated by ferroportin 1.

  2. A novel co-culture model of murine K12 osteosarcoma cells and S. aureus on common orthopedic implant materials: 'the race to the surface' studied in vitro.

    Science.gov (United States)

    McConda, David B; Karnes, Jonathan M; Hamza, Therwa; Lindsey, Brock A

    2016-07-01

    Infection is a major cause of orthopedic implant failure. There are few studies assessing both tissue cell and bacterial adherence on common orthopedic implant materials in a co-culture environment. An in vitro co-culture model was created using K12 osteosarcoma cells and Staphylococcus aureus in a medium incubated over metal disks for 48 h. The results showed that, in the presence of S. aureus, there were fewer osteosarcoma cells attached to the disks for all substrata tested. There were significantly more osteosarcoma cells adhering to the cobalt chrome than the stainless steel and titanium disks. Overall, in the presence of osteosarcoma cells, there were more bacteria adhering to the disks for all the substrata tested, with significantly more bacteria adhering to the stainless steel disks compared to cobalt chrome and titanium disks. Scanning electron microscopy verified that osteosarcoma cells and bacteria were adherent to the metal disks after incubation for 48 h. Furthermore, the observation that more bacteria were in the co-culture than in the control sample suggests that the osteosarcoma cells serve as a nutrient source for the bacteria. Future models assessing the interaction of osteogenic cells with bacteria on a substratum would be improved if the model accounted for the role of the immune system in secondary bone healing.

  3. Low-temperature bonded glass-membrane microfluidic device for in vitro organ-on-a-chip cell culture models

    Science.gov (United States)

    Pocock, Kyall J.; Gao, Xiaofang; Wang, Chenxi; Priest, Craig; Prestidge, Clive A.; Mawatari, Kazuma; Kitamori, Takehiko; Thierry, Benjamin

    2015-12-01

    The integration of microfluidics with living biological systems has paved the way to the exciting concept of "organson- a-chip", which aims at the development of advanced in vitro models that replicate the key features of human organs. Glass based devices have long been utilised in the field of microfluidics but the integration of alternative functional elements within multi-layered glass microdevices, such as polymeric membranes, remains a challenge. To this end, we have extended a previously reported approach for the low-temperature bonding of glass devices that enables the integration of a functional polycarbonate porous membrane. The process was initially developed and optimised on specialty low-temperature bonding equipment (μTAS2001, Bondtech, Japan) and subsequently adapted to more widely accessible hot embosser units (EVG520HE Hot Embosser, EVG, Austria). The key aspect of this method is the use of low temperatures compatible with polymeric membranes. Compared to borosilicate glass bonding (650 °C) and quartz/fused silica bonding (1050 °C) processes, this method maintains the integrity and functionality of the membrane (Tg 150 °C for polycarbonate). Leak tests performed showed no damage or loss of integrity of the membrane for up to 150 hours, indicating sufficient bond strength for long term cell culture. A feasibility study confirmed the growth of dense and functional monolayers of Caco-2 cells within 5 days.

  4. Progress toward forecasting product quality and quantity of mammalian cell culture processes by performance-based modeling.

    Science.gov (United States)

    Schmidberger, Timo; Posch, Christoph; Sasse, Alexandra; Gülch, Carina; Huber, Robert

    2015-01-01

    The production of biopharmaceuticals requires highly sophisticated, complex cell based processes. Once a process has been developed, acceptable ranges for various control parameters are typically defined based on process characterization studies often comprising several dozens of small scale bioreactor cultivations. A lot of data is generated during these studies and usually only the information needed to define acceptable ranges is processed in more detail. Making use of the wealth of information contained in such data sets, we present here a methodology that uses performance data (such as metabolite profiles) to forecast the product quality and quantity of mammalian cell culture processes based on a toolbox of advanced statistical methods. With this performance based modeling (PBM) the final product concentration and 12 quality attributes (QAs) for two different biopharmaceutical products were predicted in daily intervals throughout the main stage process. The best forecast was achieved for product concentration in a very early phase of the process. Furthermore, some glycan isoforms were predicted with good accuracy several days before the bioreactor was harvested. Overall, PBM clearly demonstrated its capability of early process endpoint prediction by only using commonly available data, even though it was not possible to predict all QAs with the desired accuracy. Knowing the product quality prior to the harvest allows the manufacturer to take counter measures in case the forecasted quality or quantity deviates from what is expected. This would be a big step towards real-time release, an important element of the FDA's PAT initiative. © 2015 American Institute of Chemical Engineers.

  5. Metabolism of ibuprofen in higher plants: A model Arabidopsis thaliana cell suspension culture system

    Czech Academy of Sciences Publication Activity Database

    Maršík, Petr; Šíša, Miroslav; Lacina, O.; Moťková, Kateřina; Langhansová, Lenka; Rezek, Jan; Vaněk, Tomáš

    2017-01-01

    Roč. 220, JAN (2017), s. 383-392 ISSN 0269-7491 R&D Projects: GA ČR(CZ) GA14-22593S Grant - others:European Regional Development Fund(XE) CZ.2.16/3.1.00/24014 Institutional support: RVO:61389030 Keywords : Arabidopsis thaliana * Ibuprofen * Metabolism * Plant cells * Sequestration Subject RIV: CE - Biochemistry OBOR OECD: Plant sciences, botany Impact factor: 5.099, year: 2016

  6. Electric cell-substrate impedance sensing (ECIS) based real-time measurement of titer dependent cytotoxicity induced by adenoviral vectors in an IPI-2I cell culture model.

    Science.gov (United States)

    Müller, Jakob; Thirion, Christian; Pfaffl, Michael W

    2011-01-15

    Recombinant viral vectors are widespread tools for transfer of genetic material in various modern biotechnological applications like for example RNA interference (RNAi). However, an accurate and reproducible titer assignment represents the basic step for most downstream applications regarding a precise multiplicity of infection (MOI) adjustment. As necessary scaffold for the studies described in this work we introduce a quantitative real-time PCR (qPCR) based approach for viral particle measurement. Still an implicated problem concerning physiological effects is that the appliance of viral vectors is often attended by toxic effects on the individual target. To determine the critical viral dose leading to cell death we developed an electric cell-substrate impedance sensing (ECIS) based assay. With ECIS technology the impedance change of a current flow through the cell culture medium in an array plate is measured in a non-invasive manner, visualizing effects like cell attachment, cell-cell contacts or proliferation. Here we describe the potential of this online measurement technique in an in vitro model using the porcine ileal epithelial cell line IPI-2I in combination with an adenoviral transfection vector (Ad5-derivate). This approach shows a clear dose-depending toxic effect, as the amount of applied virus highly correlates (p<0.001) with the level of cell death. Thus this assay offers the possibility to discriminate the minimal non-toxic dose of the individual transfection method. In addition this work suggests that the ECIS-device bears the feasibility to transfer this assay to multiple other cytotoxicological questions. Copyright © 2010 Elsevier B.V. All rights reserved.

  7. 9 CFR 101.6 - Cell cultures.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Cell cultures. 101.6 Section 101.6..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to as tissue cultures...

  8. Effects of anti-inflammatory compounds on sulfur mustard injured cells: Recommendations and caveats suggested by in vitro cell culture models.

    Science.gov (United States)

    Menacher, Georg; Steinritz, Dirk; Schmidt, Annette; Popp, Tanja; Worek, Franz; Gudermann, Thomas; Thiermann, Horst; Balszuweit, Frank

    2018-09-01

    Sulfur mustard (SM) is a vesicant agent who had its first military use 100 years ago, in Ypres. Since then it has been used in several conflicts like the Iran-Iraq war in the 1980s. The use of SM in Syria 2015 indicated the still existing threat. Despite decades of research no causal antidote against SM intoxication is available, so far. A SM intoxication is accompanied by necrosis, apoptosis and inflammation. To counteract the SM-induced inflammation, glucocorticoids and non-steroidal anti-inflammatory compounds (NSAIDs) are recommended. Aim of this study was to evaluate the efficacy of the anti-inflammatory compounds dexamethasone, ibuprofen and diclofenac in vitro. For that purpose, two different cell culture models were used. Firstly, a monoculture of keratinocytes (HaCaT) and secondly, an established co-culture of keratinocytes (HaCaT) and immunocompetent cells (THP-1) to identify the role of immune cells in the process and to mimic the dermal physiology more closely. Both models were challenged with different SM concentrations (100, 200 and 300μM) and treated with different anti-inflammatory compounds one hour after the SM exposure. Analytical analysis of necrosis (ToxiLight), apoptosis (CDDE) and inflammation (IL-6 and -8 ELISAs) followed 24h thereafter. Dexamethasone provided small but consistent protective effects in the monoculture. For the reduction of apoptosis, 3μM dexamethasone was sufficient. The most effective reduction regarding interleukin (IL) production was found with 6μM dexamethasone. Protective effects were less pronounced in co-culture, which implies, that the protective effects of dexamethasone are rather generic and not due to a modulation of the immune cells. Against our expectations, ibuprofen strongly amplified apoptosis and necrosis in SM exposed cells in the monoculture as well as the co-culture. Therefore, use of ibuprofen for treatment of SM intoxication should at least be considered most critically, if not even regarded as

  9. A spore counting method and cell culture model for chlorine disinfection studies of Encephalitozoon syn. Septata intestinalis.

    Science.gov (United States)

    Wolk, D M; Johnson, C H; Rice, E W; Marshall, M M; Grahn, K F; Plummer, C B; Sterling, C R

    2000-04-01

    The microsporidia have recently been recognized as a group of pathogens that have potential for waterborne transmission; however, little is known about the effects of routine disinfection on microsporidian spore viability. In this study, in vitro growth of Encephalitozoon syn. Septata intestinalis, a microsporidium found in the human gut, was used as a model to assess the effect of chlorine on the infectivity and viability of microsporidian spores. Spore inoculum concentrations were determined by using spectrophotometric measurements (percent transmittance at 625 nm) and by traditional hemacytometer counting. To determine quantitative dose-response data for spore infectivity, we optimized a rabbit kidney cell culture system in 24-well plates, which facilitated calculation of a 50% tissue culture infective dose (TCID(50)) and a minimal infective dose (MID) for E. intestinalis. The TCID(50) is a quantitative measure of infectivity and growth and is the number of organisms that must be present to infect 50% of the cell culture wells tested. The MID is as a measure of a system's permissiveness to infection and a measure of spore infectivity. A standardized MID and a standardized TCID(50) have not been reported previously for any microsporidian species. Both types of doses are reported in this paper, and the values were used to evaluate the effects of chlorine disinfection on the in vitro growth of microsporidia. Spores were treated with chlorine at concentrations of 0, 1, 2, 5, and 10 mg/liter. The exposure times ranged from 0 to 80 min at 25 degrees C and pH 7. MID data for E. intestinalis were compared before and after chlorine disinfection. A 3-log reduction (99.9% inhibition) in the E. intestinalis MID was observed at a chlorine concentration of 2 mg/liter after a minimum exposure time of 16 min. The log(10) reduction results based on percent transmittance-derived spore counts were equivalent to the results based on hemacytometer-derived spore counts. Our data

  10. Bio-Physicochemical Interactions of Engineered Nanomaterials in in Vitro Cell Culture Model

    Science.gov (United States)

    2014-10-11

    properties of these manufactured materials (Edwards-Jones, 2009). The principal reason for producing and exploiting NMs is that their behaviour is...butanol layer was collected and read at 532 nm. The MDA level of the sample was calculated using an extinction coefficient of 1.56x105 M-1cm-1. The...glutathione content in the cell samples was expressed as µmoles GSH/mg protein and calculated using extinction coefficient of 13600 M-1cm-1. 2.12

  11. Autophagy and Neurodegeneration: Insights from a Cultured Cell Model of ALS

    Directory of Open Access Journals (Sweden)

    Francesca Navone

    2015-08-01

    Full Text Available Autophagy plays a major role in the elimination of cellular waste components, the renewal of intracellular proteins and the prevention of the build-up of redundant or defective material. It is fundamental for the maintenance of homeostasis and especially important in post-mitotic neuronal cells, which, without competent autophagy, accumulate protein aggregates and degenerate. Many neurodegenerative diseases are associated with defective autophagy; however, whether altered protein turnover or accumulation of misfolded, aggregate-prone proteins is the primary insult in neurodegeneration has long been a matter of debate. Amyotrophic lateral sclerosis (ALS is a fatal disease characterized by selective degeneration of motor neurons. Most of the ALS cases occur in sporadic forms (SALS, while 10%–15% of the cases have a positive familial history (FALS. The accumulation in the cell of misfolded/abnormal proteins is a hallmark of both SALS and FALS, and altered protein degradation due to autophagy dysregulation has been proposed to contribute to ALS pathogenesis. In this review, we focus on the main molecular features of autophagy to provide a framework for discussion of our recent findings about the role in disease pathogenesis of the ALS-linked form of the VAPB gene product, a mutant protein that drives the generation of unusual cytoplasmic inclusions.

  12. Youth Culture and Cell Phone

    Directory of Open Access Journals (Sweden)

    mohammad saeed zokaei

    2009-11-01

    Full Text Available Iranian youth’s leisure culture has been immediately affected by the digital media culture. As a communicative media, cell phone has crossed borders of youth norms and identity; and in addition to facilitating their communication, has changed its patterns. Applying Bourdieu’s concepts of habitus and field, and relied on the qualitative and quantitative data gathered from the mobile youth users, the present study argues that mobile has produced a new field in which youth’s opportunities for leisure, entertainment, communication, and independence have extended. In addition, cell phone has facilitated and compensated for some defects in public sphere, and therefore empowered youth agency, individuality, and power. Despite this strengthening, cell phone does not cross borders of gender and class differences, or the levels of social capital.

  13. Saposin B-dependent Reconstitution of Arylsulfatase A Activity in Vitro and in Cell Culture Models of Metachromatic Leukodystrophy*

    Science.gov (United States)

    Matzner, Ulrich; Breiden, Bernadette; Schwarzmann, Günter; Yaghootfam, Afshin; Fluharty, Arvan L.; Hasilik, Andrej; Sandhoff, Konrad; Gieselmann, Volkmar

    2009-01-01

    Arylsulfatase A (ASA) catalyzes the intralysosomal desulfation of 3-O-sulfogalactosylceramide (sulfatide) to galactosylceramide. The reaction requires saposin B (Sap B), a non-enzymatic proteinaceous cofactor which presents sulfatide to the catalytic site of ASA. The lack of either ASA or Sap B results in a block of sulfatide degradation, progressive intralysosomal accumulation of sulfatide, and the fatal lysosomal storage disease metachromatic leukodystrophy. We studied the coupled Sap B-ASA reaction in vitro using detergent-free micellar and liposomal assay systems and in vivo using cell culture models of metachromatic leukodystrophy. Under in vitro conditions, the reaction had a narrow pH optimum around pH 4.3 and was inhibited by mono- and divalent cations, phosphate and sulfite. Bis(monoacylglycero) phosphate and phosphatidic acid were activators of the reaction, underscoring a significant role of acidic phosphoglycerolipids in sphingolipid degradation. Desulfation was negligible when Sap B was substituted by Sap A, C, or D. Up to a molar ratio between Sap B and sulfatide of 1:5, an elevation of Sap B concentrations caused a sharp increase of sulfatide hydrolysis, indicating the requirement of unexpected high Sap B levels for maximum turnover. Feeding of ASA-deficient, sulfatide-storing primary mouse kidney cells with ASA caused partial clearance of sulfatide. Co-feeding of Sap B or its precursor prosaposin resulted in the lysosomal uptake of the cofactor but did not promote ASA-catalyzed sulfatide hydrolysis. This suggests that Sap B is not a limiting factor of the coupled Sap B-ASA reaction in mouse kidney cells even if sulfatide has accumulated to unphysiologically high levels. PMID:19224915

  14. A cell culture model for Nosema ceranae and Nosema apis allows new insights into the life cycle of these important honey bee-pathogenic microsporidia.

    Science.gov (United States)

    Gisder, Sebastian; Möckel, Nadine; Linde, Andreas; Genersch, Elke

    2011-02-01

    The population of managed honey bees has been dramatically declining in the recent past in many regions of the world. Consensus now seems to be that pathogens and parasites (e.g. the ectoparasitic mite Varroa destructor, the microsporidium Nosema ceranae and viruses) play a major role in this demise. However, little is known about host-pathogen interactions for bee pathogens and attempts to develop novel strategies to combat bee diseases have been hampered by this gap in our knowledge. One reason for this dire situation is the complete lack of cell cultures for the propagation and study of bee pathogens. Here we present a cell culture model for two honey bee-pathogenic microsporidian species, Nosema apis and N. ceranae. Our cell culture system is based on a lepidopteran cell line, which proved to be susceptible to infection by both N. ceranae and N. apis and enabled us to illustrate the entire life cycle of these microsporidia. We observed hitherto undescribed spindle-shaped meronts and confirmed our findings in infected bees. Our cell culture model provides a previously unavailable means to explore the nature of interactions between the honey bee and its pathogen complex at a mechanistic level and will allow the development of novel treatment strategies.

  15. Utility and translatability of mathematical modeling, cell culture and small and large animal models in magnetic nanoparticle hyperthermia cancer treatment research

    Science.gov (United States)

    Hoopes, P. J.; Petryk, Alicia A.; Misra, Adwiteeya; Kastner, Elliot J.; Pearce, John A.; Ryan, Thomas P.

    2015-03-01

    For more than 50 years, hyperthermia-based cancer researchers have utilized mathematical models, cell culture studies and animal models to better understand, develop and validate potential new treatments. It has been, and remains, unclear how and to what degree these research techniques depend on, complement and, ultimately, translate accurately to a successful clinical treatment. In the past, when mathematical models have not proven accurate in a clinical treatment situation, the initiating quantitative scientists (engineers, mathematicians and physicists) have tended to believe the biomedical parameters provided to them were inaccurately determined or reported. In a similar manner, experienced biomedical scientists often tend to question the value of mathematical models and cell culture results since those data typically lack the level of biologic and medical variability and complexity that are essential to accurately study and predict complex diseases and subsequent treatments. Such quantitative and biomedical interdependence, variability, diversity and promise have never been greater than they are within magnetic nanoparticle hyperthermia cancer treatment. The use of hyperthermia to treat cancer is well studied and has utilized numerous delivery techniques, including microwaves, radio frequency, focused ultrasound, induction heating, infrared radiation, warmed perfusion liquids (combined with chemotherapy), and, recently, metallic nanoparticles (NP) activated by near infrared radiation (NIR) and alternating magnetic field (AMF) based platforms. The goal of this paper is to use proven concepts and current research to address the potential pathobiology, modeling and quantification of the effects of treatment as pertaining to the similarities and differences in energy delivered by known external delivery techniques and iron oxide nanoparticles.

  16. Drug-releasing nano-engineered titanium implants: therapeutic efficacy in 3D cell culture model, controlled release and stability

    Energy Technology Data Exchange (ETDEWEB)

    Gulati, Karan [School of Chemical Engineering, The University of Adelaide, SA 5005 (Australia); Kogawa, Masakazu; Prideaux, Matthew; Findlay, David M. [Discipline of Orthopaedics and Trauma, The University of Adelaide, SA 5005 (Australia); Atkins, Gerald J., E-mail: gerald.atkins@adelaide.edu.au [Discipline of Orthopaedics and Trauma, The University of Adelaide, SA 5005 (Australia); Losic, Dusan, E-mail: dusan.losic@adelaide.edu.au [School of Chemical Engineering, The University of Adelaide, SA 5005 (Australia)

    2016-12-01

    There is an ongoing demand for new approaches for treating localized bone pathologies. Here we propose a new strategy for treatment of such conditions, via local delivery of hormones/drugs to the trauma site using drug releasing nano-engineered implants. The proposed implants were prepared in the form of small Ti wires/needles with a nano-engineered oxide layer composed of array of titania nanotubes (TNTs). TNTs implants were inserted into a 3D collagen gel matrix containing human osteoblast-like, and the results confirmed cell migration onto the implants and their attachment and spread. To investigate therapeutic efficacy, TNTs/Ti wires loaded with parathyroid hormone (PTH), an approved anabolic therapeutic for the treatment of severe bone fractures, were inserted into 3D gels containing osteoblast-like cells. Gene expression studies revealed a suppression of SOST (sclerostin) and an increase in RANKL (receptor activator of nuclear factor kappa-B ligand) mRNA expression, confirming the release of PTH from TNTs at concentrations sufficient to alter cell function. The performance of the TNTs wire implants using an example of a drug needed at relatively higher concentrations, the anti-inflammatory drug indomethacin, is also demonstrated. Finally, the mechanical stability of the prepared implants was tested by their insertion into bovine trabecular bone cores ex vivo followed by retrieval, which confirmed the robustness of the TNT structures. This study provides proof of principle for the suitability of the TNT/Ti wire implants for localized bone therapy, which can be customized to cater for specific therapeutic requirements. - Highlights: • Ti wire with titania nanotubes (TNTs) are proposed as ‘in-bone’ therapeutic implants. • 3D cell culture model is used to confirm therapeutic efficacy of drug releasing implants. Osteoblasts migrated and firmly attached to the TNTs and the micro-scale cracks. • Tailorable drug loading from few nanograms to several hundred

  17. Multistage carcinogenesis in cell culture.

    Science.gov (United States)

    Rubin, H

    2001-01-01

    Rodent fibroblasts explanted from embryos to culture undergo a period of declining growth rate in serial passages leading to crisis, followed by the appearance of variants which can multiply indefinitely. If the "immortal" cell line was established by low density passage, i.e., 3T3 cells, it has a low saturation density and is non-tumorigenic. If it was established by high density passage, it has a high saturation density and is tumorigenic. The establishment of cells goes through successive stages, including increased capacity to multiply in low serum concentration, growth to high saturation density, growth in suspension, assisted tumour formation in susceptible hosts and unassisted tumour formation. Chromosome aberrations and aneuploidy occur long before the capacity to produce tumours appears. Contrary to conventional belief, human fibroblast populations also undergo a continuous loss of capacity to multiply from the time of explantation, with only the longest surviving clone reaching the Hayflick limit. Neoplastic transformation of rodent cells is strongly favoured by maintaining them in a quiescent state at confluence for prolonged periods, which results in genetic damage to the cells. It also produces a large variety of chromosomal aberrations in human cells and extends their replicative lifespan. Individual clones are more susceptible to spontaneous transformation than their heterogeneous parental cultures. The implications of these results for tumour development in vivo are that oncogenic genetic changes may be common under stressful conditions which restrict replication, and that such changes are maximized when a rogue clone reaches a critical size that reduces stabilizing interactions with neighbouring clones. An alternative explanation, described in the Addendum, which we retrospectively favor is that the easily transformed clones are a minority in the uncloned parental population. The reason they transform before the parental population is that when

  18. Neuroglial cells in long-term primary cultures from the gilthead sea bream (Sparus aurata L.: new functional in vitro model from bony fish brain

    Directory of Open Access Journals (Sweden)

    Gerardo Centoducati

    2013-01-01

    Full Text Available Neuroglia has been historically considered the “glue” of the nervous system, as the ancient Greek name suggests, being simply referred as non-neuronal cells, with supporting functions for neurons in the CNS of mammalian and lower vertebrates. All around the world, approximately 283 cell lines were obtained from fish, yet none of these was from the brain of Sparus aurata, neither in cell lines nor as primary culture. Here we describe a novel in vitro reproducible neuroglial marine model for establishing primary neuroglial cell cultures, by dissociating the whole brain of seabream juveniles. We showed that proliferating neural stem cells produced alongside three generating lineages, such as neuronal precursor cells, astroglial precursor cells and oligodendroglia precursor cells, which developed respectively neurons, astrocytes and oligodendrocytes. The radial glia, finely described by morphological studies and immunochemical antigen expression, showed a peculiar spatial distribution, giving rise simultaneously both to astrocytes and neuronal precursors within a highly proliferative assemblate. Radial glia cells were assessed by glial fibrillary acidic protein (GFAP and vimentin reactivity, astrocytes by GFAP, neurons by the neuron-specific markers for ubiquitin carboxy-terminal hydrolase 1 (UCHL1 and intermediate filament associated protein (NF, whereas myelinating oligodendrocytes were immunostained with anti-myelin basic protein (MBP and anti-O4. Our findings suggest that seabream neuroglial cells gain in 3-4 weeks of culturing proliferation, neuroglial differentiation, and oligodendrocyte maturation with myelination, thus disclosing on the possibility that mixed neuroglial cultures can accelerate the maturation of oligodendrocytes and the regeneration of CNS injury in fish.

  19. Quantifying the correlation between spatially defined oxygen gradients and cell fate in an engineered three-dimensional culture model

    OpenAIRE

    Ardakani, Amir G.; Cheema, Umber; Brown, Robert A.; Shipley, Rebecca J.

    2014-01-01

    A challenge in three-dimensional tissue culture remains the lack of quantitative information linking nutrient delivery and cellular distribution. Both in vivo and in vitro, oxygen is delivered by diffusion from its source (blood vessel or the construct margins). The oxygen level at a defined distance from its source depends critically on the balance of diffusion and cellular metabolism. Cells may respond to this oxygen environment through proliferation, death and chemotaxis, resulting in spat...

  20. A three-dimensional cell culture model to study the mechano-biological behavior in periodontal ligament regeneration

    NARCIS (Netherlands)

    Oortgiesen, D.A.W.; Yu, N.; Bronckers, A.L.J.J.; Yang, F.; Walboomers, X.F.; Jansen, J.A.

    2012-01-01

    Periodontitis is a disease affecting the supporting structures of the teeth, which can eventually result in tooth loss. A three-dimensional (3D) tissue culture model was developed that may serve to grow a 3D construct that not only transplants into defective periodontal sites, but also allows to

  1. Polylysine-modified polyethylenimine (PEI-PLL) mediated VEGF gene delivery protects dopaminergic neurons in cell culture and in rat models of Parkinson's Disease (PD).

    Science.gov (United States)

    Sheikh, Muhammad Abid; Malik, Yousra Saeed; Xing, Zhenkai; Guo, Zhaopei; Tian, Huayu; Zhu, Xiaojuan; Chen, Xuesi

    2017-05-01

    Parkinson's Disease (PD) is a chronic neurodegenerative disorder characterized by motor deficits which result from the progressive loss of dopaminergic neurons. Gene therapy using growth factors such as VEGF seems to be a viable approach for potential therapeutic treatment of PD. In this study, we utilized a novel non-viral gene carrier designated as PEI-PLL synthesized by our laboratory to deliver VEGF gene to study its effect by using both cell culture as well as animal models of PD. For cell culture experiments, we utilized 6-hydroxydopamine (6-OHDA) mediated cell death model of MN9D cells following transfection with either a control plasmid or VEGF expressing plasmid. As compared to control transfected cells, PEI-PLL mediated VEGF gene delivery to MN9D cells resulted in increased cell viability, increase in the number of Tyrosine hydroxylase (TH) positive cells and decreased apoptosis following 6-OHDA insult. Next, we studied the therapeutic potential of PEI-PLL mediated VEGF gene delivery in SNPc by using unilateral 6-OHDA Medial forebrain bundle (MFB) lesion model of PD in rats. VEGF administration prevented the loss of motor functions induced by 6-OHDA as determined by behavior analysis. Similarly, VEGF inhibited the 6-OHDA mediated loss of DA neurons in Substantia Nigra Pars Compacta (SNPc) as well as DA nerve fibers in striatum as determined by TH immunostaining. In addition, PEI-PLL mediated VEGF gene delivery also prevented apoptosis and microglial activation in PD rat models. Together, these results clearly demonstrated the beneficial effects of PEI-PLL mediated VEGF gene delivery on dopaminergic system in both cell culture and animal models of PD. In this report, we exploited the potential of PEI-PLL to deliver VEGF gene for the potential therapeutic treatment of PD by using both cell culture and animal models of PD. To the best of our knowledge, this is the first report describing the use of novel polymeric gene carriers for the delivery of VEGF gene

  2. Characterization of a cultured human T-cell line with genetically altered ribonucleotide reductase activity. Model for immunodeficiency.

    Science.gov (United States)

    Waddell, D; Ullman, B

    1983-04-10

    From human CCRF-CEM T-cells growing in continuous culture, we have selected, isolated, and characterized a clonal cell line, APHID-D2, with altered ribonucleotide reductase activity. In comparative growth rate experiments, the APHID-D2 cell line is less sensitive than the parental cell line to growth inhibition by deoxyadenosine in the presence of 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase. The APHID-D2 cell line has elevated levels of all four dNTPs. The resistance of the APHID-D2 cell line to growth inhibition by deoxyadenosine and the abnormal dNTP levels can be explained by the fact that the APHID-D2 ribonucleotide reductase, unlike the parental ribonucleotide reductase, is not normally sensitive to inhibition by dATP. These results suggest that the allosteric site of ribonucleotide reductase which binds both dATP and ATP is altered in the APHID-D2 line. The isolation of a mutant clone of human T-cells which contains a ribonucleotide reductase that has lost its normal sensitivity to dATP and which is resistant to deoxyadenosine-mediated growth inhibition suggests that a primary pathogenic target of accumulated dATP in lymphocytes from patients with adenosine deaminase deficiency may be the cellular ribonucleotide reductase.

  3. The evolution of chicken stem cell culture methods.

    Science.gov (United States)

    Farzaneh, M; Attari, F; Mozdziak, P E; Khoshnam, S E

    2017-12-01

    1. The avian embryo is an excellent model for studying embryology and the production of pharmaceutical proteins in transgenic chickens. Furthermore, chicken stem cells have the potential for proliferation and differentiation and emerged as an attractive tool for various cell-based technologies. 2. The objective of these studies is the derivation and culture of these stem cells is the production of transgenic birds for recombinant biomaterials and vaccine manufacture, drug and cytotoxicity testing, as well as to gain insight into basic science, including cell tracking. 3. Despite similarities among the established chicken stem cell lines, fundamental differences have been reported between their culture conditions and applications. Recent conventional protocols used for expansion and culture of chicken stem cells mostly depend on feeder cells, serum-containing media and static culture. 4. Utilising chicken stem cells for generation of cell-based transgenic birds and a variety of vaccines requires large-scale cell production. However, scaling up the conventional adherent chicken stem cells is challenging and labour intensive. Development of a suspension cell culture process for chicken embryonic stem cells (cESCs), chicken primordial germ cells (PGCs) and chicken induced pluripotent stem cells (ciPSCs) will be an important advance for increasing the growth kinetics of these cells. 6. This review describes various approaches and suggestions to achieve optimal cell growth for defined chicken stem cells cultures and use in future manufacturing applications.

  4. PRGF exerts more potent proliferative and anti-inflammatory effects than autologous serum on a cell culture inflammatory model.

    Science.gov (United States)

    Anitua, E; Muruzabal, F; de la Fuente, M; Riestra, A; Merayo-Lloves, J; Orive, G

    2016-10-01

    Ocular graft versus host disease (oGVHD) is part of a systemic inflammatory disease that usually affects ocular surface tissues manifesting as a dry eye syndrome. Current treatments provide unsatisfactory results. Blood-derived products, like plasma rich in growth factors (PRGF) emerge as a potential therapy for this disease. The purpose of this study was to evaluate the tissue regeneration and anti-inflammatory capability of PRGF, an autologous platelet enriched plasma eye-drop, compared to autologous serum (AS) obtained from oGVHD patients on ocular surface cells cultured in a pro-inflammatory environment. PRGF and AS were obtained from four GVHD patients. Cell proliferation and inflammation markers, intercellular adhesion molecule-1 (ICAM-1) and cyclooxygenase-2 (COX-2), were measured in corneal and conjunctival fibroblastic cells cultured under pro-inflammatory conditions and after treatment with PRGF or AS eye drops. Moreover, cell proliferation increased after treatment with PRGF and AS, though this enhancement in the case of keratocytes was significantly higher with PRGF. PRGF eye drops showed a significant reduction of both inflammatory markers with respect to the initial inflammatory situation and to the AS treatment. Our results concluded that PRGF exerts more potent regenerative and anti-inflammatory effects than autologous serum on ocular surface fibroblasts treated with pro-inflammatory IL-1β and TNFα. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Establishment of primary cell culture and an intracranial xenograft model of pediatric ependymoma: a prospect for therapy development and understanding of tumor biology.

    Science.gov (United States)

    Pavon, Lorena Favaro; Sibov, Tatiana Tais; Caminada de Toledo, Silvia Regina; Mara de Oliveira, Daniela; Cabral, Francisco Romero; Gabriel de Souza, Jean; Boufleur, Pamela; Marti, Luciana C; Malheiros, Jackeline Moraes; Ferreira da Cruz, Edgar; Paiva, Fernando F; Malheiros, Suzana M F; de Paiva Neto, Manoel A; Tannús, Alberto; Mascarenhas de Oliveira, Sérgio; Silva, Nasjla Saba; Cappellano, Andrea Maria; Petrilli, Antonio Sérgio; Chudzinski-Tavassi, Ana Marisa; Cavalheiro, Sérgio

    2018-04-24

    Ependymoma (EPN), the third most common pediatric brain tumor, is a central nervous system (CNS) malignancy originating from the walls of the ventricular system. Surgical resection followed by radiation therapy has been the primary treatment for most pediatric intracranial EPNs. Despite numerous studies into the prognostic value of histological classification, the extent of surgical resection and adjuvant radiotherapy, there have been relatively few studies into the molecular and cellular biology of EPNs. We elucidated the ultrastructure of the cultured EPN cells and characterized their profile of immunophenotypic pluripotency markers (CD133, CD90, SSEA-3, CXCR4). We established an experimental EPN model by the intracerebroventricular infusion of EPN cells labeled with multimodal iron oxide nanoparticles (MION), thereby generating a tumor and providing a clinically relevant animal model. MRI analysis was shown to be a valuable tool when combined with effective MION labeling techniques to accompany EPN growth. We demonstrated that GFAP/CD133+CD90+/CD44+ EPN cells maintained key histopathological and growth characteristics of the original patient tumor. The characterization of EPN cells and the experimental model could facilitate biological studies and preclinical drug screening for pediatric EPNs. In this work, we established notoriously challenging primary cell culture of anaplastic EPNs (WHO grade III) localized in the posterior fossa (PF), using EPNs obtained from 1 to 10-year-old patients ( n = 07), and then characterized their immunophenotype and ultrastructure to finally develop a xenograft model.

  6. Cell Culture Assay for Human Noroviruses [response

    Energy Technology Data Exchange (ETDEWEB)

    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

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

    Science.gov (United States)

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

    2000-01-01

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

  8. Zinc tolerance and accumulation in stable cell suspension cultures and in vitro regenerated plants of the emerging model plant Arabidopsis halleri (Brassicaceae).

    Science.gov (United States)

    Vera-Estrella, Rosario; Miranda-Vergara, Maria Cristina; Barkla, Bronwyn J

    2009-03-01

    Arabidopsis halleri is increasingly employed as a model plant for studying heavy metal hyperaccumulation. With the aim of providing valuable tools for studies on cellular physiology and molecular biology of metal tolerance and transport, this study reports the development of successful and highly efficient methods for the in vitro regeneration of A. halleri plants and production of stable cell suspension lines. Plants were regenerated from leaf explants of A. halleri via a three-step procedure: callus induction, somatic embryogenesis and shoot development. Efficiency of callus proliferation and regeneration depended on the initial callus induction media and was optimal in the presence of 1 mg L(-1) 2,4-dichlorophenoxyacetic acid, and 0.05 mg L(-1) benzylaminopurine. Subsequent shoot and root regeneration from callus initiated under these conditions reached levels of 100% efficiency. High friability of the callus supported the development of cell suspension cultures with minimal cellular aggregates. Characterization of regenerated plants and cell cultures determined that they maintained not only the zinc tolerance and requirement of the whole plant but also the ability to accumulate zinc; with plants accumulating up to 50.0 micromoles zinc g(-1) FW, and cell suspension cultures 30.9 micromoles zinc g(-1) DW. Together this work will provide the experimental basis for furthering our knowledge of A. halleri as a model heavy metal hyperaccumulating plant.

  9. Transplantation of mesenchymal stem cells cultured on biomatrix support induces repairing of digestive tract defects, in animal model.

    Science.gov (United States)

    Sîrbu-Boeţi, Mirela-Patricia; Chivu, Mihaela; Pâslaru, Liliana Livia; Efrimescu, C; Herlea, V; Pecheanu, C; Moldovan, Lucia; Dragomir, Laura; Bleotu, Coralia; Ciucur, Elena; Vidulescu, Cristina; Vasilescu, Mihaela; Boicea, Anişoara; Mănoiu, S; Ionescu, M I; Popescu, I

    2009-01-01

    Transplanted mesenchymal stem cells (MSCs) appear to play a significant role in adult tissue repair. The aim of this research was to obtain MSCs enriched, three dimensional (3D) patches for transplant, and to test their ability to induce repair of iatrogenic digestive tract defects in rats. MSCs were obtained from human and rat bone marrow, cultured in vitro, and seeded in a collagen-agarose scaffold, where they showed enhanced viability and proliferation. The phenotype of the cultured cells was representative for MSCs (CD105+, CD90+, and CD34-, CD45-, CD3-, CD14-). The 3D patch was obtained by laying the MSCs enriched collagen-agarose scaffold on a human or swine aortic fragment. After excision of small portions of the rat digestive tract, the 3D patches were sutured at the edge of the defect using micro-surgical techniques. The rats were sacrificed at time-points and the regeneration of the digestive wall was investigated by immunofluorescence, light and electron microscopy. The MSCs enriched 3D patches were biocompatible, biodegradable, and prompted the regeneration of the four layers of the stomach and intestine wall in rats. Human cells were identified in the rat regenerated digestive wall as a hallmark of the transplanted MSCs. For the first time we constructed 3D patches made of cultured bone marrow MSCs, embedded into a collagen-rich biomatrix, on vascular bio-material support, and transplanted them in order to repair iatrogenic digestive tract defects. The result was a complete repair with preservation of the four layered structure of the digestive wall.

  10. Computational Modeling of Culture's Consequences

    NARCIS (Netherlands)

    Hofstede, G.J.; Jonker, C.M.; Verwaart, T.

    2010-01-01

    This paper presents an approach to formalize the influence of culture on the decision functions of agents in social simulations. The key components are (a) a definition of the domain of study in the form of a decision model, (b) knowledge acquisition based on a dimensional theory of culture,

  11. Delineating Cultural Models

    Science.gov (United States)

    2010-08-25

    APPENDIX A: Bibliography Albert , D. (1994). Knowledge structures. New York: Springer-Verlag. Amblard, F., & Deffuant, G. (2004). The role...Cambridge, MA: The MIT Press. Groeber, P., Schweitzer , F., & Press, K. (2009). How groups can foster consensus: The case of local cultures. Journal of...narratives. Journal of Knowledge Management, 8(4), 65-74. Stark, H. U., Tessone, C. J., & Schweitzer , F. (2008). Slower is faster: Fostering

  12. ORGANISATIONAL CULTURE ANALYSIS MODEL

    OpenAIRE

    Mihaela Simona Maracine

    2012-01-01

    The studies and researches undertaken have demonstrated the importance of studying organisational culture because of the practical valences it presents and because it contributes to increasing the organisation’s performance. The analysis of the organisational culture’s dimensions allows observing human behaviour within the organisation and highlighting reality, identifying the strengths and also the weaknesses which have an impact on its functionality and development. In this paper, we try to...

  13. Mouse cell culture - Methods and protocols

    Directory of Open Access Journals (Sweden)

    CarloAlberto Redi

    2010-12-01

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

  14. Protein hydrolysate from canned sardine and brewing by-products improves TNF-α-induced inflammation in an intestinal-endothelial co-culture cell model.

    Science.gov (United States)

    Vieira, Elsa F; Van Camp, John; Ferreira, Isabel M P L V O; Grootaert, Charlotte

    2017-07-17

    The anti-inflammatory activity of sardine protein hydrolysates (SPH) obtained by hydrolysis with proteases from brewing yeast surplus was ascertained. For this purpose, a digested and desalted SPH fraction with molecular weight lower than 10 kDa was investigated using an endothelial cell line (EA.hy926) as such and in a co-culture model with an intestinal cell line (Caco-2). Effects of SPH <10 kDa on nitric oxide (NO) production, reactive oxygen species (ROS) inhibition and secretion of monocyte chemoattractant protein 1 (MCP-1), vascular endothelial growth factor (VEGF), chemokine IL-8 (IL-8) and intercellular adhesion molecule-1 (ICAM-1) were evaluated in TNF-α-treated and untreated cells. Upon TNF-α treatment, levels of NO, MCP-1, VEGF, IL-8, ICAM-1 and endothelial ROS were significantly increased in both mono- and co-culture models. Treatment with SPH <10 kDa (2.0 mg peptides/mL) significantly decreased all the inflammation markers when compared to TNF-α-treated control. This protective effect was more pronounced in the co-culture model, suggesting that SPH <10 kDa Caco-2 cells metabolites produced in the course of intestinal absorption may provide a more relevant protective effect against endothelial dysfunction. Additionally, indirect cross-talk between two cell types was established, suggesting that SPH <10 kDa may also bind to receptors on the Caco-2 cells, thereby triggering a pathway to secrete the pro-inflammatory compounds. Overall, these in vitro screening results, in which intestinal digestion, absorption and endothelial bioactivity are simulated, show the potential of SPH to be used as a functional food with anti-inflammatory properties.

  15. Cell Culture as an Alternative in Education.

    Science.gov (United States)

    Nardone, Roland M.

    1990-01-01

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

  16. Quantifying the correlation between spatially defined oxygen gradients and cell fate in an engineered three-dimensional culture model.

    Science.gov (United States)

    Ardakani, Amir G; Cheema, Umber; Brown, Robert A; Shipley, Rebecca J

    2014-09-06

    A challenge in three-dimensional tissue culture remains the lack of quantitative information linking nutrient delivery and cellular distribution. Both in vivo and in vitro, oxygen is delivered by diffusion from its source (blood vessel or the construct margins). The oxygen level at a defined distance from its source depends critically on the balance of diffusion and cellular metabolism. Cells may respond to this oxygen environment through proliferation, death and chemotaxis, resulting in spatially resolved gradients in cellular density. This study extracts novel spatially resolved and simultaneous data on tissue oxygenation, cellular proliferation, viability and chemotaxis in three-dimensional spiralled, cellular collagen constructs. Oxygen concentration gradients drove preferential cellular proliferation rates and viability in the higher oxygen zones and induced chemotaxis along the spiral of the collagen construct; an oxygen gradient of 1.03 mmHg mm(-1) in the spiral direction induced a mean migratory speed of 1015 μm day(-1). Although this movement was modest, it was effective in balancing the system to a stable cell density distribution, and provided insights into the natural cell mechanism for adapting cell number and activity to a prevailing oxygen regime.

  17. Optimization of micro-fabricated porous membranes for intestinal epithelial cell culture and in vitro modeling of the human intestinal barrier

    Science.gov (United States)

    Nair Gourikutty Sajay, Bhuvanendran; Yin, Chiam Su; Ramadan, Qasem

    2017-12-01

    In vitro modeling of organs could provide a controlled platform for studying physiological events and has great potential in the field of pharmaceutical development. Here, we describe the characterization of in vitro modeling of the human intestinal barrier mimicked using silicon porous membranes as a substrate. To mimic an intestinal in vivo setup as closely as possible, a porous substrate is required in a dynamic environment for the cells to grow rather than a static setup with an impermeable surface such as a petri dish. In this study, we focus on the detailed characterization of Caco-2 cells cultured on a silicon membrane with different pore sizes as well as the effect of dynamic fluid flow on the model. The porous silicon membrane together with continuous perfusion of liquid applying shear stress on the cells enhances the differentiation of polarized cells by providing access to the both their basal and apical surfaces. Membranes with pore sizes of 0.5-3 µm were used and a shear stress of ~0.03 dyne cm-2 was created by applying a low flow rate of 20 nl s-1. By providing these optimized conditions, cells were able to differentiate with columnar morphology, which developed microvilli structures on their apical side and tight junctions between adjacent cells like those in a healthy human intestinal barrier. In this setup, it is possible to study the important cellular functions of the intestine such as transport, absorption and secretion, and thus this model has great potential in drug screening.

  18. Expression patterns of tight junction components induced by CD24 in an oral epithelial cell-culture model correlated to affected periodontal tissues.

    Science.gov (United States)

    Ye, P; Yu, H; Simonian, M; Hunter, N

    2014-04-01

    Previously we demonstrated uniformly strong expression of CD24 in the epithelial attachment to the tooth and in the migrating epithelium of the periodontitis lesion. Titers of serum antibodies autoreactive with CD24 peptide correlated with reduced severity of periodontal disease. Ligation of CD24 expressed by oral epithelial cells induced formation of tight junctions that limited paracellular diffusion. In this study, we aimed to reveal that the lack of uniform expression of tight junction components in the pocket epithelium of periodontitis lesions is likely to contribute to increased paracellular permeability to bacterial products. This is proposed as a potential driver of the immunopathology of periodontitis. An epithelial culture model with close correspondence for expression patterns for tight junction components in periodontal epithelia was used. Immunohistochemical staining and confocal laser scanning microscopy were used to analyse patterns of expression of gingival epithelial tight junction components. The minimally inflamed gingival attachment was characterized by uniformly strong staining at cell contacts for the tight junction components zona occludens-1, zona occludens-2, occludin, junction adhesion molecule-A, claudin-4 and claudin-15. In contrast, the pocket epithelium of the periodontal lesion showed scattered, uneven staining for these components. This pattern correlated closely with that of unstimulated oral epithelial cells in culture. Following ligation of CD24 expressed by these cells, the pattern of tight junction component expression of the minimally inflamed gingival attachment developed rapidly. There was evidence for non-uniform and focal expression only of tight junction components in the pocket epithelium. In the cell-culture model, ligation of CD24 induced a tight junction expression profile equivalent to that observed for the minimally inflamed gingival attachment. Ligation of CD24 expressed by gingival epithelial cells by lectin

  19. Development of a soft-sensor based on multi-wavelength fluorescence spectroscopy and a dynamic metabolic model for monitoring mammalian cell cultures.

    Science.gov (United States)

    Ohadi, Kaveh; Legge, Raymond L; Budman, Hector M

    2015-01-01

    A soft-sensor based on an Extended Kalman Filter (EKF) that combines data obtained using a fluorescence-based soft-sensor with a dynamic mechanistic model, was investigated as a tool for continuous monitoring of a Chinese hamster ovary (CHO) cell cultivation process. A standalone fluorescence based soft-sensor, which uses a combination of an empirical multivariate statistical model and measured spectra, was designed for predicting key culture variables including viable and dead cells, recombinant protein, glucose, and ammonia concentrations. The standalone fluorescence sensor was then combined with a dynamic mechanistic model within an EKF framework, for improving the prediction accuracy and generating predictions in-between sampling instances. The dynamic model used for the EKF framework was based on a structured metabolic flux analysis and mass balances. In order to calibrate the fluorescence-based empirical model and the dynamic mechanistic model, cells were grown in batch mode with different initial glucose and glutamine concentrations. To mitigate the uncertainty associated with the model structure and parameters, non-stationary disturbances were accounted for in the EKF by parameter-adaptation. It was demonstrated that the implementation of the EKF along with the dynamic model could improve the accuracy of the fluorescence-based predictions at the sampling instances. Additionally, it was shown that the major advantage of the EKF-based soft-sensor, compared to the standalone fluorescence-based counterpart, was its capability to track the temporal evolution of key process variables between measurement instances obtained by the fluorescence-based soft-sensor. This is crucial for designing control strategies of CHO cell cultures with the aim of guaranteeing product quality. © 2014 Wiley Periodicals, Inc.

  20. The impact of cell culture equipment on energy loss.

    Science.gov (United States)

    Davies, Lleucu B; Kiernan, Michael N; Bishop, Joanna C; Thornton, Catherine A; Morgan, Gareth

    2014-01-01

    Light energy of discrete wavelengths supplied via lasers and broadband intense pulsed light have been used therapeutically for many years. In vitro models complement clinical studies, especially for the elucidation of underlying mechanisms of action. Clarification that light energy reaches the cells is necessary when developing protocols for the treatment of cells using in vitro models. Few studies report on energy loss in cell culture equipment. The ability of energy from light with therapeutic potential to reach cells in culture needs to be determined; this includes determining the proportion of light energy lost within standard cell culture media and cell culture vessels. The energy absorption of cell culture media, with/without the pH indicator dye phenol red, and the loss of energy within different plastics and glassware used typically for in vitro cell culture were investigated using intense pulsed light and a yellow pulsed dye laser. Media containing phenol red have a distinctive absorption peak (560 nm) absent in phenol red-free media and restored by the addition of phenol red. For both light sources, energy loss was lowest in standard polystyrene tissue culture flasks or multi-well plates and highest in polypropylene vessels or glass tubes. The effects of phenol red-free media on the absorption of energy varied with the light source used. Phenol red-free media are the media of choice; polystyrene vessels with flat surfaces such as culture flasks or multi-well plates should be used in preference to polypropylene or glass vessels.

  1. Effect of adipose-derived mesenchymal stromal cells on tendon healing in aging and estrogen deficiency: an in vitro co-culture model.

    Science.gov (United States)

    Veronesi, Francesca; Della Bella, Elena; Torricelli, Paola; Pagani, Stefania; Fini, Milena

    2015-11-01

    Aging and estrogen deficiency play a pivotal role in reducing tenocyte proliferation, collagen turnover and extracellular matrix remodeling. Mesenchymal stromal cells are being studied as an alternative for tendon regeneration, but little is known about the molecular events of adipose-derived mesenchymal stromal cells (ADSCs) on tenocytes in tendons compromised by aging and estrogen deficiency. The present in vitro study aims to compare the potential therapeutic effects of ADSCs, harvested from healthy young (sham) and aged estrogen-deficient (OVX) subjects, for tendon healing. An indirect co-culture system was set up with ADSCs, isolated from OVX or sham rats, and tenocytes from OVX rats. Cell proliferation, healing rate and gene expression were evaluated in both a standard culture condition and a microwound-healing model. It was observed that tenocyte proliferation, healing rate and collagen expression improved after the addition of sham ADSCs in both culture situations. OVX ADSCs also increased tenocyte proliferation and healing rate but less compared with sham ADSCs. Decorin and Tenascin C expression increased in the presence of OVX ADSCs. Findings suggest that ADSCs might be a promising treatment for tendon regeneration in advanced age and estrogen deficiency. However, some differences between allogenic and autologous cells were found and should be investigated in further in vivo studies. It appears that allogenic ADSCs improve tenocyte proliferation, collagen expression and the healing rate more than autologous cells. Autologous cells increase collagen expression only in the absence of an injury and increase Decorin and Tenascin C more than allogenic cells. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  2. A brain slice culture model for studies of endogenous and exogenous precursor cell migration in the rostral migratory stream

    DEFF Research Database (Denmark)

    Tanvig, Mette; Blaabjerg, Morten; Andersen, Rikke K

    2009-01-01

    The rostral migratory stream (RMS) is the main pathway by which newly born subventricular zone (SVZ) cells reach the olfactory bulb (OB) in rodents. This migration has been well studied in vivo, but an organotypic in vitro model would facilitate more experimental investigations. Here we introduce...

  3. Optimization of a pH-shift control strategy for producing monoclonal antibodies in Chinese hamster ovary cell cultures using a pH-dependent dynamic model.

    Science.gov (United States)

    Hogiri, Tomoharu; Tamashima, Hiroshi; Nishizawa, Akitoshi; Okamoto, Masahiro

    2018-02-01

    To optimize monoclonal antibody (mAb) production in Chinese hamster ovary cell cultures, culture pH should be temporally controlled with high resolution. In this study, we propose a new pH-dependent dynamic model represented by simultaneous differential equations including a minimum of six system component, depending on pH value. All kinetic parameters in the dynamic model were estimated using an evolutionary numerical optimization (real-coded genetic algorithm) method based on experimental time-course data obtained at different pH values ranging from 6.6 to 7.2. We determined an optimal pH-shift schedule theoretically. We validated this optimal pH-shift schedule experimentally and mAb production increased by approximately 40% with this schedule. Throughout this study, it was suggested that the culture pH-shift optimization strategy using a pH-dependent dynamic model is suitable to optimize any pH-shift schedule for CHO cell lines used in mAb production projects. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  4. Cardiac tissue geometry as a determinant of unidirectional conduction block: assessment of microscopic excitation spread by optical mapping in patterned cell cultures and in a computer model.

    Science.gov (United States)

    Fast, V G; Kléber, A G

    1995-05-01

    Unidirectional conduction block (UCB) and reentry may occur as a consequence of an abrupt tissue expansion and a related change in the electrical load. The aim of this study was to evaluate critical dimensions of the tissue necessary for establishing UCB in heart cell culture. Neonatal rat heart cell cultures with cell strands of variable width emerging into a large cell area were grown using a technique of patterned cell growth. Action potential upstrokes were measured using a voltage sensitive dye (RH-237) and a linear array of 10 photodiodes with a 15 microns resolution. A mathematical model was used to relate action potential wave shapes to underlying ionic currents. UCB (block of a single impulse in anterograde direction - from a strand to a large area - and conduction in the retrograde direction) occurred in narrow cell strands with a width of 15(SD 4) microns (1-2 cells in width, n = 7) and there was no conduction block in strands with a width of 31(8) microns (n = 9, P multiple rising phases. Mathematical modelling showed that two rising phases were caused by electronic current flow, whereas local ionic current did not coincide with the rising portions of the upstrokes. (1) High resolution optical mapping shows multiphasic action potential upstrokes at the region of abrupt expansion. At the site of the maximum decrement in conduction, these peaks were largely determined by the electrotonus and not by the local ionic current. (2) Unidirectional conduction block occurred in strands with a width of 15(4) microns (1-2 cells).

  5. Modeling the ischemic blood-brain barrier; the effects of oxygen-glucose deprivation (OGD) on endothelial cells in culture

    DEFF Research Database (Denmark)

    Tornabene, Erica; Helms, Hans Christian Cederberg; Berndt, Philipp

    Introduction - The blood-brain barrier (BBB) is a physical, transport and metabolic barrier which plays a key role in preventing uncontrolled exchanges between blood and brain, ensuring an optimal environment for neurons activity. This extent interface is created by the endothelial cells forming...... pathways across the barrier in ischemic and postischemic brain endothelium is important for developing new medical therapies capable to exploit the barrier changes occurring during/after ischemia to permeate in the brain and treat this devastating disease. Materials and Methods - Primary cultures...... the wall of brain capillaries. The restrictive nature of the BBB is due to the tight junctions (TJs), which seal the intercellular clefts, limiting the paracellular diffusion, efflux transporters, which extrude xenobiotics, and metabolizing enzymes, which may break down or convert molecules during...

  6. Radiosensitivity of normal human epidermal cells in culture

    International Nuclear Information System (INIS)

    Dover, R.; Potten, C.S.

    1983-01-01

    Using an in vitro culture system the authors have derived #betta#-radiation survival curves over a dose range 0-8 Gy for the clonogenic cells of normal human epidermis. The culture system used allows the epidermal cells to stratify and form a multi-layered sheet of keratinizing cells. The cultures appear to be a very good model for epidermis in vivo. The survival curves show a population which is apparently more sensitive than murine epidermis in vivo. It remains unclear whether this is an intrinsic difference between the species or is a consequence of the in vitro cultivation of the human cells. (author)

  7. Poly(vinyl alcohol/gelatin Hydrogels Cultured with HepG2 Cells as a 3D Model of Hepatocellular Carcinoma: A Morphological Study

    Directory of Open Access Journals (Sweden)

    Stefania Moscato

    2015-01-01

    Full Text Available It has been demonstrated that three-dimensional (3D cell culture models represent fundamental tools for the comprehension of cellular phenomena both for normal and cancerous tissues. Indeed, the microenvironment affects the cellular behavior as well as the response to drugs. In this study, we performed a morphological analysis on a hepatocarcinoma cell line, HepG2, grown for 24 days inside a bioartificial hydrogel composed of poly(vinyl alcohol (PVA and gelatin (G to model a hepatocellular carcinoma (HCC in 3D. Morphological features of PVA/G hydrogels were investigated, resulting to mimic the trabecular structure of liver parenchyma. A histologic analysis comparing the 3D models with HepG2 cell monolayers and tumor specimens was performed. In the 3D setting, HepG2 cells were viable and formed large cellular aggregates showing different morphotypes with zonal distribution. Furthermore, β-actin and α5β1 integrin revealed a morphotype-related expression; in particular, the frontline cells were characterized by a strong immunopositivity on a side border of their membrane, thus suggesting the formation of lamellipodia-like structures apt for migration. Based on these results, we propose PVA/G hydrogels as valuable substrates to develop a long term 3D HCC model that can be used to investigate important aspects of tumor biology related to migration phenomena.

  8. Free Publishing Culture. Sustainable Models?

    Directory of Open Access Journals (Sweden)

    Silvia Nanclares Escudero

    2013-03-01

    Full Text Available As a result of the collective research on the possibilities for publishing production and distribution offered nowadays by the Free Culture scenario, we present here a mapping of symptoms in order to propose a transitory diagnostic of the question: Is it possible to generate an economically sustainable publishing model based on the uses and customs generated and provided by Free Culture? Data, intuitions, experiences and ideas attempt to back up our affirmative answer.

  9. 21st Century Cell Culture for 21st Century Toxicology.

    Science.gov (United States)

    Pamies, David; Hartung, Thomas

    2017-01-17

    There is no good science in bad models. Cell culture is especially prone to artifacts. A number of novel cell culture technologies have become more broadly available in the 21st century, which allow overcoming limitations of traditional culture and are more physiologically relevant. These include the use of stem-cell derived human cells, cocultures of different cell types, scaffolds and extracellular matrices, perfusion platforms (such as microfluidics), 3D culture, organ-on-chip technologies, tissue architecture, and organ functionality. The physiological relevance of such models is further enhanced by the measurement of biomarkers (e.g., key events of pathways), organ specific functionality, and more comprehensive assessment cell responses by high-content methods. These approaches are still rarely combined to create microphysiological systems. The complexity of the combination of these technologies can generate results closer to the in vivo situation but increases the number of parameters to control, bringing some new challenges. In fact, we do not argue that all cell culture needs to be that sophisticated. The efforts taken are determined by the purpose of our experiments and tests. If only a very specific molecular target to cell response is of interest, a very simple model, which reflects this, might be much more suited to allow standardization and high-throughput. However, the less defined the end point of interest and cellular response are, the better we should approximate organ- or tissue-like culture conditions to make physiological responses more probable. Besides these technologic advances, important progress in the quality assurance and reporting on cell cultures as well as the validation of cellular test systems brings the utility of cell cultures to a new level. The advancement and broader implementation of Good Cell Culture Practice (GCCP) is key here. In toxicology, this is a major prerequisite for meaningful and reliable results, ultimately

  10. Association between expression of cumulus expansion markers and real-time proliferation of porcine follicular granulosa cells in a primary cell culture model.

    Science.gov (United States)

    Ciesiółka, S; Budna, J; Bryja, A; Kranc, W; Chachuła, A; Dyszkiewicz-Konwińska, M; Piotrowska, H; Bukowska, D; Antosik, P; Bruska, M; Brüssow, K P; Nowicki, M; Zabel, M; Kempisty, B

    2016-01-01

    Folliculogenesis is a compound process that involves both ovarian follicle growth and oocyte development, which is tightly attached to the follicular wall. During this process, cells that form the follicle structure undergo substantial morphological and molecular modifications that finally lead to differentiation and specialization of ovarian follicular cells. The differentiation of ovarian cells encompasses formation of follicle, which is composed of theca (TCs), mural granulosa (GCs), and cumulus cells (CCs). It was previously hypothesized that GCs and CCs represent undifferentiated and highly specialized follicular cells, respectively, which may have similar primordial cell origins. In this study, we investigated the expression pattern of cumulus expansion markers such as COX2, HAS2, PTX3, and TSG6 in porcine GCs during short-term, in vitro culture. We hypothesized that these genes may display an important function in GCs in relation to cellular real-time proliferation. The expression pattern of COX2, HAS2, PTX3, and TSG6 was evaluated after using RT-qPCR in relation to confocal microscopy observations of protein expression and distribution during real-time proliferation of porcine follicular GCs. The COX2 and HAS2 mRNAs were highly expressed after 120 h of in vitro culture (IVC), whereas PTX3 and TSG6 mRNAs were increased during the first 24-48 h of IVC (P less than 0.001, P less than 0.01). Conversely, all of the encoded proteins were highly expressed after 144-168 h of IVC as compared to other culture periods (P less than 0.001, P less than 0.01). When analyzing the realtime proliferation of GCs in vitro, we observed a logarithmic increase of cell proliferation between 0 h and 120 h of IVC. However, after 120-168 h of IVC, the cells reached the lag phase of proliferation. Since it is well accepted that porcine GCs undergo luteinization shortly after 24-48 h of IVC, the expression pattern of investigated genes indicated that Cox2 and Has2 are independent from

  11. Δ⁹-tetrahydrocannabinol (Δ⁹-THC) exerts a direct neuroprotective effect in a human cell culture model of Parkinson's disease.

    Science.gov (United States)

    Carroll, C B; Zeissler, M-L; Hanemann, C O; Zajicek, J P

    2012-10-01

    Δ⁹-tetrahydrocannabinol (Δ⁹-THC) is neuroprotective in models of Parkinson's disease (PD). Although CB1 receptors are increased within the basal ganglia of PD patients and animal models, current evidence suggests a role for CB1 receptor-independent mechanisms. Here, we utilized a human neuronal cell culture PD model to further investigate the protective properties of Δ⁹-THC. Differentiated SH-SY5Y neuroblastoma cells were exposed to PD-relevant toxins: 1-methyl-4-phenylpyridinium (MPP+), lactacystin and paraquat. Changes in CB1 receptor level were determined by quantitative polymerase chain reaction and Western blotting. Cannabinoids and modulatory compounds were co-administered with toxins for 48 h and the effects on cell death, viability, apoptosis and oxidative stress assessed. We found CB1 receptor up-regulation in response to MPP+, lactacystin and paraquat and a protective effect of Δ⁹-THC against all three toxins. This neuroprotective effect was not reproduced by the CB1 receptor agonist WIN55,212-2 or blocked by the CB1 antagonist AM251. Furthermore, the antioxidants α-tocopherol and butylhydroxytoluene as well as the antioxidant cannabinoids, nabilone and cannabidiol were unable to elicit the same neuroprotection as Δ⁹-THC. However, the peroxisome proliferator-activated receptor-gamma (PPARγ) antagonist T0070907 dose-dependently blocked the neuroprotective, antioxidant and anti-apoptotic effects of Δ⁹-THC, while the PPARγ agonist pioglitazone resulted in protection from MPP+-induced neurotoxicity. Furthermore, Δ⁹-THC increased PPARγ expression in MPP+-treated SH-SY5Y cells, another indicator of PPARγ activation. We have demonstrated up-regulation of the CB1 receptor in direct response to neuronal injury in a human PD cell culture model, and a direct neuronal protective effect of Δ⁹-THC that may be mediated through PPARγ activation. © 2011 The Authors. Neuropathology and Applied Neurobiology © 2011 British Neuropathological

  12. Impairment of mitochondrial calcium handling in a mtSOD1 cell culture model of motoneuron disease

    Directory of Open Access Journals (Sweden)

    Zippelius Annette

    2009-06-01

    Full Text Available Abstract Background Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons (MN in the brain stem and spinal cord. Intracellular disruptions of cytosolic and mitochondrial calcium have been associated with selective MN degeneration, but the underlying mechanisms are not well understood. The present evidence supports a hypothesis that mitochondria are a target of mutant SOD1-mediated toxicity in familial amyotrophic lateral sclerosis (fALS and intracellular alterations of cytosolic and mitochondrial calcium might aggravate the course of this neurodegenerative disease. In this study, we used a fluorescence charged cool device (CCD imaging system to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells in an established cellular model of ALS. Results To gain insights into the molecular mechanisms of SOD1G93A associated motor neuron disease, we simultaneously monitored cytosolic and mitochondrial calcium concentrations in individual cells. Voltage – dependent cytosolic Ca2+ elevations and mitochondria – controlled calcium release mechanisms were monitored after loading cells with fluorescent dyes fura-2 and rhod-2. Interestingly, comparable voltage-dependent cytosolic Ca2+ elevations in WT (SH-SY5YWT and G93A (SH-SY5YG93A expressing cells were observed. In contrast, mitochondrial intracellular Ca2+ release responses evoked by bath application of the mitochondrial toxin FCCP were significantly smaller in G93A expressing cells, suggesting impaired calcium stores. Pharmacological experiments further supported the concept that the presence of G93A severely disrupts mitochondrial Ca2+ regulation. Conclusion In this study, by fluorescence measurement of cytosolic calcium and using simultaneous [Ca2+]i and [Ca2+]mito measurements, we are able to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations

  13. Basic study on a lower-energy defibrillation method using computer simulation and cultured myocardial cell models.

    Science.gov (United States)

    Yaguchi, A; Nagase, K; Ishikawa, M; Iwasaka, T; Odagaki, M; Hosaka, H

    2006-01-01

    Computer simulation and myocardial cell models were used to evaluate a low-energy defibrillation technique. A generated spiral wave, considered to be a mechanism of fibrillation, and fibrillation were investigated using two myocardial sheet models: a two-dimensional computer simulation model and a two-dimensional experimental model. A new defibrillation technique that has few side effects, which are induced by the current passing into the patient's body, on cardiac muscle is desired. The purpose of the present study is to conduct a basic investigation into an efficient defibrillation method. In order to evaluate the defibrillation method, the propagation of excitation in the myocardial sheet is measured during the normal state and during fibrillation, respectively. The advantages of the low-energy defibrillation technique are then discussed based on the stimulation timing.

  14. Replication of cultured lung epithelial cells

    International Nuclear Information System (INIS)

    Guzowski, D.; Bienkowski, R.

    1986-01-01

    The authors have investigated the conditions necessary to support replication of lung type 2 epithelial cells in culture. Cells were isolated from mature fetal rabbit lungs (29d gestation) and cultured on feeder layers of mitotically inactivated 3T3 fibroblasts. The epithelial nature of the cells was demonstrated by indirect immunofluorescent staining for keratin and by polyacid dichrome stain. Ultrastructural examination during the first week showed that the cells contained myofilaments, microvilli and lamellar bodies (markers for type 2 cells). The following changes were observed after the first week: increase in cell size; loss of lamellar bodies and appearance of multivesicular bodies; increase in rough endoplasmic reticulum and golgi; increase in tonafilaments and well-defined junctions. General cell morphology was good for up to 10 wk. Cells cultured on plastic surface degenerated after 1 wk. Cell replication was assayed by autoradiography of cultures exposed to ( 3 H)-thymidine and by direct cell counts. The cells did not replicate during the first week; however, between 2-10 wk the cells incorporated the label and went through approximately 6 population doublings. They have demonstrated that lung alveolar epithelial cells can replicate in culture if they are maintained on an appropriate substrate. The coincidence of ability to replicate and loss of markers for differentiation may reflect the dichotomy between growth and differentiation commonly observed in developing systems

  15. Application of cell co-culture system to study fat and muscle cells.

    Science.gov (United States)

    Pandurangan, Muthuraman; Hwang, Inho

    2014-09-01

    Animal cell culture is a highly complex process, in which cells are grown under specific conditions. The growth and development of these cells is a highly unnatural process in vitro condition. Cells are removed from animal tissues and artificially cultured in various culture vessels. Vitamins, minerals, and serum growth factors are supplied to maintain cell viability. Obtaining result homogeneity of in vitro and in vivo experiments is rare, because their structure and function are different. Living tissues have highly ordered complex architecture and are three-dimensional (3D) in structure. The interaction between adjacent cell types is quite distinct from the in vitro cell culture, which is usually two-dimensional (2D). Co-culture systems are studied to analyze the interactions between the two different cell types. The muscle and fat co-culture system is useful in addressing several questions related to muscle modeling, muscle degeneration, apoptosis, and muscle regeneration. Co-culture of C2C12 and 3T3-L1 cells could be a useful diagnostic tool to understand the muscle and fat formation in animals. Even though, co-culture systems have certain limitations, they provide a more realistic 3D view and information than the individual cell culture system. It is suggested that co-culture systems are useful in evaluating the intercellular communication and composition of two different cell types.

  16. Effects of ethanol and acetaldehyde on tight junction integrity: in vitro study in a three dimensional intestinal epithelial cell culture model.

    Directory of Open Access Journals (Sweden)

    Elhaseen Elamin

    Full Text Available 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

  17. Biased Type 1 Cannabinoid Receptor Signaling Influences Neuronal Viability in a Cell Culture Model of Huntington Disease.

    Science.gov (United States)

    Laprairie, Robert B; Bagher, Amina M; Kelly, Melanie E M; Denovan-Wright, Eileen M

    2016-03-01

    Huntington disease (HD) is an inherited, autosomal dominant, neurodegenerative disorder with limited treatment options. Prior to motor symptom onset or neuronal cell loss in HD, levels of the type 1 cannabinoid receptor (CB1) decrease in the basal ganglia. Decreasing CB1 levels are strongly correlated with chorea and cognitive deficit. CB1 agonists are functionally selective (biased) for divergent signaling pathways. In this study, six cannabinoids were tested for signaling bias in in vitro models of medium spiny projection neurons expressing wild-type (STHdh(Q7/Q7)) or mutant huntingtin protein (STHdh(Q111/Q111)). Signaling bias was assessed using the Black and Leff operational model. Relative activity [ΔlogR (τ/KA)] and system bias (ΔΔlogR) were calculated relative to the reference compound WIN55,212-2 for Gαi/o, Gαs, Gαq, Gβγ, and β-arrestin1 signaling following treatment with 2-arachidonoylglycerol (2-AG), anandamide (AEA), CP55,940, Δ(9)-tetrahydrocannabinol (THC), cannabidiol (CBD), and THC+CBD (1:1), and compared between wild-type and HD cells. The Emax of Gαi/o-dependent extracellular signal-regulated kinase (ERK) signaling was 50% lower in HD cells compared with wild-type cells. 2-AG and AEA displayed Gαi/o/Gβγ bias and normalized CB1 protein levels and improved cell viability, whereas CP55,940 and THC displayed β-arrestin1 bias and reduced CB1 protein levels and cell viability in HD cells. CBD was not a CB1 agonist but inhibited THC-dependent signaling (THC+CBD). Therefore, enhancing Gαi/o-biased endocannabinoid signaling may be therapeutically beneficial in HD. In contrast, cannabinoids that are β-arrestin-biased--such as THC found at high levels in modern varieties of marijuana--may be detrimental to CB1 signaling, particularly in HD where CB1 levels are already reduced. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  18. Fluvoxamine stimulates oligodendrogenesis of cultured neural stem cells and attenuates inflammation and demyelination in an animal model of multiple sclerosis.

    Science.gov (United States)

    Ghareghani, Majid; Zibara, Kazem; Sadeghi, Heibatollah; Dokoohaki, Shima; Sadeghi, Hossein; Aryanpour, Roya; Ghanbari, Amir

    2017-07-07

    Multiple Sclerosis (MS) require medications controlling severity of the pathology and depression, affecting more than half of the patients. In this study, the effect of antidepressant drug fluvoxamine, a selective serotonin reuptake inhibitor, was investigated in vitro and in vivo. Nanomolar concentrations of fluvoxamine significantly increased cell viability and proliferation of neural stem cells (NSCs) through increasing mRNA expression of Notch1, Hes1 and Ki-67, and protein levels of NICD. Also, physiological concentrations of fluvoxamine were optimal for NSC differentiation toward oligodendrocytes, astrocytes and neurons. In addition, fluvoxamine attenuated experimental autoimmune encephalomyelitis (EAE) severity, a rat MS model, by significantly decreasing its clinical scores. Moreover, fluvoxamine treated EAE rats showed a decrease in IFN-γ serum levels and an increase in IL-4, pro- and anti-inflammatory cytokines respectively, compared to untreated EAE rats. Furthermore, immune cell infiltration and demyelination plaque significantly decreased in spinal cords of fluvoxamine-treated rats, which was accompanied by an increase in protein expression of MBP and GFAP positive cells and a decrease in lactate serum levels, a new biomarker of MS progression. In summary, besides its antidepressant activity, fluvoxamine stimulates proliferation and differentiation of NSCs particularly toward oligodendrocytes, a producer of CNS myelin.

  19. Multizone Paper Platform for 3D Cell Cultures

    Science.gov (United States)

    Derda, Ratmir; Hong, Estrella; Mwangi, Martin; Mammoto, Akiko; Ingber, Donald E.; Whitesides, George M.

    2011-01-01

    In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other environmental factors (temperature, mechanical stress, etc). The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously (“cells-in-gels-in-paper” or CiGiP), this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM) gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers) containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D culture, by peeling apart the sheets of paper, “sections” all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures. PMID:21573103

  20. An in vitro 3D bone metastasis model by using a human bone tissue culture and human sex-related cancer cells.

    Science.gov (United States)

    Salamanna, Francesca; Borsari, Veronica; Brogini, Silvia; Giavaresi, Gianluca; Parrilli, Annapaola; Cepollaro, Simona; Cadossi, Matteo; Martini, Lucia; Mazzotti, Antonio; Fini, Milena

    2016-11-22

    One of the main limitations, when studying cancer-bone metastasis, is the complex nature of the native bone environment and the lack of reliable, simple, inexpensive models that closely mimic the biological processes occurring in patients and allowing the correct translation of results. To enhance the understanding of the mechanisms underlying human bone metastases and in order to find new therapies, we developed an in vitro three-dimensional (3D) cancer-bone metastasis model by culturing human breast or prostate cancer cells with human bone tissue isolated from female and male patients, respectively. Bone tissue discarded from total hip replacement surgery was cultured in a rolling apparatus system in a normoxic or hypoxic environment. Gene expression profile, protein levels, histological, immunohistochemical and four-dimensional (4D) micro-CT analyses showed a noticeable specificity of breast and prostate cancer cells for bone colonization and ingrowth, thus highlighting the species-specific and sex-specific osteotropism and the need to widen the current knowledge on cancer-bone metastasis spread in human bone tissues. The results of this study support the application of this model in preclinical studies on bone metastases and also follow the 3R principles, the guiding principles, aimed at replacing/reducing/refining (3R) animal use and their suffering for scientific purposes.

  1. 3D Cell Culture in Alginate Hydrogels

    Directory of Open Access Journals (Sweden)

    Therese Andersen

    2015-03-01

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

  2. Diclofenac protects cultured human corneal epithelial cells against hyperosmolarity and ameliorates corneal surface damage in a rat model of dry eye.

    Science.gov (United States)

    Sawazaki, Ryoichi; Ishihara, Tomoaki; Usui, Shinya; Hayashi, Erika; Tahara, Kayoko; Hoshino, Tatsuya; Higuchi, Akihiro; Nakamura, Shigeru; Tsubota, Kazuo; Mizushima, Tohru

    2014-04-21

    Dry eye syndrome (DES) is characterized by an increase in tear osmolarity and induction of the expression and nuclear localization of an osmoprotective transcription factor (nuclear factor of activated T-cells 5 [NFAT5]) that plays an important role in providing protection against hyperosmotic tears. In this study, we screened medicines already in clinical use with a view of finding compounds that protect cultured human corneal epithelial cells against hyperosmolarity-induced cell damage. Viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and cellular NFAT5 level was measured by immunoblotting. The rat model for DES was developed by removal of the lacrimal glands, with an assessment of corneal surface damage based on levels of fluorescein staining and epithelial apoptosis. Some nonsteroidal anti-inflammatory drugs (NSAIDs), including diclofenac sodium (diclofenac), were identified during the screening procedure. These NSAIDs were able to suppress hyperosmolarity-induced apoptosis and cell growth arrest. In contrast, other NSAIDs, including bromfenac sodium (bromfenac), did not exert such a protective action. Treatment of cells with diclofenac, but not bromfenac, stimulated both the nuclear localization and expression of NFAT5 under hyperosmotic conditions. In the rat model for DES, topical administration of diclofenac (but not bromfenac) to eyes reduced corneal surface damage without affecting the volume of tear fluid. Diclofenac appears to protect cells against hyperosmolarity-induced cell damage and NFAT5 would play an important role in this protective action. The findings reported here may also indicate that the topical administration of diclofenac to eyes may be therapeutically beneficial for DES patients.

  3. Anti-osteoporotic activity of harpagide by regulation of bone formation in osteoblast cell culture and ovariectomy-induced bone loss mouse models.

    Science.gov (United States)

    Chung, Hwa-Jin; Kyung Kim, Won; Joo Park, Hyen; Cho, Lan; Kim, Me-Riong; Kim, Min Jeong; Shin, Joon-Shik; Ho Lee, Jin; Ha, In-Hyuk; Kook Lee, Sang

    2016-02-17

    Harpagide, an iridoid glucoside, is a constituent of the root of Harpagophytum procumbens var. sublobatum (Engl.) Stapf, Devil's claw which has been used in patients with osteoarthritis (OA). In the present study, we investigated the anti-osteoporotic potential of harpagide and its underlying mechanism of action in in vitro cell culture and in vivo bone loss animal models. Harpagide was obtained from the alkalic hydrolysis of harpagoside, a major constituent of H. procumbens var. sublobatum Analysis of biomarkers for bone formation in osteoblastic MC3T3-E1 cells and bone resorption in osteoclast cells derived from mouse bone marrow cells was performed to evaluate the mechanism of action. The protective activity of harpagide against bone loss was also evaluated in ovariectomized (OVX) mouse model. Harpagide improved bone properties by stimulating the process of differentiation and maturation of osteoblast cells and suppressing the process of RANKL-induced differentiation of osteoclast cells. In OVX-induced bone loss mouse model, oral administration of harpagide significantly improved recovery of bone mineral density, trabecular bone volume, and trabecular number in the femur. Harpagide also prevented increase of trabecular separation and structure model index induced by OVX. Harpagide effectively inhibited the serum levels of biochemical markers of bone loss, including alkaline phosphatase, osteocalcin, C-terminal telopeptide, and tartrate-resistant acid phosphatase. Taken together, the present study demonstrates that harpagide has a potential for prevention of bone loss in OVX mice by regulating the stimulation of osteoblast differentiation and the suppression of osteoclast formation. Therefore, these findings suggest that harpagide might serve as a bioactive compound derived from H. procumbens var. sublobatum for improvement of age-dependent bone destruction disease. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  4. Viral epidemics in a cell culture: novel high resolution data and their interpretation by a percolation theory based model.

    Directory of Open Access Journals (Sweden)

    Balázs Gönci

    2010-12-01

    Full Text Available Because of its relevance to everyday life, the spreading of viral infections has been of central interest in a variety of scientific communities involved in fighting, preventing and theoretically interpreting epidemic processes. Recent large scale observations have resulted in major discoveries concerning the overall features of the spreading process in systems with highly mobile susceptible units, but virtually no data are available about observations of infection spreading for a very large number of immobile units. Here we present the first detailed quantitative documentation of percolation-type viral epidemics in a highly reproducible in vitro system consisting of tens of thousands of virtually motionless cells. We use a confluent astroglial monolayer in a Petri dish and induce productive infection in a limited number of cells with a genetically modified herpesvirus strain. This approach allows extreme high resolution tracking of the spatio-temporal development of the epidemic. We show that a simple model is capable of reproducing the basic features of our observations, i.e., the observed behaviour is likely to be applicable to many different kinds of systems. Statistical physics inspired approaches to our data, such as fractal dimension of the infected clusters as well as their size distribution, seem to fit into a percolation theory based interpretation. We suggest that our observations may be used to model epidemics in more complex systems, which are difficult to study in isolation.

  5. Viral epidemics in a cell culture: novel high resolution data and their interpretation by a percolation theory based model.

    Science.gov (United States)

    Gönci, Balázs; Németh, Valéria; Balogh, Emeric; Szabó, Bálint; Dénes, Ádám; Környei, Zsuzsanna; Vicsek, Tamás

    2010-12-20

    Because of its relevance to everyday life, the spreading of viral infections has been of central interest in a variety of scientific communities involved in fighting, preventing and theoretically interpreting epidemic processes. Recent large scale observations have resulted in major discoveries concerning the overall features of the spreading process in systems with highly mobile susceptible units, but virtually no data are available about observations of infection spreading for a very large number of immobile units. Here we present the first detailed quantitative documentation of percolation-type viral epidemics in a highly reproducible in vitro system consisting of tens of thousands of virtually motionless cells. We use a confluent astroglial monolayer in a Petri dish and induce productive infection in a limited number of cells with a genetically modified herpesvirus strain. This approach allows extreme high resolution tracking of the spatio-temporal development of the epidemic. We show that a simple model is capable of reproducing the basic features of our observations, i.e., the observed behaviour is likely to be applicable to many different kinds of systems. Statistical physics inspired approaches to our data, such as fractal dimension of the infected clusters as well as their size distribution, seem to fit into a percolation theory based interpretation. We suggest that our observations may be used to model epidemics in more complex systems, which are difficult to study in isolation.

  6. Advances in cell culture: anchorage dependence

    Science.gov (United States)

    Merten, Otto-Wilhelm

    2015-01-01

    Anchorage-dependent cells are of great interest for various biotechnological applications. (i) They represent a formidable production means of viruses for vaccination purposes at very large scales (in 1000–6000 l reactors) using microcarriers, and in the last decade many more novel viral vaccines have been developed using this production technology. (ii) With the advent of stem cells and their use/potential use in clinics for cell therapy and regenerative medicine purposes, the development of novel culture devices and technologies for adherent cells has accelerated greatly with a view to the large-scale expansion of these cells. Presently, the really scalable systems—microcarrier/microcarrier-clump cultures using stirred-tank reactors—for the expansion of stem cells are still in their infancy. Only laboratory scale reactors of maximally 2.5 l working volume have been evaluated because thorough knowledge and basic understanding of critical issues with respect to cell expansion while retaining pluripotency and differentiation potential, and the impact of the culture environment on stem cell fate, etc., are still lacking and require further studies. This article gives an overview on critical issues common to all cell culture systems for adherent cells as well as specifics for different types of stem cells in view of small- and large-scale cell expansion and production processes. PMID:25533097

  7. A fetal whole ovarian culture model for the evaluation of CrVI-induced developmental toxicity during germ cell nest breakdown

    International Nuclear Information System (INIS)

    Stanley, Jone A.; Arosh, Joe A.; Burghardt, Robert C.; Banu, Sakhila K.

    2015-01-01

    Prenatal exposure to endocrine disrupting chemicals (EDCs), including bisphenol A, dioxin, pesticides, and cigarette smoke, has been linked to several ovarian diseases such as premature ovarian failure (POF) and early menopause in women. Hexavalent chromium (CrVI), one of the more toxic heavy metals, is widely used in more than 50 industries. As one of the world's leading producers of Cr compounds, the U.S. is facing growing challenges in protecting human health against adverse effects of CrVI. Our recent findings demonstrated that in vivo CrVI exposure during gestational period caused POF in F1 offspring. Our current research focus is three-fold: (i) to identify the effect of CrVI on critical windows of great vulnerability of fetal ovarian development; (ii) to understand the molecular mechanism of CrVI-induced POF; (iii) to identify potential intervention strategies to mitigate or inhibit CrVI effects. In order to accomplish these goals we used a fetal whole ovarian culture system. Fetuses were removed from the normal pregnant rats on gestational day 13.5. Fetal ovaries were cultured in vitro for 12 days, and treated with or without 0.1 ppm potassium dichromate (CrVI) from culture day 2–8, which recapitulated embryonic day 14.5–20.5, in vivo. Results showed that CrVI increased germ cell/oocyte apoptosis by increasing caspase 3, BAX, p53 and PUMA; decreasing BCL2, BMP15, GDF9 and cKIT; and altering cell cycle regulatory genes and proteins. This model system may serve as a potential tool for high throughput testing of various drugs and/or EDCs in particular to assess developmental toxicity of the ovary. - Highlights: • CrVI (0.1 ppm, a regulatory dose) increased germ cell apoptosis of fetal ovaries. • CrVI (0.1 ppm) increased pro-apoptotic proteins. • CrVI (0.1 ppm) decreased cyclins and CDK1 and cell survival proteins. • CrVI (0.1 ppm) increased oxidative stress during fetal ovarian development. • We propose fetal ovarian culture model for high

  8. A fetal whole ovarian culture model for the evaluation of CrVI-induced developmental toxicity during germ cell nest breakdown

    Energy Technology Data Exchange (ETDEWEB)

    Stanley, Jone A.; Arosh, Joe A.; Burghardt, Robert C.; Banu, Sakhila K., E-mail: skbanu@cvm.tamu.edu

    2015-11-15

    Prenatal exposure to endocrine disrupting chemicals (EDCs), including bisphenol A, dioxin, pesticides, and cigarette smoke, has been linked to several ovarian diseases such as premature ovarian failure (POF) and early menopause in women. Hexavalent chromium (CrVI), one of the more toxic heavy metals, is widely used in more than 50 industries. As one of the world's leading producers of Cr compounds, the U.S. is facing growing challenges in protecting human health against adverse effects of CrVI. Our recent findings demonstrated that in vivo CrVI exposure during gestational period caused POF in F1 offspring. Our current research focus is three-fold: (i) to identify the effect of CrVI on critical windows of great vulnerability of fetal ovarian development; (ii) to understand the molecular mechanism of CrVI-induced POF; (iii) to identify potential intervention strategies to mitigate or inhibit CrVI effects. In order to accomplish these goals we used a fetal whole ovarian culture system. Fetuses were removed from the normal pregnant rats on gestational day 13.5. Fetal ovaries were cultured in vitro for 12 days, and treated with or without 0.1 ppm potassium dichromate (CrVI) from culture day 2–8, which recapitulated embryonic day 14.5–20.5, in vivo. Results showed that CrVI increased germ cell/oocyte apoptosis by increasing caspase 3, BAX, p53 and PUMA; decreasing BCL2, BMP15, GDF9 and cKIT; and altering cell cycle regulatory genes and proteins. This model system may serve as a potential tool for high throughput testing of various drugs and/or EDCs in particular to assess developmental toxicity of the ovary. - Highlights: • CrVI (0.1 ppm, a regulatory dose) increased germ cell apoptosis of fetal ovaries. • CrVI (0.1 ppm) increased pro-apoptotic proteins. • CrVI (0.1 ppm) decreased cyclins and CDK1 and cell survival proteins. • CrVI (0.1 ppm) increased oxidative stress during fetal ovarian development. • We propose fetal ovarian culture model for high

  9. [Safety culture: definition, models and design].

    Science.gov (United States)

    Pfaff, Holger; Hammer, Antje; Ernstmann, Nicole; Kowalski, Christoph; Ommen, Oliver

    2009-01-01

    Safety culture is a multi-dimensional phenomenon. Safety culture of a healthcare organization is high if it has a common stock in knowledge, values and symbols in regard to patients' safety. The article intends to define safety culture in the first step and, in the second step, demonstrate the effects of safety culture. We present the model of safety behaviour and show how safety culture can affect behaviour and produce safe behaviour. In the third step we will look at the causes of safety culture and present the safety-culture-model. The main hypothesis of this model is that the safety culture of a healthcare organization strongly depends on its communication culture and its social capital. Finally, we will investigate how the safety culture of a healthcare organization can be improved. Based on the safety culture model six measures to improve safety culture will be presented.

  10. Advances in 3D neuronal cell culture

    NARCIS (Netherlands)

    Frimat, Jean Philippe; Xie, Sijia; Bastiaens, Alex; Schurink, Bart; Wolbers, Floor; Den Toonder, Jaap; Luttge, Regina

    2015-01-01

    In this contribution, the authors present our advances in three-dimensional (3D) neuronal cell culture platform technology contributing to controlled environments for microtissue engineering and analysis of cellular physiological and pathological responses. First, a micromachined silicon sieving

  11. The use of real-time cell analyzer technology in drug discovery: defining optimal cell culture conditions and assay reproducibility with different adherent cellular models.

    Science.gov (United States)

    Atienzar, Franck A; Tilmant, Karen; Gerets, Helga H; Toussaint, Gaelle; Speeckaert, Sebastien; Hanon, Etienne; Depelchin, Olympe; Dhalluin, Stephane

    2011-07-01

    The use of impedance-based label-free technology applied to drug discovery is nowadays receiving more and more attention. Indeed, such a simple and noninvasive assay that interferes minimally with cell morphology and function allows one to perform kinetic measurements and to obtain information on proliferation, migration, cytotoxicity, and receptor-mediated signaling. The objective of the study was to further assess the usefulness of a real-time cell analyzer (RTCA) platform based on impedance in the context of quality control and data reproducibility. The data indicate that this technology is useful to determine the best coating and cellular density conditions for different adherent cellular models including hepatocytes, cardiomyocytes, fibroblasts, and hybrid neuroblastoma/neuronal cells. Based on 31 independent experiments, the reproducibility of cell index data generated from HepG2 cells exposed to DMSO and to Triton X-100 was satisfactory, with a coefficient of variation close to 10%. Cell index data were also well reproduced when cardiomyocytes and fibroblasts were exposed to 21 compounds three times (correlation >0.91, p technology appears to be a powerful and reliable tool in drug discovery because of the reasonable throughput, rapid and efficient performance, technical optimization, and cell quality control.

  12. Surface modified alginate microcapsules for 3D cell culture

    Science.gov (United States)

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

    2016-06-01

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

  13. Integrated Safety Culture Model and Application

    Institute of Scientific and Technical Information of China (English)

    汪磊; 孙瑞山; 刘汉辉

    2009-01-01

    A new safety culture model is constructed and is applied to analyze the correlations between safety culture and SMS. On the basis of previous typical definitions, models and theories of safety culture, an in-depth analysis on safety culture's structure, composing elements and their correlations was conducted. A new definition of safety culture was proposed from the perspective of sub-cuhure. 7 types of safety sub-culture, which are safety priority culture, standardizing culture, flexible culture, learning culture, teamwork culture, reporting culture and justice culture were defined later. Then integrated safety culture model (ISCM) was put forward based on the definition. The model divided safety culture into intrinsic latency level and extrinsic indication level and explained the potential relationship between safety sub-culture and all safety culture dimensions. Finally in the analyzing of safety culture and SMS, it concluded that positive safety culture is the basis of im-plementing SMS effectively and an advanced SMS will improve safety culture from all around.

  14. Culture of Mouse Neural Stem Cell Precursors

    OpenAIRE

    Currle, D. Spencer; Hu, Jia Sheng; Kolski-Andreaco, Aaron; Monuki, Edwin S.

    2007-01-01

    Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the...

  15. Sponge cell culture? A molecular identification method for sponge cells

    NARCIS (Netherlands)

    Sipkema, D.; Heilig, G.H.J.; Akkermans, A.D.L.; Osinga, R.; Tramper, J.; Wijffels, R.H.

    2003-01-01

    Dissociated sponge cells are easily confused with unicellular organisms. This has been an obstacle in the development of sponge-cell lines. We developed a molecular detection method to identify cells of the sponge Dysidea avara in dissociated cell cultures. The 18S ribosomal RNA gene from a Dysidea

  16. miRNA-431 Prevents Amyloid-β-Induced Synapse Loss in Neuronal Cell Culture Model of Alzheimer's Disease by Silencing Kremen1.

    Science.gov (United States)

    Ross, Sean P; Baker, Kelly E; Fisher, Amanda; Hoff, Lee; Pak, Elena S; Murashov, Alexander K

    2018-01-01

    Synapse loss is well regarded as the underlying cause for the progressive decline of memory function over the course of Alzheimer's disease (AD) development. Recent observations suggest that the accumulation of the Wnt antagonist Dickkopf-1 (Dkk1) in the AD brain plays a critical role in triggering synaptic degeneration. Mechanistically, Dkk1 cooperates with Kremen1 (Krm1), its transmembrane receptor, to block the Wnt/β-catenin signaling pathway. Here, we show that silencing Krm1 with miR-431 prevents amyloid-β-mediated synapse loss in cortico-hippocampal cultures isolated from triple transgenic 3xTg-AD mice. Exposure to AβDDL (an amyloid-β derived diffusive ligand) or Dkk1 reduced the number of pre- and post-synaptic puncta in primary neuronal cultures, while treatment with miR-431 prevented synapse loss. In addition, treatment with miR-431 also prevented neurite degeneration. Our findings demonstrate that miR-431 protects synapses and neurites from Aβ-toxicity in an AD cell culture model and may be a promising therapeutic target.

  17. 8-prenylnaringenin and tamoxifen inhibit the shedding of irradiated epithelial cells and increase the latency period of radiation-induced oral mucositis. Cell culture and murine model

    Energy Technology Data Exchange (ETDEWEB)

    Ryck, Tine de; Impe, Annouchka van; Bracke, Marc E. [Ghent University, Laboratory of Experimental Cancer Research, Department Radiation Oncology and Experimental Cancer Research, Ghent (Belgium); Vanhoecke, Barbara W. [Ghent University, Laboratory of Experimental Cancer Research, Department Radiation Oncology and Experimental Cancer Research, Ghent (Belgium); Ghent University, Laboratory of Microbial Ecology and Technology (LabMET), Ghent (Belgium); Heyerick, Arne [Ghent University, Laboratory of Pharmacognosy and Phytochemistry, Ghent (Belgium); Vakaet, Luc; Neve, Wilfried de [Ghent University Hospital, Department of Radiation Oncology, Ghent (Belgium); Mueller, Doreen [Medical Faculty and University Hospital Carl Gustav Carus, Technische Universitaet Dresden, Department of Radiotherapy and Radiation Oncology, OncoRay-National Center for Radiation Research in Oncology, Dresden (Germany); Schmidt, Margret [Medical Faculty and University Hospital Carl Gustav Carus, Technische Universitaet Dresden, Department of Radiotherapy and Radiation Oncology, OncoRay-National Center for Radiation Research in Oncology, Dresden (Germany); German Cancer Consortium (DKTK) partner site Dresden and German Cancer Center (DKFZ), Heidelberg (Germany); Doerr, Wolfgang [Medical Faculty and University Hospital Carl Gustav Carus, Technische Universitaet Dresden, Department of Radiotherapy and Radiation Oncology, OncoRay-National Center for Radiation Research in Oncology, Dresden (Germany); Medical University, Department of Radiation Oncology, CCC, and CD-Laboratory RadOnc, Vienna (Austria)

    2015-05-01

    The major component in the pathogenesis of oral radiation-induced mucositis is progressive epithelial hypoplasia and eventual ulceration. Irradiation inhibits cell proliferation, while cell loss at the surface continues. We conceived to slow down this desquamation by increasing intercellular adhesion, regulated by the E-cadherin/catenin complex. We investigated if 8-prenylnaringenin (8-PN) or tamoxifen (TAM) decrease the shedding of irradiated human buccal epithelial cells in vitro and thus delay the ulcerative phase of radiation-induced mucositis in vivo. In vitro, aggregates of buccal epithelial cells were irradiated and cultured in suspension for 11 days. 8-PN or TAM were investigated regarding their effect on cell shedding. In vivo, the lower tongue surface of mice was irradiated with graded single doses of 25 kV X-rays. The incidence, latency, and duration of the resulting mucosal ulcerations were analyzed after topical treatment with 8-PN, TAM or solvent. 8-PN or TAM prevented the volume reduction of the irradiated cell aggregates during the incubation period. This was the result of a higher residual cell number in the treated versus the untreated irradiated aggregates. In vivo, topical treatment with 8-PN or TAM significantly increased the latency of mucositis from 10.9 to 12.1 and 12.4 days respectively, while the ulcer incidence was unchanged. 8-PN and TAM prevent volume reduction of irradiated cell aggregates in suspension culture. In the tongues of mice, these compounds increase the latency period. This suggests a role for these compounds for the amelioration of radiation-induced mucositis in the treatment of head and neck tumors. (orig.) [German] Die wesentliche Komponente in der Pathogenese der radiogenen Mukositis ist eine progressive epitheliale Hypoplasie und letztendlich Ulzeration. Die Bestrahlung hemmt die Zellproliferation, waehrend der Zellverlust an der Oberflaeche fortbesteht. Wir versuchten, diese Desquamation durch eine Stimulation der

  18. A Single Amino Acid Change in the Marburg Virus Glycoprotein Arises during Serial Cell Culture Passages and Attenuates the Virus in a Macaque Model of Disease.

    Science.gov (United States)

    Alfson, Kendra J; Avena, Laura E; Delgado, Jenny; Beadles, Michael W; Patterson, Jean L; Carrion, Ricardo; Griffiths, Anthony

    2018-01-01

    Marburg virus (MARV) causes disease with high case fatality rates, and there are no approved vaccines or therapies. Licensing of MARV countermeasures will likely require approval via the FDA's Animal Efficacy Rule, which requires well-characterized animal models that recapitulate human disease. This includes selection of the virus used for exposure and ensuring that it retains the properties of the original isolate. The consequences of amplification of MARV for challenge studies are unknown. Here, we serially passaged and characterized MARV through 13 passes from the original isolate. Surprisingly, the viral genome was very stable, except for a single nucleotide change that resulted in an amino acid substitution in the hydrophobic region of the signal peptide of the glycoprotein (GP). The particle/PFU ratio also decreased following passages, suggesting a role for the amino acid in viral infectivity. To determine if amplification introduces a phenotype in an animal model, cynomolgus macaques were exposed to either 100 or 0.01 PFU of low- and high-passage-number MARV. All animals succumbed when exposed to 100 PFU of either passage 3 or 13 viruses, although animals exposed to the high-passage-number virus survived longer. However, none of the passage 13 MARV-exposed animals succumbed to 0.01-PFU exposure compared to 75% of passage 3-exposed animals. This is consistent with other filovirus studies that show some particles that are unable to yield a plaque in cell culture can cause lethal disease in vivo . These results have important consequences for the design of experiments that investigate MARV pathogenesis and that test the efficacy of MARV countermeasures. IMPORTANCE Marburg virus (MARV) causes disease with a high case fatality rate, and there are no approved vaccines or therapies. Serial amplification of viruses in cell culture often results in accumulation of mutations, but the effect of such cell culture passage on MARV is unclear. Serial passages of MARV

  19. A novel three-dimensional cell culture method enhances antiviral drug screening in primary human cells.

    Science.gov (United States)

    Koban, Robert; Neumann, Markus; Daugs, Aila; Bloch, Oliver; Nitsche, Andreas; Langhammer, Stefan; Ellerbrok, Heinz

    2018-02-01

    Gefitinib is a specific inhibitor of the epidermal growth factor receptor (EGFR) and FDA approved for treatment of non-small cell lung cancer. In a previous study we could show the in vitro efficacy of gefitinib for treatment of poxvirus infections in monolayer (2D) cultivated cell lines. Permanent cell lines and 2D cultures, however, are known to be rather unphysiological; therefore it is difficult to predict whether determined effective concentrations or the drug efficacy per se are transferable to the in vivo situation. 3D cell cultures, which meanwhile are widely distributed across all fields of research, are a promising tool for more predictive in vitro investigations of antiviral compounds. In this study the spreading of cowpox virus and the antiviral efficacy of gefitinib were analyzed in primary human keratinocytes (NHEK) grown in a novel 3D extracellular matrix-based cell culture model and compared to the respective monolayer culture. 3D-cultivated NHEK grew in a polarized and thus a more physiological manner with altered morphology and close cell-cell contact. Infected cultures showed a strongly elevated sensitivity towards gefitinib. EGFR phosphorylation, cell proliferation, and virus replication were significantly reduced in 3D cultures at gefitinib concentrations which were at least 100-fold lower than those in monolayer cultures and well below the level of cytotoxicity. Our newly established 3D cell culture model with primary human cells is an easy-to-handle alternative to conventional monolayer cell cultures and previously described more complex 3D cell culture systems. It can easily be adapted to other cell types and a broad spectrum of viruses for antiviral drug screening and many other aspects of virus research under more in vivo-like conditions. In consequence, it may contribute to a more targeted realization of necessary in vivo experiments. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Characterization of A Three-Dimensional Organotypic Co-Culture Skin Model for Epidermal Differentiation of Rat Adipose-Derived Stem Cells.

    Science.gov (United States)

    Ghanavati, Zeinab; Orazizadeh, Mahmoud; Bayati, Vahid; Abbaspour, Mohammad Reza; Khorsandi, Layasadat; Mansouri, Esrafil; Neisi, Niloofar

    2016-01-01

    The organotypic co-culture is a well-known technique to examine cellular interactions and their roles in stem cell proliferation and differentiation. This study aims to evaluate the effects of dermal fibroblasts (DFs) on epidermal differentiation of adipose-derived stem cells (ASCs) using a three-dimensional (3D) organotypic co- culture technique. In this experimental research study, rat DFs and ASCs were isolated and cultured separately on electrospun polycaprolactone (PCL) matrices. The PCL matrices seeded by ASCs were superimposed on to the matrices seeded by DFs in order to create a 3D organotypic co-culture. In the control groups, PCL matrices seeded by ASCs were placed on matrices devoid of DFs. After 10 days, we assessed the expressions of keratinocyte-related genes by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and expression of pan-cytokeratin protein by immunofluorescence in the differentiated keratinocyte-like cells from co- culture and control groups. Keratinocyte-like cell morphologies were also observed by scanning electron microscopy (SEM). The early, intermediate, and terminal differentiation keratinocyte markers-Cytokeratin14, Filaggrin, and Involucrin significantly expressed in the co-culture groups com- pared to the control ones (P<0.05). We observed pan-cytokeratin in keratinocyte-like cells of both groups by immunofluorescence. SEM observation of the co-culture groups showed that the differentiated keratinocyte-like cells developed a polygonal cobblestone shape, considered characteristic of keratinocytes. The 3D organotypic co-culture bilayered construct that consisted of DFs and ASCs was an effective technique for epidermal differentiation of ASCs. This co-culture might be useful for epidermal differentiation of stem cells for future applications in skin regeneration.

  1. Melphalan metabolism in cultured cells

    International Nuclear Information System (INIS)

    Seagrave, J.C.; Valdez, J.G.; Tobey, R.A.; Gurley, L.R.

    1985-06-01

    Procedures are presented for the adaptation of reversed-phase-HPLC methods to accomplish separation and isolation of the cancer therapeutic drug melphalan (L-phenylalanine mustard) and its metabolic products from whole cells. Five major degradation products of melphalan were observed following its hydrolysis in phosphate buffer in vitro. The two most polar of these products (or modifications of them) were also found in the cytosol of Chinese hamster CHO cells. The amounts of these two polar products (shown not to be mono- or dihydroxymelphalan) were significantly changed by the pretreatment of cells with ZnC1 2 , one being increased in amount while the other was reduced to an insignificant level. In ZnC1 2 -treated cells, there was also an increased binding of melphalan (or its derivatives) to one protein fraction resolved by gel filtration-HPLC. These observations suggest that changes in polar melphalan products, and perhaps their interaction with a protein, may by involved in the reduction of melphalan cytotoxicity observed in ZnC1 2 -treated cells. While ZnC1 2 is also known to increase the level of glutathione in cells, no significant amounts of glutathione-melphalan derivatives of the type formed non-enzymatically in vitro could be detected in ZnC1 2 -treated or untreated cells. Formation of derivatives of melphalan with glutathione catabolic products in ZnC1 2 -treated cells has not yet been eliminated, however. 17 refs., 5 figs., 1 tab

  2. Flux analysis of mammalian cell culture

    NARCIS (Netherlands)

    Martens, D.E.; Tramper, J.

    2010-01-01

    Animal cells are used for the production of vaccines and pharmaceutical proteins. The increase in demand for these products requires an increase in volumetric productivity of animal cell culture processes, which can be attained through an increase in biomass concentration and/or specific

  3. Quantitative volumetric Raman imaging of three dimensional cell cultures

    KAUST Repository

    Kallepitis, Charalambos; Bergholt, Mads S.; Mazo, Manuel M.; Leonardo, Vincent; Skaalure, Stacey C.; Maynard, Stephanie A.; Stevens, Molly M.

    2017-01-01

    in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in cell shape, cytoplasm, nucleus, lipid bodies

  4. Mechanism for multiplicity of steady states with distinct cell concentration in continuous culture of mammalian cells.

    Science.gov (United States)

    Yongky, Andrew; Lee, Jongchan; Le, Tung; Mulukutla, Bhanu Chandra; Daoutidis, Prodromos; Hu, Wei-Shou

    2015-07-01

    Continuous culture for the production of biopharmaceutical proteins offers the possibility of steady state operations and thus more consistent product quality and increased productivity. Under some conditions, multiplicity of steady states has been observed in continuous cultures of mammalian cells, wherein with the same dilution rate and feed nutrient composition, steady states with very different cell and product concentrations may be reached. At those different steady states, cells may exhibit a high glycolysis flux with high lactate production and low cell concentration, or a low glycolysis flux with low lactate and high cell concentration. These different steady states, with different cell concentration, also have different productivity. Developing a mechanistic understanding of the occurrence of steady state multiplicity and devising a strategy to steer the culture toward the desired steady state is critical. We establish a multi-scale kinetic model that integrates a mechanistic intracellular metabolic model and cell growth model in a continuous bioreactor. We show that steady state multiplicity exists in a range of dilution rate in continuous culture as a result of the bistable behavior in glycolysis. The insights from the model were used to devise strategies to guide the culture to the desired steady state in the multiple steady state region. The model provides a guideline principle in the design of continuous culture processes of mammalian cells. © 2015 Wiley Periodicals, Inc.

  5. Systems Biology for Organotypic Cell Cultures

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-08-04

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

  6. Dopaminergic neurotoxicant 6-OHDA induces oxidative damage through proteolytic activation of PKCδ in cell culture and animal models of Parkinson's disease

    International Nuclear Information System (INIS)

    Latchoumycandane, Calivarathan; Anantharam, Vellareddy; Jin, Huajun; Kanthasamy, Anumantha; Kanthasamy, Arthi

    2011-01-01

    The neurotoxicant 6-hydroxydopamine (6-OHDA) is used to investigate the cellular and molecular mechanisms underlying selective degeneration of dopaminergic neurons in Parkinson's disease (PD). Oxidative stress and caspase activation contribute to the 6-OHDA-induced apoptotic cell death of dopaminergic neurons. In the present study, we sought to systematically characterize the key downstream signaling molecule involved in 6-OHDA-induced dopaminergic degeneration in cell culture and animal models of PD. Treatment of mesencephalic dopaminergic neuronal N27 cells with 6-OHDA (100 μM) for 24 h significantly reduced mitochondrial activity and increased cytosolic cytochrome c, followed by sequential activation of caspase-9 and caspase-3. Co-treatment with the free radical scavenger MnTBAP (10 μM) significantly attenuated 6-OHDA-induced caspase activities. Interestingly, 6-OHDA induced proteolytic cleavage and activation of protein kinase C delta (PKCδ) was completely suppressed by treatment with a caspase-3-specific inhibitor, Z-DEVD-FMK (50 μM). Furthermore, expression of caspase-3 cleavage site-resistant mutant PKCδ D327A and kinase dead PKCδ K376R or siRNA-mediated knockdown of PKCδ protected against 6-OHDA-induced neuronal cell death, suggesting that caspase-3-dependent PKCδ promotes oxidative stress-induced dopaminergic degeneration. Suppression of PKCδ expression by siRNA also effectively protected N27 cells from 6-OHDA-induced apoptotic cell death. PKCδ cleavage was also observed in the substantia nigra of 6-OHDA-injected C57 black mice but not in control animals. Viral-mediated delivery of PKCδ D327A protein protected against 6-OHDA-induced PKCδ activation in mouse substantia nigra. Collectively, these results strongly suggest that proteolytic activation of PKCδ is a key downstream event in dopaminergic degeneration, and these results may have important translational value for development of novel treatment strategies for PD.

  7. Long-term culture and differentiation of porcine red bone marrow hematopoietic cells co-cultured with immortalized mesenchymal cells.

    Science.gov (United States)

    Garba, Abubakar; Acar, Delphine D; Roukaerts, Inge D M; Desmarets, Lowiese M B; Devriendt, Bert; Nauwynck, Hans J

    2017-09-01

    Mesenchymal cells are multipotent stromal cells with self-renewal, differentiation and immunomodulatory capabilities. We aimed to develop a co-culture model for differentiating hematopoietic cells on top of immortalized mesenchymal cells for studying interactions between hematopoietic and mesenchymal cells, useful for adequately exploring the therapeutic potential of mesenchymal cells. In this study, we investigated the survival, proliferation and differentiation of porcine red bone marrow hematopoietic cells co-cultured with immortalized porcine bone marrow mesenchymal cells for a period of five weeks. Directly after collection, primary porcine bone marrow mesenchymal cells adhered firmly to the bottom of the culture plates and showed a fibroblast-like appearance, one week after isolation. Upon immortalization, porcine bone marrow mesenchymal cells were continuously proliferating. They were positive for simian virus 40 (SV40) large T antigen and the mesenchymal cell markers CD44 and CD55. Isolated red bone marrow cells were added to these immortalized mesenchymal cells. Five weeks post-seeding, 92±6% of the red bone marrow hematopoietic cells were still alive and their number increased 3-fold during five weekly subpassages on top of the immortalized mesenchymal cells. The red bone marrow hematopoietic cells were originally small and round; later, the cells increased in size. Some of them became elongated, while others remained round. Tiny dendrites appeared attaching hematopoietic cells to the underlying immortalized mesenchymal cells. Furthermore, weekly differential-quick staining of the cells indicated the presence of monoblasts, monocytes, macrophages and lymphocytes in the co-cultures. At three weeks of co-culture, flow cytometry analysis showed an increased surface expression of CD172a, CD14, CD163, CD169, CD4 and CD8 up to 37±0.8%, 40±8%, 41±4%, 23±3% and 19±5% of the hematopoietic cells, respectively. In conclusion, continuous mesenchymal cell

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

    Science.gov (United States)

    Stampfer, Martha R; Garbe, James C

    2015-02-24

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

  9. Frankincense essential oil prepared from hydrodistillation of Boswellia sacra gum resins induces human pancreatic cancer cell death in cultures and in a xenograft murine model

    Science.gov (United States)

    2012-01-01

    Background Regardless of the availability of therapeutic options, the overall 5-year survival for patients diagnosed with pancreatic cancer remains less than 5%. Gum resins from Boswellia species, also known as frankincense, have been used as a major ingredient in Ayurvedic and Chinese medicine to treat a variety of health-related conditions. Both frankincense chemical extracts and essential oil prepared from Boswellia species gum resins exhibit anti-neoplastic activity, and have been investigated as potential anti-cancer agents. The goals of this study are to identify optimal condition for preparing frankincense essential oil that possesses potent anti-tumor activity, and to evaluate the activity in both cultured human pancreatic cancer cells and a xenograft mouse cancer model. Methods Boswellia sacra gum resins were hydrodistilled at 78°C; and essential oil distillate fractions were collected at different durations (Fraction I at 0–2 h, Fraction II at 8–10 h, and Fraction III at 11–12 h). Hydrodistillation of the second half of gum resins was performed at 100°C; and distillate was collected at 11–12 h (Fraction IV). Chemical compositions were identified by gas chromatography–mass spectrometry (GC-MS); and total boswellic acids contents were quantified by high-performance liquid chromatography (HPLC). Frankincense essential oil-modulated pancreatic tumor cell viability and cytotoxicity were determined by colorimetric assays. Levels of apoptotic markers, signaling molecules, and cell cycle regulators expression were characterized by Western blot analysis. A heterotopic (subcutaneous) human pancreatic cancer xenograft nude mouse model was used to evaluate anti-tumor capability of Fraction IV frankincense essential oil in vivo. Frankincense essential oil-induced tumor cytostatic and cytotoxic activities in animals were assessed by immunohistochemistry. Results Longer duration and higher temperature hydrodistillation produced more abundant high molecular

  10. 5-Fluorouracil-induced apoptosis in cultured oral cancer cells.

    Science.gov (United States)

    Tong, D; Poot, M; Hu, D; Oda, D

    2000-03-01

    Chemotherapy is commonly used to treat advanced oral squamous cell carcinoma (SCC) and is known to kill cancer cells through apoptosis. Our hypothesis states that 5-fluorouracil (5FU) also kills cultured oral epithelial cells through programmed cell death or apoptosis. Cultured oral cancer cells were exposed to an optimum dose of 20 mg/ml of 5FU. Cells were analyzed for changes in cell cycle distribution and induction of cell death including apoptosis. Normal control, human papilloma virus-immortalized (PP), ATCC SCC cell line (CA1) and two primary oral SCC cell lines (CA3 and -4) were studied. Inhibition of apoptosis by a pan-caspase inhibitor was used. SYTO 11 flow cytometry showed increased apoptosis in all 5FU-treated cell cultures compared to untreated controls. The results show biological variation in apoptotic response. CA1 had the lowest apoptotic rate of the cancer cell lines at 1.5%. Next lowest was CA3, followed by CA4 and PP. In addition, alteration in the G1 and S phase fractions were found. Untreated CA1 showed 28% G1, 53% S compared to 43% G1, and 40% S of treated. We investigated the pathway of apoptosis using the pan-caspase inhibitor IDN-1529 by methylthiazolyl diphenyl tetrazolium bromide (MTT) colorimetric analysis. Results showed mild inhibition of cell death when cells were incubated with 50 microM IDN-1529 for 24 h. This suggests a probable caspase-dependent apoptotic pathway. In conclusion, our data suggest that 5FU induces oral cancer cell death through apoptosis and that biological variation exists between normal and cancer cells and between different types of cancer cells themselves. Our data indicate that cultures of a useful in vitro model for chemosensitivity assays are possible. Our results also suggest a caspase-dependent pathway for chemocytotoxicity in oral SCC.

  11. Substrate utilisation by plant-cell cultures

    Energy Technology Data Exchange (ETDEWEB)

    Fowler, M W

    1982-01-01

    Plant cell cultures have been grown on a wide range of carbon sources in addition to the traditional ones of sucrose and glucose. Biomass yields and growth rates vary greatly between the different carbon sources and there is a variation in response between different cell cultures to individual carbon sources. Some attempts have been made to grow cell cultures on 'waste' and related carbon sources, such as lactose, maltose, starch, molasses and milk whey. Only maltose was found to support growth to anything near the levels observed with glucose and sucrose. In the case of molasses carbon source cell growth was either non-existent or only just measurable. All the data point to glucose as being the most suitable carbon source, principally on the grounds of biomass yield and growth rate. It should be noted, however, that other carbon sources do appear to have a major (positive) influence on natural product synthesis. Uptake into the cell is an important aspect of carbohydrate utilisation. There is strong evidence that from disaccharides upwards, major degradation to smaller units occurs before uptake. In some cases the necessary enzymes appear to be excreted into the culture broth, in others they may be located within the cell wall; invertase that hydrolyses sucrose is a good example. Once the products of carbohydrate degradation and mobilisation enter the cell they may suffer one of two fates, oxidation or utilisation for biosynthesis. The precise split between these two varies depending on such factors as cell growth rate, cell size, nutrient broth composition and carbohydrate status of the cells. In general rapidly growing cells have a high rate of oxidation, whereas cells growing more slowly tend to be more directed towards biosynthesis. Carbohydrate utilisation is a key area of study, underpinning as it does both biomass yield and natural product synthesis. (Refs. 13).

  12. Long-term culture of organotypic multicellular glioma spheroids: a good culture model for studying gliomas

    NARCIS (Netherlands)

    Kaaijk, P.; Troost, D.; Das, P. K.; Leenstra, S.; Bosch, D. A.

    1995-01-01

    Gliomas, as well as other solid tumours, contain tumour stroma composed of connective tissue, macrophages, capillaries and other non-cellular constituents. Therefore, a homogeneous culture of tumour cells alone, as is often used as a culture model for gliomas, is not ideal to study all aspects of

  13. Cell culture experiments planned for the space bioreactor

    Science.gov (United States)

    Morrison, Dennis R.; Cross, John H.

    1987-01-01

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

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

    NARCIS (Netherlands)

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

    2001-01-01

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

  15. Huh-7 cell line as an alternative cultural model for the production of human like erythropoietin (EPO

    Directory of Open Access Journals (Sweden)

    Kausar Humera

    2011-11-01

    Full Text Available Abstract Background and Aims Erythropoietin (EPO is a glycoprotein hormone which is required to regulate the production of red blood cells. Deficiency of EPO is known to cause anemia in chronically infected renal patients and they require regular blood transfusion. Availability of recombinant EPO has eliminated the need for blood transfusion and now it is extensively used for the treatment of anemia. Glycosylation of erythropoietin is essential for its secretion, stability, protein conformation and biological activity. However, maintenance of human like glycosylation pattern during manufacturing of EPO is a major challenge in biotechnology. Currently, Chinese hamster ovary (CHO cell line is used for the commercial production of erythropoietin but this cell line does not maintain glycosylation resembling human system. With the trend to eliminate non-human constituent from biopharmaceutical products, as a preliminary approach, we have investigated the potential of human hepatoma cell line (Huh-7 to produce recombinant EPO. Materials and methods Initially, the secretory signal and Kozak sequences was added before the EPO mature protein sequence using overlap extension PCR technique. PCR-amplified cDNA fragments of EPO was inserted into mammalian expression vector under the control of the cytomegalovirus (CMV promoter and transiently expressed in CHO and Huh-7 cell lines. After RT-PCR analysis, ELISA and Western blotting was performed to verify the immunochemical properties of secreted EPO. Results Addition of secretory signal and Kozak sequence facilitated the extra-cellular secretion and enhanced the expression of EPO protein. Significant expression (P Conclusion Huh-7 cell line has a great potential to produce glycosylated EPO, suggesting the use of this cell line to produce glycoproteins of the therapeutic importance resembling to the natural human system.

  16. 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

    International Nuclear Information System (INIS)

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

    1987-01-01

    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 [ 125 I]-labelled insulin (INS) and insulin-like growth factor 1(IGF-1) by BBCEC. After 2 hrs at 23 0 C, the specific binding of INS and IGF-1 was 1.6% and 13.6%, respectively. At 37 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 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

  17. Cultural relativism: maintenance of genomic imprints in pluripotent stem cell culture systems.

    Science.gov (United States)

    Greenberg, Maxim Vc; Bourc'his, Déborah

    2015-04-01

    Pluripotent stem cells (PSCs) in culture have become a widely used model for studying events occurring during mammalian development; they also present an exciting avenue for therapeutics. However, compared to their in vivo counterparts, cultured PSC derivatives have unique properties, and it is well established that their epigenome is sensitive to medium composition. Here we review the specific effects on genomic imprints in various PSC types and culture systems. Imprinted gene regulation is developmentally important, and imprinting defects have been associated with several human diseases. Therefore, imprint abnormalities in PSCs may have considerable consequences for downstream applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Characterization of primary human mammary epithelial cells isolated and propagated by conditional reprogrammed cell culture.

    Science.gov (United States)

    Jin, Liting; Qu, Ying; Gomez, Liliana J; Chung, Stacey; Han, Bingchen; Gao, Bowen; Yue, Yong; Gong, Yiping; Liu, Xuefeng; Amersi, Farin; Dang, Catherine; Giuliano, Armando E; Cui, Xiaojiang

    2018-02-20

    Conditional reprogramming methods allow for the inexhaustible in vitro proliferation of primary epithelial cells from human tissue specimens. This methodology has the potential to enhance the utility of primary cell culture as a model for mammary gland research. However, few studies have systematically characterized this method in generating in vitro normal human mammary epithelial cell models. We show that cells derived from fresh normal breast tissues can be propagated and exhibit heterogeneous morphologic features. The cultures are composed of CK18, desmoglein 3, and CK19-positive luminal cells and vimentin, p63, and CK14-positive myoepithelial cells, suggesting the maintenance of in vivo heterogeneity. In addition, the cultures contain subpopulations with different CD49f and EpCAM expression profiles. When grown in 3D conditions, cells self-organize into distinct structures that express either luminal or basal cell markers. Among these structures, CK8-positive cells enclosing a lumen are capable of differentiation into milk-producing cells in the presence of lactogenic stimulus. Furthermore, our short-term cultures retain the expression of ERα, as well as its ability to respond to estrogen stimulation. We have investigated conditionally reprogrammed normal epithelial cells in terms of cell type heterogeneity, cellular marker expression, and structural arrangement in two-dimensional (2D) and three-dimensional (3D) systems. The conditional reprogramming methodology allows generation of a heterogeneous culture from normal human mammary tissue in vitro . We believe that this cell culture model will provide a valuable tool to study mammary cell function and malignant transformation.

  19. Construction of human induced pluripotent stem cell-derived oriented bone matrix microstructure by using in vitro engineered anisotropic culture model.

    Science.gov (United States)

    Ozasa, Ryosuke; Matsugaki, Aira; Isobe, Yoshihiro; Saku, Taro; Yun, Hui-Suk; Nakano, Takayoshi

    2018-02-01

    Bone tissue has anisotropic microstructure based on collagen/biological apatite orientation, which plays essential roles in the mechanical and biological functions of bone. However, obtaining an appropriate anisotropic microstructure during the bone regeneration process remains a great challenging. A powerful strategy for the control of both differentiation and structural development of newly-formed bone is required in bone tissue engineering, in order to realize functional bone tissue regeneration. In this study, we developed a novel anisotropic culture model by combining human induced pluripotent stem cells (hiPSCs) and artificially-controlled oriented collagen scaffold. The oriented collagen scaffold allowed hiPSCs-derived osteoblast alignment and further construction of anisotropic bone matrix which mimics the bone tissue microstructure. To the best of our knowledge, this is the first report showing the construction of bone mimetic anisotropic bone matrix microstructure from hiPSCs. Moreover, we demonstrated for the first time that the hiPSCs-derived osteoblasts possess a high level of intact functionality to regulate cell alignment. © 2017 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 360-369, 2018. © 2017 The Authors Journal of Biomedical Materials Research Part A Published by Wiley Periodicals, Inc.

  20. Anaerobic Co-Culture of Mesenchymal Stem Cells and Anaerobic Pathogens - A New In Vitro Model System

    OpenAIRE

    Kriebel, Katja; Biedermann, Anne; Kreikemeyer, Bernd; Lang, Hermann

    2013-01-01

    BACKGROUND: Human mesenchymal stem cells (hMSCs) are multipotent by nature and are originally isolated from bone marrow. In light of a future application of hMSCs in the oral cavity, a body compartment with varying oxygen partial pressures and an omnipresence of different bacterial species i.e. periodontitis pathogens, we performed this study to gain information about the behavior of hMSC in an anaerobic system and the response in interaction with oral bacterial pathogens. METHODOLOGY/PRINCIP...

  1. Administration of Protein kinase D1 induce an immunomodulatory effect on lipopolysaccharide-induced intestinal inflammation in a co-culture model of intestinal epithelial Caco-2 cells and RAW 264.7 macrophage cells

    DEFF Research Database (Denmark)

    Nielsen, Ditte Søvsø Gundelund; Fredborg, Marlene; Andersen, Vibeke

    2017-01-01

    the effects of human PKD1 in relation to intestinal inflammation, using a co-culture model of intestinal epithelial Caco-2 cells and RAW264.7 macrophages. An inflammatory response was induced in the macrophages by lipopolysaccharide (LPS), upregulating the expression of tumour necrosis factor alpha (TNF......-α), interleukin- (IL-) 1β, and IL-6 besides increasing the secretion of TNF-α protein. The effect of administering PKD1 to Caco-2 was evaluated in relation to both amelioration of inflammation and the ability to suppress inflammation initiation. Administration of PKD1 (10–100 ng/ml) following induction...

  2. Safety Cultural Competency Modeling in Nuclear Organizations

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sa Kil; Oh, Yeon Ju; Luo, Meiling; Lee, Yong Hee [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2014-05-15

    The nuclear safety cultural competency model should be supplemented through a bottom-up approach such as behavioral event interview. The developed model, however, is meaningful for determining what should be dealt for enhancing safety cultural competency of nuclear organizations. The more details of the developing process, results, and applications will be introduced later. Organizational culture include safety culture in terms of its organizational characteristics.

  3. Organizational culture diagnosis - a new model

    Directory of Open Access Journals (Sweden)

    Ph. D. Ionuţ Constantin

    2010-05-01

    Full Text Available Organizational culture is a key source of competitive advantage. There is a demonstrated relation between organizational culture and organizational performance. This paper reviews previous research in the field and introduce a new model for understanding, diagnosing and changing organizational culture. The main advantage of the new model is based on regarding culture as the management and work practices that are either hindering or helping an organization's bottom line performance.

  4. Organizational culture diagnosis - a new model

    OpenAIRE

    Ph. D. Ionuţ Constantin; Ph. D. Doru Curteanu

    2010-01-01

    Organizational culture is a key source of competitive advantage. There is a demonstrated relation between organizational culture and organizational performance. This paper reviews previous research in the field and introduce a new model for understanding , diagnosing and changing organizational culture. The main advantage of the new model is based on regarding culture as the management and work practices that are either hindering or helping an organization's bottom line performance.

  5. Evaluation of the osteogenic differentiation of gingiva-derived stem cells grown on culture plates or in stem cell spheroids: Comparison of two- and three-dimensional cultures.

    Science.gov (United States)

    Lee, Sung-Il; Ko, Youngkyung; Park, Jun-Beom

    2017-09-01

    Three-dimensional cell culture systems provide a convenient in vitro model for the study of complex cell-cell and cell-matrix interactions in the absence of exogenous substrates. The current study aimed to evaluate the osteogenic differentiation potential of gingiva-derived stem cells cultured in two-dimensional or three-dimensional systems. To the best of our knowledge, the present study is the first to compare the growth of gingiva-derived stem cells in monolayer culture to a three-dimensional culture system with microwells. For three-dimensional culture, gingiva-derived stem cells were isolated and seeded into polydimethylsiloxane-based concave micromolds. Alkaline phosphatase activity and alizarin red S staining assays were then performed to evaluate osteogenesis and the degree of mineralization, respectively. Stem cell spheroids had a significantly increased level of alkaline phosphatase activity and mineralization compared with cells from the two-dimensional culture. In addition, an increase in mineralized deposits was observed with an increase in the loading cell number. The results of present study indicate that gingiva-derived stem cell spheroids exhibit an increased osteogenic potential compared with stem cells from two-dimensional culture. This highlights the potential of three-dimensional culture systems using gingiva-derived stem cells for regenerative medicine applications requiring stem cells with osteogenic potential.

  6. The Evolution of Polystyrene as a Cell Culture Material.

    Science.gov (United States)

    Lerman, Max J; Lembong, Josephine; Muramoto, Shin; Gillen, Greg; Fisher, John P

    2018-04-10

    Polystyrene (PS) has brought in vitro cell culture from its humble beginnings to the modern era, propelling dozens of research fields along the way. This review discusses the development of the material, fabrication, and treatment approaches to create the culture material. However, native PS surfaces poorly facilitate cell adhesion and growthin vitro. To overcome this, liquid surface deposition, energetic plasma activation, and emerging functionalization methods transform the surface chemistry. This review seeks to highlight the many potential applications of the first widely accepted polymer growth surface. Although the majority of in vitro research occurs on 2D surfaces, the importance of 3D culture models cannot be overlooked. Here the methods to transition PS to specialized 3D culture surfaces are also reviewed. Specifically, casting, electrospinning, 3D printing, and microcarrier approaches to shift PS to a 3D culture surface are highlighted. The breadth of applications of the material makes it impossible to highlight every use, but the aim remains to demonstrate the versatility and potential as both a general and custom cell culture surface. The review concludes with emerging scaffolding approaches and, based on the findings, presents our insights on the future steps for PS as a tissue culture platform.

  7. A transgenic rat hepatocyte - Kupffer cell co-culture model for evaluation of direct and macrophage-related effect of poly(amidoamine) dendrimers.

    Science.gov (United States)

    Jemnitz, Katalin; Bátai-Konczos, Attila; Szabó, Mónika; Ioja, Enikő; Kolacsek, Orsolya; Orbán, Tamás I; Török, György; Homolya, László; Kovács, Eszter; Jablonkai, István; Veres, Zsuzsa

    2017-02-01

    Increasing number of papers demonstrate that Kupffer cells (KCs) play a role in the development of drug induced liver injury (DILI). Furthermore, elevated intracellular Ca 2+ level of hepatocytes is considered as a common marker of DILI. Here we applied an in vitro model based on hepatocyte mono- and hepatocyte/KC co-cultures (H/KC) isolated from transgenic rats stably expressing the GCaMP2 fluorescent Ca 2+ sensor protein to investigate the effects of polycationic (G5), polyanionic (G4.5) and polyethylene-glycol coated neutral (G5 Peg) dendrimers known to accumulate in the liver, primarily in KCs. Following dendrimer exposure, hepatocyte homeostasis was measured by MTT cytotoxicity assay and by Ca 2+ imaging, while hepatocyte functions were studied by CYP2B1/2 inducibility, and bilirubin and taurocholate transport. G5 was significantly more cytotoxic than G4.5 for hepatocytes and induced Ca 2+ oscillation and sustained Ca 2+ signals at 1μM and10 μM, respectively both in hepatocytes and KCs. Dendrimer-induced Ca 2+ signals in hepatocytes were attenuated by macrophages. Activation of KCs by lipopolysaccharide and G5 decreased the inducibility of CYP2B1/2, which was restored by depleting the KCs with gadolinium-chloride and pentoxyphylline, suggesting a role of macrophages in the hindrance of CYP2B1/2 induction by G5 and lipopolysaccharide. In the H/KC, but not in the hepatocyte mono-culture, G5 reduced the canalicular efflux of bilirubin and stimulated the uptake and canalicular efflux of taurocholate. In conclusion, H/KC provides a good model for the prediction of hepatotoxic potential of drugs, especially of nanomaterials known to be trapped by macrophages, activation of which presumably contributes to DILI. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Transfection in Primary Cultured Neuronal Cells.

    Science.gov (United States)

    Marwick, Katie F M; Hardingham, Giles E

    2017-01-01

    Transfection allows the introduction of foreign nucleic acid into eukaryotic cells. It is an important tool in understanding the roles of NMDARs in neurons. Here, we describe using lipofection-mediated transfection to introduce cDNA encoding NMDAR subunits into postmitotic rodent primary cortical neurons maintained in culture.

  9. Cell Culture Microfluidic Biochips: Experimental Throughput Maximization

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  10. Plant Cell Culture Initiation: practical tips

    NARCIS (Netherlands)

    Hall, R.D.

    2001-01-01

    The use of cultured plant cells in either organized or unorganized form has increased vey considerably in the last 10-15 yr. Many new technologies have been developed and applications in both fundamental and applied research have led to the development of some powerful tools for improving our

  11. Cell culture from sponges: pluripotency and immortality

    NARCIS (Netherlands)

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

    2007-01-01

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

  12. Nanotechnology, Cell Culture and Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Kazutoshi Haraguchi

    2011-01-01

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

  13. Bridging the gap between cell culture and live tissue

    Directory of Open Access Journals (Sweden)

    Stefan Przyborski

    2017-11-01

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

  14. Frequency and magnitude of intermittent hypoxia modulate endothelial wound healing in a cell culture model of sleep apnea.

    Science.gov (United States)

    Campillo, Noelia; Falcones, Bryan; Montserrat, Josep M; Gozal, David; Obeso, Ana; Gallego-Martin, Teresa; Navajas, Daniel; Almendros, Isaac; Farré, Ramon

    2017-11-01

    Intermittent hypoxia (IH) has been implicated in the cardiovascular consequences of obstructive sleep apnea (OSA). However, the lack of suitable experimental systems has precluded assessment as to whether IH is detrimental, protective, or both for the endothelium. The aim of the work was to determine the effects of frequency and amplitude of IH oxygenation swings on aortic endothelial wound healing. Monolayers of human primary endothelial cells were wounded and subjected to constant oxygenation (1%, 4%, 13%, or 20% O 2 ) or IH at different frequencies (0.6, 6, or 60 cycles/h) and magnitude ranges (13-4% O 2 or 20-1% O 2 ), using a novel well-controlled system, with wound healing being measured after 24 h. Cell monolayer repair was similar at 20% O 2 and 13% O 2 , but was considerably increased (approximately twofold) in constant hypoxia at 4% O 2 The magnitude and frequency of IH considerably modulated wound healing. Cycles ranging 13-4% O 2 at the lowest frequency (0.6 cycles/h) accelerated endothelial wound healing by 102%. However, for IH exposures consisting of 20% to 1% O 2 oscillations, wound closure was reduced compared with oscillation in the 13-4% range (by 74% and 44% at 6 cycles/h and 0.6 cycles/h, respectively). High-frequency IH patterns simulating severe OSA (60 cycles/h) did not significantly modify endothelial wound closure, regardless of the oxygenation cycle amplitude. In conclusion, the frequency and magnitude of hypoxia cycling in IH markedly alter wound healing responses and emerge as key factors determining how cells will respond in OSA. NEW & NOTEWORTHY Intermittent hypoxia (IH) induces cardiovascular consequences in obstructive sleep apnea (OSA) patients. However, the vast array of frequencies and severities of IH previously employed in OSA-related experimental studies has led to controversial results on the effects of IH. By employing an optimized IH experimental system here, we provide evidence that the frequency and magnitude of IH

  15. Dopaminergic neurotoxicant 6-OHDA induces oxidative damage through proteolytic activation of PKC{delta} in cell culture and animal models of Parkinson's disease

    Energy Technology Data Exchange (ETDEWEB)

    Latchoumycandane, Calivarathan; Anantharam, Vellareddy; Jin, Huajun; Kanthasamy, Anumantha; Kanthasamy, Arthi, E-mail: arthik@iastate.edu

    2011-11-15

    The neurotoxicant 6-hydroxydopamine (6-OHDA) is used to investigate the cellular and molecular mechanisms underlying selective degeneration of dopaminergic neurons in Parkinson's disease (PD). Oxidative stress and caspase activation contribute to the 6-OHDA-induced apoptotic cell death of dopaminergic neurons. In the present study, we sought to systematically characterize the key downstream signaling molecule involved in 6-OHDA-induced dopaminergic degeneration in cell culture and animal models of PD. Treatment of mesencephalic dopaminergic neuronal N27 cells with 6-OHDA (100 {mu}M) for 24 h significantly reduced mitochondrial activity and increased cytosolic cytochrome c, followed by sequential activation of caspase-9 and caspase-3. Co-treatment with the free radical scavenger MnTBAP (10 {mu}M) significantly attenuated 6-OHDA-induced caspase activities. Interestingly, 6-OHDA induced proteolytic cleavage and activation of protein kinase C delta (PKC{delta}) was completely suppressed by treatment with a caspase-3-specific inhibitor, Z-DEVD-FMK (50 {mu}M). Furthermore, expression of caspase-3 cleavage site-resistant mutant PKC{delta}{sup D327A} and kinase dead PKC{delta}{sup K376R} or siRNA-mediated knockdown of PKC{delta} protected against 6-OHDA-induced neuronal cell death, suggesting that caspase-3-dependent PKC{delta} promotes oxidative stress-induced dopaminergic degeneration. Suppression of PKC{delta} expression by siRNA also effectively protected N27 cells from 6-OHDA-induced apoptotic cell death. PKC{delta} cleavage was also observed in the substantia nigra of 6-OHDA-injected C57 black mice but not in control animals. Viral-mediated delivery of PKC{delta}{sup D327A} protein protected against 6-OHDA-induced PKC{delta} activation in mouse substantia nigra. Collectively, these results strongly suggest that proteolytic activation of PKC{delta} is a key downstream event in dopaminergic degeneration, and these results may have important translational value for

  16. Characterization of the inflammatory phenotype of Mycobacterium avium subspecies paratuberculosis using a novel cell culture passage model

    Science.gov (United States)

    Understanding the pathogenic mechanisms and host responses to Johne’s disease, a chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP), is complicated by the multifaceted disease progression, late-onset host reaction, and the lack of ex vivo infection models ...

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

    Science.gov (United States)

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

    1998-01-01

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

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

    DEFF Research Database (Denmark)

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

    2006-01-01

    culture chip compared to cell culture flasks. The cell culture chip could without further modification support cell growth of two other cell lines. Light coming from the microscope lamp during optical recordings of the cells was the only external factor identified, that could have a negative effect...... on cell survival. Low grade light exposure was however compatible with optical recordings as well as cell viability. These results strongly indicate that a cell culture chip could be constructed that allowed for on-line optical recording of cellular events without affecting the cell culturing condition...

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

    DEFF Research Database (Denmark)

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

    2006-01-01

    culture chip compared to cell culture flasks. The cell culture chip could without further modification support cell growth of two other cell lines. Light coming from the microscope lamp during optical recordings of the cells was the only external factor identified, that could have a negative effect...... on cell survival. Low grade light exposure was however compatible with optical recordings as well as cell viability. These results strongly indicate that a cell culture chip could be constructed that allowed for on-line optical recording of cellular events without affecting the cell culturing condition...

  20. Lipoprotein receptors in cultured bovine endothelial cells

    International Nuclear Information System (INIS)

    Struempfer, A.E.M.

    1983-07-01

    In this study, receptors that may be involved in the uptake of low density lipoproteins (LDL) and low density lipoproteins which have been modified by acetylation (AcLDL), were characterized. Aortic epithelial cells were used and a cell culture system which closely resembled the in vivo monolayer was established. Endothelial cell and lipoprotein interactions were examined by incubating the cells with 125 l-labelled lipoproteins under various conditions. The receptor affinity of bovine aortic endothelial cells was higher for AcLDL than that for LDL. Competition studies demonstrated that there were two distinct receptors for LDL and AcLDL on the endothelial cells. AcLDL did not compete with LDL for the LDL receptor, and conversely LDL did not compete with AcLDL for the AcLDL receptor. The receptor activities for LDL and AcLDL were examined as a function of culture age. Whereas the LDL receptor could be regulated, the AcLDL receptor was not as susceptible to regulation. Upon exposing endothelial cells for 72 h to either LDL or AcLDL, it was found that the total amount of cellular cholesterol increased by about 50%. However, the increase of total cholesterol was largely in the form of free cholesterol. This is in contrast to macrophages, where the increase in total cholesterol upon exposure to AcLDL is largely in the form cholesteryl esters

  1. A Spore Counting Method and Cell Culture Model for Chlorine Disinfection Studies of Encephalitozoon syn. Septata intestinalis

    OpenAIRE

    Wolk, D. M.; Johnson, C. H.; Rice, E. W.; Marshall, M. M.; Grahn, K. F.; Plummer, C. B.; Sterling, C. R.

    2000-01-01

    The microsporidia have recently been recognized as a group of pathogens that have potential for waterborne transmission; however, little is known about the effects of routine disinfection on microsporidian spore viability. In this study, in vitro growth of Encephalitozoon syn. Septata intestinalis, a microsporidium found in the human gut, was used as a model to assess the effect of chlorine on the infectivity and viability of microsporidian spores. Spore inoculum concentrations were determine...

  2. A comparison of the embryonic stem cell test and whole embryo culture assay combined with the BeWo placental passage model for predicting the embryotoxicity of azoles.

    NARCIS (Netherlands)

    Dimopoulou, Myrto; Verhoef, Aart; Gomes, Caroline A; van Dongen, Catharina W; Rietjens, Ivonne M C M; Piersma, Aldert H; van Ravenzwaay, Bennard

    2018-01-01

    In the present study, we show the value of combining toxico-dynamic and -kinetic in vitro approaches for embryotoxicity testing of azoles. Both the whole embryo culture (WEC) and the embryonic stem cells test (EST) predicted the in vivo potency ranking of twelve tested azoles with moderate accuracy.

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

    Science.gov (United States)

    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...

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

    Science.gov (United States)

    Cogoli, Augusto

    1992-01-01

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

  5. 21 CFR 864.2280 - Cultured animal and human cells.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cultured animal and human cells. 864.2280 Section... Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro cultivated cell lines from the tissue of humans or other animals which are used in various diagnostic...

  6. Modeling culture in intelligent virtual agents

    OpenAIRE

    Mascarenhas, S.; Degens, N.; Paiva, A.; Prada, R.; Hofstede, G.J.; Beulens, A.J.M.; Aylett, R.

    2016-01-01

    This work addresses the challenge of creating virtual agents that are able to portray culturally appropriate behavior when interacting with other agents or humans. Because culture influences how people perceive their social reality it is important to have agent models that explicitly consider social elements, such as existing relational factors. We addressed this necessity by integrating culture into a novel model for simulating human social behavior. With this model, we operationalized a par...

  7. Evaluation of the anti-inflammatory effects of steroids and arthritis-related biotherapies in an in vitro co-culture model with immune cells and synoviocytes

    Directory of Open Access Journals (Sweden)

    Melissa Noack

    2016-11-01

    Full Text Available Background: During rheumatoid arthritis (RA, steroids and biotherapies are used alone and combined. Efficacy has been established in clinical trials but their differential effects at the cellular level are less documented. The aim was to study these cellular effects using an in vitro model with synoviocytes interacting with peripheral blood mononuclear cells (PBMC to reproduce the interactions in the RA synovium.Methods: Activated-PBMC were co-cultured with RA synoviocytes during 48h. A dose-response of methylprednisolone (MP was tested and different biotherapies (Infliximab, Etanercept, Adalimumab, Tocilizumab, Abatacept and Rituximab were added alone or in combination with MP. Cytokine production (IL-17, IL-6, IL-1β, IFN-γ and IL-10 was measured by ELISA.Results: Addition of MP to co-cultures inhibited the production of all cytokines. The response to the biotherapies alone was treatment-dependent. IL-17 production was inhibited only by Tocilizumab (p=0.004 while IL-6 was decreased only by Infliximab (p≤0.002. IL-1β level was affected in all conditions (p≤0.03. IFN-γ production was mainly decreased by Infliximab (p=0.004, and IL-10 by Infliximab and Tocilizumab (p≤0.004. The combination MP and biotherapy did not induce an additional effect on pro-inflammatory cytokine inhibition. The combination MP and biotherapies induced a higher IL-10 secretion than MP alone, mainly with Rituximab.Conclusion: Steroids inhibited the secretion of all cytokines, and low doses were as potent. The anti-inflammatory effect of biotherapies was dependent on their mechanism of action. MP and biotherapy combination did not enhance the inhibitory effect on pro-inflammatory cytokines but could have a beneficial effect by increasing IL-10 production.

  8. Effect of amino acid supplementation on titer and glycosylation distribution in hybridoma cell cultures-Systems biology-based interpretation using genome-scale metabolic flux balance model and multivariate data analysis.

    Science.gov (United States)

    Reimonn, Thomas M; Park, Seo-Young; Agarabi, Cyrus D; Brorson, Kurt A; Yoon, Seongkyu

    2016-09-01

    Genome-scale flux balance analysis (FBA) is a powerful systems biology tool to characterize intracellular reaction fluxes during cell cultures. FBA estimates intracellular reaction rates by optimizing an objective function, subject to the constraints of a metabolic model and media uptake/excretion rates. A dynamic extension to FBA, dynamic flux balance analysis (DFBA), can calculate intracellular reaction fluxes as they change during cell cultures. In a previous study by Read et al. (2013), a series of informed amino acid supplementation experiments were performed on twelve parallel murine hybridoma cell cultures, and this data was leveraged for further analysis (Read et al., Biotechnol Prog. 2013;29:745-753). In order to understand the effects of media changes on the model murine hybridoma cell line, a systems biology approach is applied in the current study. Dynamic flux balance analysis was performed using a genome-scale mouse metabolic model, and multivariate data analysis was used for interpretation. The calculated reaction fluxes were examined using partial least squares and partial least squares discriminant analysis. The results indicate media supplementation increases product yield because it raises nutrient levels extending the growth phase, and the increased cell density allows for greater culture performance. At the same time, the directed supplementation does not change the overall metabolism of the cells. This supports the conclusion that product quality, as measured by glycoform assays, remains unchanged because the metabolism remains in a similar state. Additionally, the DFBA shows that metabolic state varies more at the beginning of the culture but less by the middle of the growth phase, possibly due to stress on the cells during inoculation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1163-1173, 2016. © 2016 American Institute of Chemical Engineers.

  9. A Configuration Model of Organizational Culture

    Directory of Open Access Journals (Sweden)

    Daniel Dauber

    2012-01-01

    Full Text Available The article proposes a configuration model of organizational culture, which explores dynamic relationships between organizational culture, strategy, structure, and operations of an organization (internal environment and maps interactions with the external environment (task and legitimization environment. A major feature of the configuration model constitutes its well-defined processes, which connect the elements of the model systematically to each other, such as single- and double-loop learning, operationalization of strategies, legitimization management, and so on. The model is grounded in a large review of literature in different research areas and builds on widely recognized models in the field of organization and culture theory. It constitutes a response to the call for new models, which are able to explain and facilitate the exploration of the empirical complexity that organizations face today. The configuration model of organizational culture is of particular interest to scholars who investigate into cultural phenomena and change over time.

  10. EFFECT OF FUROSTANOL GLYCOSIDES FROM CULTURED DIOSCOREA DELTOIDEA CELLS ON REGULATORY FUNCTION OF ENDOTHELIUM IN A RAT MODEL OF HYPOESTROGEN-INDUCED ENDOTHELIAL DYSFUNCTION

    Directory of Open Access Journals (Sweden)

    E. B. Artyushkova

    2008-01-01

    Full Text Available Aim. To study the effects of furostanol glycosides from cultured Dioscorea Deltoidea cells (DM-05, Institute of Plant Physiology, RAS on physiological and biochemical markers of endothelial function in rats with hypoestrogen-induced endothelial dysfunction.Material and methods. 10 female rats of Wistar line, with body mass 200-300 g have been included in the experiment. The bilateral ovariectomy was performed in rats to produce the model of hypoestrogen-induced endothelial dysfunction. Rats were treated with the injections of DM-05 during 6 weeks. False ovariectomy was performed in rats of control group (n=10.Results. DM-05 restored the levels of stable metabolites of nitric oxide (NO which reflex endothelial NO-synthase activity. Besides DM-05 corrected blood pressure and endothelial function. Experiments on open heart showed that DM-05 protects the cardiac tissue from hypoestrogen-induced hyperadrenoreactivity.Conclusion. Treatment with plant origin substances with estrogen-like activity can be a perspective approach to the correction of endothelial function and decrease in cardiovascular risk in menopause women.

  11. Culturicon model: A new model for cultural-based emoticon

    Science.gov (United States)

    Zukhi, Mohd Zhafri Bin Mohd; Hussain, Azham

    2017-10-01

    Emoticons are popular among distributed collective interaction user in expressing their emotion, gestures and actions. Emoticons have been proved to be able to avoid misunderstanding of the message, attention saving and improved the communications among different native speakers. However, beside the benefits that emoticons can provide, the study regarding emoticons in cultural perspective is still lacking. As emoticons are crucial in global communication, culture should be one of the extensively research aspect in distributed collective interaction. Therefore, this study attempt to explore and develop model for cultural-based emoticon. Three cultural models that have been used in Human-Computer Interaction were studied which are the Hall Culture Model, Trompenaars and Hampden Culture Model and Hofstede Culture Model. The dimensions from these three models will be used in developing the proposed cultural-based emoticon model.

  12. Protein A affinity chromatography of Chinese hamster ovary (CHO) cell culture broths containing biopharmaceutical monoclonal antibody (mAb): Experiments and mechanistic transport, binding and equilibrium modeling.

    Science.gov (United States)

    Grom, Matic; Kozorog, Mirijam; Caserman, Simon; Pohar, Andrej; Likozar, Blaž

    2018-04-15

    Protein A-based affinity chromatography is a highly-efficient separation method to capture, purify and isolate biosimilar monoclonal antibodies (mAb) - an important medical product of biopharmaceutical industrial manufacturing. It is considered the most expensive step in purification downstream operations; therefore, its performance optimization offers a great cost saving in the overall production expenditure. The biochemical mixture-separating specific interaction experiments with Chinese hamster ovary (CHO) cell culture harvest, containing glycosylated extracellular immunoglobulins (Ig), were made using five different state-of-the-art commercial resins. Packing breakthrough curves were recorded at an array of prolonged residence times. A mathematical simulation model was developed, applied and validated in combination with non-linear regression algorithms on bed effluent concentrations to determine the previously-unknown binding properties of stationary phase materials. Apart from the columns' differential partitioning, the whole external system was also integrated. It was confirmed that internal pore diffusion is the global rate-limiting resistance of the compound retention process. Immobilizing substrate characteristics, obtained in this engineering study, are indispensable for the scale-up of the periodic counter-current control with mechanistic load, elution and wash reduction. Furthermore, unit's volumetric flow screening measurements revealed dynamic effect correlation to eluate quality parameters, like the presence of aggregates, the host cell-related impurities at supernatant's extended feeding, and titre. Numerical sensitivity outputs demonstrated the impacts of fluidics (e.g. axial dispersion coefficient), thermodynamics (Langmuir adsorption) and mass transfer fluxes. Copyright © 2018 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    Halldorsson, Skarphedinn; Lucumi, Edinson; Gómez-Sjöberg, Rafael; Fleming, Ronan M T

    2015-01-15

    Culture of cells using various microfluidic devices is becoming more common within experimental cell biology. At the same time, a technological radiation of microfluidic cell culture device designs is currently in progress. Ultimately, the utility of microfluidic cell culture will be determined by its capacity to permit new insights into cellular function. Especially insights that would otherwise be difficult or impossible to obtain with macroscopic cell culture in traditional polystyrene dishes, flasks or well-plates. Many decades of heuristic optimization have gone into perfecting conventional cell culture devices and protocols. In comparison, even for the most commonly used microfluidic cell culture devices, such as those fabricated from polydimethylsiloxane (PDMS), collective understanding of the differences in cellular behavior between microfluidic and macroscopic culture is still developing. Moving in vitro culture from macroscopic culture to PDMS based devices can come with unforeseen challenges. Changes in device material, surface coating, cell number per unit surface area or per unit media volume may all affect the outcome of otherwise standard protocols. In this review, we outline some of the advantages and challenges that may accompany a transition from macroscopic to microfluidic cell culture. We focus on decisive factors that distinguish macroscopic from microfluidic cell culture to encourage a reconsideration of how macroscopic cell culture principles might apply to microfluidic cell culture. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  14. High-Throughput Screening of Compounds for Anti-Transmissible Spongiform Encephalopathy Activity Using Cell-Culture and Cell-Free Models and Infected Animals

    National Research Council Canada - National Science Library

    Caughey, Byron

    2006-01-01

    .... One therapeutic approach is the inhibitors of PrPSc accumulation indeed many inhibitors of PrPSc accumulation in scrapie-infected cells also have anti-scrapie activity in rodents During This year...

  15. A Dynamic Model of Cultural Reproduction

    DEFF Research Database (Denmark)

    Jæger, Mads Meier; Breen, Richard

    2016-01-01

    The authors draw on Pierre Bourdieu’s theory of cultural reproduction to develop a formal model of the pathways through which cultural capital acts to enhance children’s educational and socioeconomic success. The authors’ approach brings conceptual and empirical clarity to an important area...... of study. Their model describes how parents transmit cultural capital to their children and how children convert cultural capital into educational success. It also provides a behavioral framework for interpreting parental investments in cultural capital. The authors review results from existing empirical...... research on the role of cultural capital in education to demonstrate the usefulness of their model for interpretative purposes, and they use National Longitudinal Survey of Youth 1979—Children and Young Adults survey data to test some of its implications....

  16. Differentiation of Human Pluripotent Stem Cells into Functional Endothelial Cells in Scalable Suspension Culture

    Directory of Open Access Journals (Sweden)

    Ruth Olmer

    2018-05-01

    Full Text Available Summary: Endothelial cells (ECs are involved in a variety of cellular responses. As multifunctional components of vascular structures, endothelial (progenitor cells have been utilized in cellular therapies and are required as an important cellular component of engineered tissue constructs and in vitro disease models. Although primary ECs from different sources are readily isolated and expanded, cell quantity and quality in terms of functionality and karyotype stability is limited. ECs derived from human induced pluripotent stem cells (hiPSCs represent an alternative and potentially superior cell source, but traditional culture approaches and 2D differentiation protocols hardly allow for production of large cell numbers. Aiming at the production of ECs, we have developed a robust approach for efficient endothelial differentiation of hiPSCs in scalable suspension culture. The established protocol results in relevant numbers of ECs for regenerative approaches and industrial applications that show in vitro proliferation capacity and a high degree of chromosomal stability. : In this article, U. Martin and colleagues show the generation of hiPSC endothelial cells in scalable cultures in up to 100 mL culture volume. The generated ECs show in vitro proliferation capacity and a high degree of chromosomal stability after in vitro expansion. The established protocol allows to generate hiPSC-derived ECs in relevant numbers for regenerative approaches. Keywords: hiPSC differentiation, endothelial cells, scalable culture

  17. How do culture media influence in vitro perivascular cell behavior?

    Science.gov (United States)

    Huber, Birgit; Volz, Ann-Cathrin; Kluger, Petra Juliane

    2015-12-01

    Perivascular cells are multilineage cells located around the vessel wall and important for wall stabilization. In this study, we evaluated a stem cell media and a perivascular cell-specific media for the culture of primary perivascular cells regarding their cell morphology, doubling time, stem cell properties, and expression of cell type-specific markers. When the two cell culture media were compared to each other, perivascular cells cultured in the stem cell medium had a more elongated morphology and a faster doubling rate and cells cultured in the pericyte medium had a more typical morphology, with several filopodia, and a slower doubling rate. To evaluate stem cell properties, perivascular cells, CD146(-) cells, and mesenchymal stem cells (MSCs) were differentiated into the adipogenic, osteogenic, and chondrogenic lineages. It was seen that perivascular cells, as well as CD146(-) cells and MSCs, cultured in stem cell medium showed greater differentiation than cells cultured in pericyte-specific medium. The expression of pericyte-specific markers CD146, neural/glial antigen 2 (NG2), platelet-derived growth factor receptor-β (PDGFR-β), myosin, and α-smooth muscle actin (α-SMA) could be found in both pericyte cultures, as well as to varying amounts in CD146(-) cells, MSCs, and endothelial cells. The here presented work shows that perivascular cells can adapt to their in vitro environment and cell culture conditions influence cell functionality, such as doubling rate or differentiation behavior. Pericyte-specific markers were shown to be expressed also from cells other than perivascular cells. We can further conclude that CD146(+) perivascular cells are inhomogeneous cell population probably containing stem cell subpopulations, which are located perivascular around capillaries. © 2015 International Federation for Cell Biology.

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

    Science.gov (United States)

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

    2000-01-01

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

  19. Predictions for optimal mitigation of paracrine inhibitory signalling in haemopoietic stem cell cultures.

    Science.gov (United States)

    Berry, Joseph D; Godara, Pankaj; Liovic, Petar; Haylock, David N

    2015-04-16

    Recent studies in the literature have highlighted the critical role played by cell signalling in determining haemopoietic stem cell (HSC) fate within ex vivo culture systems. Stimulatory signals can enhance proliferation and promote differentiation, whilst inhibitory signals can significantly limit culture output. Numerical models of various mitigation strategies are presented and applied to determine effectiveness of these strategies toward mitigation of paracrine inhibitory signalling inherent in these culture systems. The strategies assessed include mixing, media-exchange, fed-batch and perfusion. The models predict that significant spatial concentration gradients exist in typical cell cultures, with important consequences for subsequent cell expansion. Media exchange is shown to be the most effective mitigation strategy, but remains labour intensive and difficult to scale-up for large culture systems. The fed-batch strategy is only effective at very small Peclet number, and its effect is diminished as the cell culture volume grows. Conversely, mixing is effective at high Peclet number, and ineffective at low Peclet number. The models predict that cell expansion in fed-batch cultures becomes independent of increasing dilution rate, consistent with experimental results previously reported in the literature. In contrast, the models predict that increasing the flow rate in perfused cultures will lead to increased cell expansion, indicating the suitability of perfusion for use as an automated, tunable strategy. The effect of initial cell seeding density is also investigated, with the model showing that perfusion outperforms dilution for all densities considered. The models predict that the impact of inhibitory signalling in HSC cultures can be mitigated against using media manipulation strategies, with the optimal strategy dependent upon the protein diffusion time-scale relative to the media manipulation time-scale. The key messages from this study can be applied to

  20. Characterisation of an in vitro blood-brain barrier model based on primary porcine capillary endothelial cells in monoculture or co-culture with primary rat or porcine astrocytes and pericytes

    DEFF Research Database (Denmark)

    Thomsen, Louiza Bohn; Larsen, Annette Burkhart; Moos, Torben

    to in vivo such as efflux transporters, tight junction proteins, and high transendothelial electric resistance (TEER). Primary BCECs are isolated from a variety of mammals such as rats, mice, cattle and pigs. Often bovine and porcine BCECs are cultured in monoculture or in co-culture with rat astrocytes......In vitro blood-brain barrier (BBB) models based on primary brain capillary endothelial cells (BCECs) in monoculture or in co-culture with primary astrocytes and pericytes are often applied for studying physiology of the BBB. Primary BCECs retain many morphological and biochemical properties similar...... obtained from neonatal rats which have been shown to strengthen the barrier properties of the BCECs. In this study, brain endothelial cells (PBECs), astrocytes and pericytes are isolated from pig brains donated by the local abattoir. The brains are from 6 month old domestic pigs. The availability and high...

  1. A Highly Efficient Human Pluripotent Stem Cell Microglia Model Displays a Neuronal-Co-culture-Specific Expression Profile and Inflammatory Response

    Directory of Open Access Journals (Sweden)

    Walther Haenseler

    2017-06-01

    Full Text Available Microglia are increasingly implicated in brain pathology, particularly neurodegenerative disease, with many genes implicated in Alzheimer's, Parkinson's, and motor neuron disease expressed in microglia. There is, therefore, a need for authentic, efficient in vitro models to study human microglial pathological mechanisms. Microglia originate from the yolk sac as MYB-independent macrophages, migrating into the developing brain to complete differentiation. Here, we recapitulate microglial ontogeny by highly efficient differentiation of embryonic MYB-independent iPSC-derived macrophages then co-culture them with iPSC-derived cortical neurons. Co-cultures retain neuronal maturity and functionality for many weeks. Co-culture microglia express key microglia-specific markers and neurodegenerative disease-relevant genes, develop highly dynamic ramifications, and are phagocytic. Upon activation they become more ameboid, releasing multiple microglia-relevant cytokines. Importantly, co-culture microglia downregulate pathogen-response pathways, upregulate homeostatic function pathways, and promote a more anti-inflammatory and pro-remodeling cytokine response than corresponding monocultures, demonstrating that co-cultures are preferable for modeling authentic microglial physiology.

  2. Traditional and Modern Cell Culture in Virus Diagnosis.

    Science.gov (United States)

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

    2016-04-01

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

  3. Attempt to develop taste bud models in three-dimensional culture.

    Science.gov (United States)

    Nishiyama, Miyako; Yuki, Saori; Fukano, Chiharu; Sako, Hideyuki; Miyamoto, Takenori; Tomooka, Yasuhiro

    2011-09-01

    Taste buds are the end organs of taste located in the gustatory papillae, which occur on the surface of the oral cavity. The goal of the present study was to establish a culture model mimicking the lingual taste bud of the mouse. To this end, three cell lines were employed: taste bud-derived cell lines (TBD cell lines), a lingual epithelial cell-derived cell line (20A cell line), and a mesenchymal cell-derived cell line (TMD cell line). TBD cells embedded in collagen gel formed three-dimensional clusters, which had an internal cavity equipped with a tight junction-like structure, a microvilluslike structure, and a laminin-positive layer surrounding the cluster. The cells with this epitheliumlike morphology expressed marker proteins of taste cells: gustducin and NCAM. TBD cells formed a monolayer on collagen gel when they were co-cultured with TMD cells. TBD, 20A, and TMD cell lines were maintained in a triple cell co-culture, in which TBD cells were pre-seeded as aggregates or in suspension on the collagen gel containing TMD cells, and 20A cells were laid over the TBD cells. TBD cells in the triple cell co-culture expressed NCAM. This result suggests that co-cultured TBD cells exhibited a characteristic of Type III taste cells. The culture model would be useful to study morphogenesis and functions of the gustatory organ.

  4. PHYTOCHEMICAL STUDY OF CELL CULTURE JATROPHA CURCAS

    Directory of Open Access Journals (Sweden)

    KOMAR RUSLAN

    2011-01-01

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

  5. Lethal impacts of cigarette smoke in cultured tobacco cells

    Directory of Open Access Journals (Sweden)

    Kawano Tomonori

    2011-07-01

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

  6. Modeling Crowdsourcing for Cultural Heritage

    NARCIS (Netherlands)

    Noordegraaf, J.; Bartholomew, A.; Eveleigh, A.; Proctor, N.; Cherry, R.

    2014-01-01

    Despite the widespread prevalence of crowdsourcing projects in the cultural heritage domain, not all initiatives to date have been universally successful. This study has revealed that the conditions in which projects are realized, and the design properties of those projects, have a significant

  7. Good cell culture practices &in vitro toxicology.

    Science.gov (United States)

    Eskes, Chantra; Boström, Ann-Charlotte; Bowe, Gerhard; Coecke, Sandra; Hartung, Thomas; Hendriks, Giel; Pamies, David; Piton, Alain; Rovida, Costanza

    2017-12-01

    Good Cell Culture Practices (GCCP) is of high relevance to in vitro toxicology. The European Society of Toxicology In Vitro (ESTIV), the Center for Alternatives for Animal Testing (CAAT) and the In Vitro Toxicology Industrial Platform (IVTIP) joined forces to address by means of an ESTIV 2016 pre-congress session the different aspects and applications of GCCP. The covered aspects comprised the current status of the OECD guidance document on Good In Vitro Method Practices, the importance of quality assurance for new technological advances in in vitro toxicology including stem cells, and the optimized implementation of Good Manufacturing Practices and Good Laboratory Practices for regulatory testing purposes. General discussions raised the duality related to the difficulties in implementing GCCP in an academic innovative research framework on one hand, and on the other hand, the need for such GCCP principles in order to ensure reproducibility and robustness of in vitro test methods for toxicity testing. Indeed, if good cell culture principles are critical to take into consideration for all uses of in vitro test methods for toxicity testing, the level of application of such principles may depend on the stage of development of the test method as well as on the applications of the test methods, i.e., academic innovative research vs. regulatory standardized test method. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    OpenAIRE

    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 dir...

  9. Enhanced engraftment of mesenchymal stem cells in a cutaneous wound model by culture in allogenic species-specific serum and administration in fibrin constructs.

    Science.gov (United States)

    Gregory, Carl A; Reyes, Emigdio; Whitney, Mandolin J; Spees, Jeffrey L

    2006-10-01

    Human mesenchymal stem cells (hMSCs), also referred to as multipotent stromal cells, are currently being applied in clinical trials for bone diseases, graft versus host disease, and myocardial infarction. However, the standard growth medium for hMSCs contains 10%-20% fetal calf serum (FCS), and FCS is strongly immunogenic in both rodents and humans. Previously, we reported that by a sensitive fluorescence-based assay, 7-30 mg of internalized FCS is associated with 10(8) hMSCs, a dosage that will probably be needed for most therapies. We also found that a brief culture in medium containing autologous 20% adult human serum (AHS) or autologous 10% AHS supplemented with growth factors (AHS(+)) reduced the contamination by more than 99.9%. We have now extensively characterized the culture conditions and shown that hMSC expansion is possible using heterologous 20% AHS or heterologous 10% AHS(+). The uptake of FCS is an active process that acts to concentrate contamination in the cells even under low serum conditions (2% FCS) but can be actively displaced by incubation of the cells in medium with AHS. Rat MSCs (rMSCs) can be expanded under similar conditions using supplemented heterologous adult rat serum (ARS(+)). After expansion in FCS, a further 8 days of culture with ARS(+) significantly improves the viability of the rMSCs in vivo after encapsulation in fibrin followed by subcutaneous implantation in rats. Our results have the potential to dramatically improve cellular and genetic therapies using hMSCs.

  10. Cardiac Cells Beating in Culture: A Laboratory Exercise

    Science.gov (United States)

    Weaver, Debora

    2007-01-01

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

  11. Comparing soluble ferric pyrophosphate to common iron salts and chelates as sources of bioavailable iron in a Caco-2 cell culture model.

    Science.gov (United States)

    Zhu, Le; Glahn, Raymond P; Nelson, Deanna; Miller, Dennis D

    2009-06-10

    Iron bioavailability from supplements and fortificants varies depending upon the form of the iron and the presence or absence of iron absorption enhancers and inhibitors. Our objectives were to compare the effects of pH and selected enhancers and inhibitors and food matrices on the bioavailability of iron in soluble ferric pyrophosphate (SFP) to other iron fortificants using a Caco-2 cell culture model with or without the combination of in vitro digestion. Ferritin formation was the highest in cells treated with SFP compared to those treated with other iron compounds or chelates. Exposure to pH 2 followed by adjustment to pH 7 markedly decreased FeSO(4) bioavailability but had a smaller effect on bioavailabilities from SFP and sodium iron(III) ethylenediaminetetraacetate (NaFeEDTA), suggesting that chelating agents minimize the effects of pH on iron bioavailability. Adding ascorbic acid (AA) and cysteine to SFP in a 20:1 molar ratio increased ferritin formation by 3- and 2-fold, respectively, whereas adding citrate had no significant effect on the bioavailability of SFP. Adding phytic acid (10:1) and tannic acid (1:1) to iron decreased iron bioavailability from SFP by 91 and 99%, respectively. The addition of zinc had a marked inhibitory effect on iron bioavailability. Calcium and magnesium also inhibited iron bioavailability but to a lesser extent. Incorporating SFP in rice greatly reduced iron bioavailability from SFP, but this effect can be partially reversed with the addition of AA. SFP and FeSO(4) were taken up similarly when added to nonfat dry milk. Our results suggest that dietary factors known to enhance and inhibit iron bioavailability from various iron sources affect iron bioavailability from SFP in similar directions. However, the magnitude of the effects of iron absorption inhibitors on SFP iron appears to be smaller than on iron salts, such as FeSO(4) and FeCl(3). This supports the hypothesis that SFP is a promising iron source for food fortification

  12. VISION: A Model of Culture for Counselors.

    Science.gov (United States)

    Baber, W. Lorenzo; Garrett, Michael T.; Holcomb-McCoy, Cheryl

    1997-01-01

    Culture as a group phenomenon versus the need of counselors to work with the individual is addressed. The VISION model of culture, which accounts for within-group and between-group differences, the disappearance of groups, and the emergence of new ones, is presented. Two examples of multicultural interventions are reported. (Author/EMK)

  13. Modeling culture in intelligent virtual agents

    NARCIS (Netherlands)

    Mascarenhas, S.; Degens, N.; Paiva, A.; Prada, R.; Hofstede, G.J.; Beulens, A.J.M.; Aylett, R.

    2016-01-01

    This work addresses the challenge of creating virtual agents that are able to portray culturally appropriate behavior when interacting with other agents or humans. Because culture influences how people perceive their social reality it is important to have agent models that explicitly consider social

  14. Feeder Cell Type Affects the Growth of In Vitro Cultured Bovine Trophoblast Cells

    Directory of Open Access Journals (Sweden)

    Islam M. Saadeldin

    2017-01-01

    Full Text Available Trophectoderm cells are the foremost embryonic cells to differentiate with prospective stem-cell properties. In the current study, we aimed at improving the current approach for trophoblast culture by using granulosa cells as feeders. Porcine granulosa cells (PGCs compared to the conventional mouse embryonic fibroblasts (MEFs were used to grow trophectoderm cells from hatched bovine blastocysts. Isolated trophectoderm cells were monitored and displayed characteristic epithelial/cuboidal morphology. The isolated trophectoderm cells expressed mRNA of homeobox protein (CDX2, cytokeratin-8 (KRT8, and interferon tau (IFNT. The expression level was higher on PGCs compared to MEFs throughout the study. In addition, primary trophectoderm cell colonies grew faster on PGCs, with a doubling time of approximately 48 hrs, compared to MEFs. PGCs feeders produced a fair amount of 17β-estradiol and progesterone. We speculated that the supplementation of sex steroids and still-unknown factors during the trophoblasts coculture on PGCs have helped to have better trophectoderm cell’s growth than on MEFs. This is the first time to use PGCs as feeders to culture trophectoderm cells and it proved superior to MEFs. We propose PGCs as alternative feeders for long-term culture of bovine trophectoderm cells. This model will potentially benefit studies on the early trophoblast and embryonic development in bovines.

  15. DNA MUTAGENESIS IN PANAX GINSENG CELL CULTURES

    Directory of Open Access Journals (Sweden)

    Kiselev K.V.

    2012-08-01

    Full Text Available At the present time, it is well documented that plant tissue culture induces a number of mutations and chromosome rearrangements termed “somaclonal variations”. However, little is known about the nature and the molecular mechanisms of the tissue culture-induced mutagenesis and the effects of long-term subculturing on the rate and specific features of the mutagenesis. The aim of the present study was to investigate and compare DNA mutagenesis in different genes of Panax ginseng callus cultures of different age. It has previously been shown that the nucleotide sequences of the Agrobacterium rhizogenes rolC locus and the selective marker nptII developed mutations during long-term cultivation of transgenic cell cultures of P. ginseng. In the present work, we analyzed nucleotide sequences of selected plant gene families in a 2-year-old and 20-year-old P. ginseng 1c cell culture and in leaves of cultivated P. ginseng plants. We analysed sequence variability between the Actin genes, which are a family of house-keeping genes; the phenylalanine ammonia-lyase (PAL and dammarenediol synthase (DDS genes, which actively participate in the biosynthesis of ginsenosides; and the somatic embryogenesis receptor kinase (SERK genes, which control plant development. The frequency of point mutations in the Actin, PAL, DDS, and SERK genes in the 2-year-old callus culture was markedly higher than that in cultivated plants but lower than that in the 20-year-old callus culture of P. ginseng. Most of the mutations in the 2- and 20-year-old P. ginseng calli were A↔G and T↔C transitions. The number of nonsynonymous mutations was higher in the 2- and 20-year-old callus cultures than the number of nonsynonymous mutations in the cultivated plants of P. ginseng. Interestingly, the total number of N→G or N→C substitutions in the analyzed genes was 1.6 times higher than the total number of N→A or N→T substitutions. Using methylation-sensitive DNA fragmentation

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

    Science.gov (United States)

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

    2014-12-15

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

  17. Turbulent Dynamics of Epithelial Cell Cultures

    Science.gov (United States)

    Blanch-Mercader, C.; Yashunsky, V.; Garcia, S.; Duclos, G.; Giomi, L.; Silberzan, P.

    2018-05-01

    We investigate the large length and long time scales collective flows and structural rearrangements within in vitro human bronchial epithelial cell (HBEC) cultures. Activity-driven collective flows result in ensembles of vortices randomly positioned in space. By analyzing a large population of vortices, we show that their area follows an exponential law with a constant mean value and their rotational frequency is size independent, both being characteristic features of the chaotic dynamics of active nematic suspensions. Indeed, we find that HBECs self-organize in nematic domains of several cell lengths. Nematic defects are found at the interface between domains with a total number that remains constant due to the dynamical balance of nucleation and annihilation events. The mean velocity fields in the vicinity of defects are well described by a hydrodynamic theory of extensile active nematics.

  18. Magnetically levitated mesenchymal stem cell spheroids cultured with a collagen gel maintain phenotype and quiescence

    Directory of Open Access Journals (Sweden)

    Natasha S Lewis

    2017-04-01

    Full Text Available Multicellular spheroids are an established system for three-dimensional cell culture. Spheroids are typically generated using hanging drop or non-adherent culture; however, an emerging technique is to use magnetic levitation. Herein, mesenchymal stem cell spheroids were generated using magnetic nanoparticles and subsequently cultured within a type I collagen gel, with a view towards developing a bone marrow niche environment. Cells were loaded with magnetic nanoparticles, and suspended beneath an external magnet, inducing self-assembly of multicellular spheroids. Cells in spheroids were viable and compared to corresponding monolayer controls, maintained stem cell phenotype and were quiescent. Interestingly, core spheroid necrosis was not observed, even with increasing spheroid size, in contrast to other commonly used spheroid systems. This mesenchymal stem cell spheroid culture presents a potential platform for modelling in vitro bone marrow stem cell niches, elucidating interactions between cells, as well as a useful model for drug delivery studies.

  19. Comparative Proteomic Characterization of 4 Human Liver-Derived Single Cell Culture Models Reveals Significant Variation in the Capacity for Drug Disposition, Bioactivation, and Detoxication.

    Science.gov (United States)

    Sison-Young, Rowena L C; Mitsa, Dimitra; Jenkins, Rosalind E; Mottram, David; Alexandre, Eliane; Richert, Lysiane; Aerts, Hélène; Weaver, Richard J; Jones, Robert P; Johann, Esther; Hewitt, Philip G; Ingelman-Sundberg, Magnus; Goldring, Christopher E P; Kitteringham, Neil R; Park, B Kevin

    2015-10-01

    In vitro preclinical models for the assessment of drug-induced liver injury (DILI) are usually based on cryopreserved primary human hepatocytes (cPHH) or human hepatic tumor-derived cell lines; however, it is unclear how well such cell models reflect the normal function of liver cells. The physiological, pharmacological, and toxicological phenotyping of available cell-based systems is necessary in order to decide the testing purpose for which they are fit. We have therefore undertaken a global proteomic analysis of 3 human-derived hepatic cell lines (HepG2, Upcyte, and HepaRG) in comparison with cPHH with a focus on drug metabolizing enzymes and transport proteins (DMETs), as well as Nrf2-regulated proteins. In total, 4946 proteins were identified, of which 2722 proteins were common across all cell models, including 128 DMETs. Approximately 90% reduction in expression of cytochromes P450 was observed in HepG2 and Upcyte cells, and approximately 60% in HepaRG cells relative to cPHH. Drug transporter expression was also lower compared with cPHH with the exception of MRP3 and P-gp (MDR1) which appeared to be significantly expressed in HepaRG cells. In contrast, a high proportion of Nrf2-regulated proteins were more highly expressed in the cell lines compared with cPHH. The proteomic database derived here will provide a rational basis for the context-specific selection of the most appropriate 'hepatocyte-like' cell for the evaluation of particular cellular functions associated with DILI and, at the same time, assist in the construction of a testing paradigm which takes into account the in vivo disposition of a new drug. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology.

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

    Science.gov (United States)

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

    1999-01-01

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

  1. A microfluidic cell culture array with various oxygen tensions.

    Science.gov (United States)

    Peng, Chien-Chung; Liao, Wei-Hao; Chen, Ying-Hua; Wu, Chueh-Yu; Tung, Yi-Chung

    2013-08-21

    Oxygen tension plays an important role in regulating various cellular functions in both normal physiology and disease states. Therefore, drug testing using conventional in vitro cell models under normoxia often possesses limited prediction capability. A traditional method of setting an oxygen tension in a liquid medium is by saturating it with a gas mixture at the desired level of oxygen, which requires bulky gas cylinders, sophisticated control, and tedious interconnections. Moreover, only a single oxygen tension can be tested at the same time. In this paper, we develop a microfluidic cell culture array platform capable of performing cell culture and drug testing under various oxygen tensions simultaneously. The device is fabricated using an elastomeric material, polydimethylsiloxane (PDMS) and the well-developed multi-layer soft lithography (MSL) technique. The prototype device has 4 × 4 wells, arranged in the same dimensions as a conventional 96-well plate, for cell culture. The oxygen tensions are controlled by spatially confined oxygen scavenging chemical reactions underneath the wells using microfluidics. The platform takes advantage of microfluidic phenomena while exhibiting the combinatorial diversities achieved by microarrays. Importantly, the platform is compatible with existing cell incubators and high-throughput instruments (liquid handling systems and plate readers) for cost-effective setup and straightforward operation. Utilizing the developed platform, we successfully perform drug testing using an anti-cancer drug, triapazamine (TPZ), on adenocarcinomic human alveolar basal epithelial cell line (A549) under three oxygen tensions ranging from 1.4% to normoxia. The developed platform is promising to provide a more meaningful in vitro cell model for various biomedical applications while maintaining desired high throughput capabilities.

  2. Recombinant Protein Production and Insect Cell Culture and Process

    Science.gov (United States)

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

    1997-01-01

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

  3. Differential marker expression by cultures rich in mesenchymal stem cells

    Science.gov (United States)

    2013-01-01

    Background Mesenchymal stem cells have properties that make them amenable to therapeutic use. However, the acceptance of mesenchymal stem cells in clinical practice requires standardized techniques for their specific isolation. To date, there are no conclusive marker (s) for the exclusive isolation of mesenchymal stem cells. Our aim was to identify markers differentially expressed between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. We compared and contrasted the phenotype of tissue cultures in which mesenchymal stem cells are rich and rare. By initially assessing mesenchymal stem cell differentiation, we established that bone marrow and breast adipose cultures are rich in mesenchymal stem cells while, in our hands, foreskin fibroblast and olfactory tissue cultures contain rare mesenchymal stem cells. In particular, olfactory tissue cells represent non-stem cell mesenchymal cells. Subsequently, the phenotype of the tissue cultures were thoroughly assessed using immuno-fluorescence, flow-cytometry, proteomics, antibody arrays and qPCR. Results Our analysis revealed that all tissue cultures, regardless of differentiation potential, demonstrated remarkably similar phenotypes. Importantly, it was also observed that common mesenchymal stem cell markers, and fibroblast-associated markers, do not discriminate between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. Examination and comparison of the phenotypes of mesenchymal stem cell and non-stem cell mesenchymal cell cultures revealed three differentially expressed markers – CD24, CD108 and CD40. Conclusion We indicate the importance of establishing differential marker expression between mesenchymal stem cells and non-stem cell mesenchymal cells in order to determine stem cell specific markers. PMID:24304471

  4. Protection of cultured mammalian cells by rebamipide

    Energy Technology Data Exchange (ETDEWEB)

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

    1997-06-01

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

  5. Protection of cultured mammalian cells by rebamipide

    International Nuclear Information System (INIS)

    Antoku, Shigetoshi; Aramaki, Ryoji; Tanaka, Hisashi; Kusumoto, Naotoshi.

    1997-01-01

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

  6. An ovine tracheal explant culture model for allergic airway inflammation

    Directory of Open Access Journals (Sweden)

    Abeynaike Latasha

    2010-08-01

    Full Text Available Abstract Background The airway epithelium is thought to play an important role in the pathogenesis of asthmatic disease. However, much of our understanding of airway epithelial cell function in asthma has been derived from in vitro studies that may not accurately reflect the interactive cellular and molecular pathways active between different tissue constituents in vivo. Methods Using a sheep model of allergic asthma, tracheal explants from normal sheep and allergic sheep exposed to house dust mite (HDM allergen were established to investigate airway mucosal responses ex vivo. Explants were cultured for up to 48 h and tissues were stained to identify apoptotic cells, goblet cells, mast cells and eosinophils. The release of cytokines (IL-1α, IL-6 and TNF-α by cultured tracheal explants, was assessed by ELISA. Results The general morphology and epithelial structure of the tracheal explants was well maintained in culture although evidence of advanced apoptosis within the mucosal layer was noted after culture for 48 h. The number of alcian blue/PAS positive mucus-secreting cells within the epithelial layer was reduced in all cultured explants compared with pre-cultured (0 h explants, but the loss of staining was most evident in allergic tissues. Mast cell and eosinophil numbers were elevated in the allergic tracheal tissues compared to naïve controls, and in the allergic tissues there was a significant decline in mast cells after 24 h culture in the presence or absence of HDM allergen. IL-6 was released by allergic tracheal explants in culture but was undetected in cultured control explants. Conclusions Sheep tracheal explants maintain characteristics of the airway mucosa that may not be replicated when studying isolated cell populations in vitro. There were key differences identified in explants from allergic compared to control airways and in their responses in culture for 24 h. Importantly, this study establishes the potential for the

  7. Culture in Transition: A learning model

    DEFF Research Database (Denmark)

    Baca, Susan

    2010-01-01

    of organizational transition, and 3) demonstrating the efficacy of the model by using it to explain empirical research findings. It is argued that learning new cultural currency involves the use of active intelligence to locate and answer relevant questions, and further that this process requires the interplay......This paper addresses the problem of resistance to attempted changes in organizational culture, particularly those involving diversity, by 1) identifying precisely what is meant by organizational as opposed to societal culture, 2) developing a theoretical model of learning useful in contexts...... is useful for both management and labor in regulating transition processes, thus making a contribution to industrial relations....

  8. Rheological characteristics of cell suspension and cell culture of Perilla frutescens.

    Science.gov (United States)

    Zhong, J J; Seki, T; Kinoshita, S; Yoshida, T

    1992-12-05

    Physical properties such as viscosity, fluid dynamic behavior of cell suspension, and size distribution of cell aggregates of a plant, Perilla frustescens, cultured in a liquid medium were studied. As a result of investigations using cells harvester after 12 days of cultivation in a flask, it was found that the apparent viscosity of the cell suspension did not change with any variation of cell concentration below 5 g dry cell/L but markedly increased when the cell concentration increased over 12.8 g dry cell/L. The cell suspension exhibited the characteristics of a Bingham plastic fluid with a small yield stress. The size of cell aggregates in the range 74 to 500 mum did not influence the rheological characteristics of the cell suspension. The rheological characteristics of cultivation mixtures of P. frutescens cultivated in a flask and in a bioreactor were also investigated. The results showed that the flow characteristics of the cell culture could be described by a Bingham plastic model. At the later stage of cultivation, the apparent viscosity increased steadily, even though the biomass concentration (by dry weight) decreased, due to the increase of individual cell size. (c) 1992 John Wiley & Sons, Inc.

  9. Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX-Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model.

    Science.gov (United States)

    Zareba, Ilona; Surazynski, Arkadiusz; Chrusciel, Marcin; Miltyk, Wojciech; Doroszko, Milena; Rahman, Nafis; Palka, Jerzy

    2017-01-01

    The effect of impaired intracellular proline availability for proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied. We generated a constitutively knocked-down PRODH/POX MCF-7 breast cancer cell line (MCF-7shPRODH/POX) as a model to analyze the functional consequences of impaired intracellular proline levels. We have used inhibitor of proline utilization in collagen biosynthesis, 2-metoxyestradiol (MOE), inhibitor of prolidase that generate proline, rapamycin (Rap) and glycyl-proline (GlyPro), substrate for prolidase. Collagen and DNA biosynthesis were evaluated by radiometric assays. Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. PRODH/POX knockdown decreased DNA and collagen biosynthesis, whereas increased prolidase activity and intracellular proline level in MCF-7shPRODH/POX cells. All studied compounds decreased cell viability in MCF-7 and MCF-7shPRODH/POX cells. DNA biosynthesis was similarly inhibited by Rap and MOE in both cell lines, but GlyPro inhibited the process only in MCF-7shPRODH/POX and MOE+GlyPro only in MCF-7 cells. All the compounds inhibited collagen biosynthesis, increased prolidase activity and cytoplasmic proline level in MCF-7shPRODH/POX cells and contributed to the induction of pro-survival mode only in MCF-7shPRODH/POX cells. In contrast, all studied compounds upregulated expression of pro-apoptotic protein only in MCF-7 cells. PRODH/POX was confirmed as a driver of apoptosis and proved the eligibility of MCF-7shPRODH/POX cell line as a highly effective model to elucidate the different mechanisms underlying proline utilization or generation in PRODH/POX-dependent pro-apoptotic pathways. © 2017 The Author(s). Published by S. Karger AG, Basel.

  10. Cross-Cultural Impression Management: A Cultural Knowledge Audit Model

    Science.gov (United States)

    Spong, Abigail; Kamau, Caroline

    2012-01-01

    Purpose: Many people moving into a new culture for work or study do so without prior cross-cultural training, yet successful cultural adaptation has important ramifications. The purpose of this paper is to focus on cross-cultural impression management as an element of cultural adaptation. Does cultural adaptation begin by paying strong attention…

  11. Light transfer in agar immobilized microalgae cell cultures

    Science.gov (United States)

    Kandilian, Razmig; Jesus, Bruno; Legrand, Jack; Pilon, Laurent; Pruvost, Jérémy

    2017-09-01

    This paper experimentally and theoretically investigates light transfer in agar-immobilized cell cultures. Certain biotechnological applications such as production of metabolites secreted by photosynthetic microorganisms require cells to be immobilized in biopolymers to minimize contamination and to facilitate metabolite recovery. In such applications, light absorption by cells is one of the most important parameters affecting cell growth or metabolite productivity. Modeling light transfer therein can aid design and optimize immobilized-cell reactors. In this study, Parachlorella kessleri cells with areal biomass concentrations ranging from 0.36 to 16.9 g/m2 were immobilized in 2.6 mm thick agar gels. The average absorption and scattering cross-sections as well as the scattering phase function of P. kessleri cells were measured. Then, the absorption and transport scattering coefficients of the agar gel were determined using an inverse method based on the modified two-flux approximation. The forward model was used to predict the normal-hemispherical transmittance and reflectance of the immobilized-cell films accounting for absorption and scattering by both microalgae and the agar gel. Good agreement was found between the measured and predicted normal-hemispherical transmittance and reflectance provided absorption and scattering by agar were taken into account. Moreover, good agreement was found between experimentally measured and predicted mean rate of photon absorption. Finally, optimal areal biomass concentration was determined to achieve complete absorption of the incident radiation.

  12. Long-Term Oocyte-Like Cell Development in Cultures Derived from Neonatal Marmoset Monkey Ovary

    Directory of Open Access Journals (Sweden)

    Bentolhoda Fereydouni

    2016-01-01

    Full Text Available We use the common marmoset monkey (Callithrix jacchus as a preclinical nonhuman primate model to study reproductive and stem cell biology. The neonatal marmoset monkey ovary contains numerous primitive premeiotic germ cells (oogonia expressing pluripotent stem cell markers including OCT4A (POU5F1. This is a peculiarity compared to neonatal human and rodent ovaries. Here, we aimed at culturing marmoset oogonia from neonatal ovaries. We established a culture system being stable for more than 20 passages and 5 months. Importantly, comparative transcriptome analysis of the cultured cells with neonatal ovary, embryonic stem cells, and fibroblasts revealed a lack of germ cell and pluripotency genes indicating the complete loss of oogonia upon initiation of the culture. From passage 4 onwards, however, the cultured cells produced large spherical, free-floating cells resembling oocyte-like cells (OLCs. OLCs strongly expressed several germ cell genes and may derive from the ovarian surface epithelium. In summary, our novel primate ovarian cell culture initially lacked detectable germ cells but then produced OLCs over a long period of time. This culture system may allow a deeper analysis of early phases of female primate germ cell development and—after significant refinement—possibly also the production of monkey oocytes.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    from porcine embryos or induced pluripotent stem cells is presented. The neural induction is performed in coculture and the isolation of rosette structures is carried out manually to ensure a homogenous population of NPCs. Using this method, multipotent NPCs can be obtained in approximately 1 month......The isolation and culture of neural progenitor cells (NPCs) from pluripotent stem cells has facilitated in vitro mechanistic studies of diseases related to the nervous system, as well as discovery of new medicine. In addition, NPCs are envisioned to play a crucial role in future cell replacement...... therapy. The pig has become recognized as an important large animal model and establishment of in vitro-derived porcine NPCs would allow for preclinical safety testing by transplantation in a porcine biomedical model. In this chapter, a detailed method for isolation and in vitro culture of porcine NPCs...

  14. Primary culture of glial cells from mouse sympathetic cervical ganglion: a valuable tool for studying glial cell biology.

    Science.gov (United States)

    de Almeida-Leite, Camila Megale; Arantes, Rosa Maria Esteves

    2010-12-15

    Central nervous system glial cells as astrocytes and microglia have been investigated in vitro and many intracellular pathways have been clarified upon various stimuli. Peripheral glial cells, however, are not as deeply investigated in vitro despite its importance role in inflammatory and neurodegenerative diseases. Based on our previous experience of culturing neuronal cells, our objective was to standardize and morphologically characterize a primary culture of mouse superior cervical ganglion glial cells in order to obtain a useful tool to study peripheral glial cell biology. Superior cervical ganglia from neonatal C57BL6 mice were enzymatically and mechanically dissociated and cells were plated on diluted Matrigel coated wells in a final concentration of 10,000cells/well. Five to 8 days post plating, glial cell cultures were fixed for morphological and immunocytochemical characterization. Glial cells showed a flat and irregular shape, two or three long cytoplasm processes, and round, oval or long shaped nuclei, with regular outline. Cell proliferation and mitosis were detected both qualitative and quantitatively. Glial cells were able to maintain their phenotype in our culture model including immunoreactivity against glial cell marker GFAP. This is the first description of immunocytochemical characterization of mouse sympathetic cervical ganglion glial cells in primary culture. This work discusses the uses and limitations of our model as a tool to study many aspects of peripheral glial cell biology. Copyright © 2010 Elsevier B.V. All rights reserved.

  15. X-ray microanalysis of single and cultured cells

    International Nuclear Information System (INIS)

    Wroblewski, J.; Roomans, G.M.

    1984-01-01

    X-ray microanalysis of single or cultured cells is often a useful alternative or complement to the analysis of the corresponding tissue. It also allows the analysis of individual cells in a cell population. Preparation for X-ray microanalysis poses a number of typical problems. Suspensions of single cells can be prepared by either of two pathways: (1) washing - mounting - drying, or (2) centrifugation - freezing or fixation - sectioning. The washing step in the preparation of single or cultured cells presents the most severe problems. Cultured cells are generally grown on a substrate that is compatible with both the analysis and the culture, washed and dried. In some cases, sectioning of cultured cell monolayers has been performed. Special problems in quantitative analysis occur in those cases where the cells are analyzed on a thick substrate, since the substrate contributes to the spectral background

  16. Radiobilogical cell survival models

    International Nuclear Information System (INIS)

    Zackrisson, B.

    1992-01-01

    A central issue in clinical radiobiological research is the prediction of responses to different radiation qualities. The choice of cell survival and dose-response model greatly influences the results. In this context the relationship between theory and model is emphasized. Generally, the interpretations of experimental data depend on the model. Cell survival models are systematized with respect to their relations to radiobiological theories of cell kill. The growing knowlegde of biological, physical, and chemical mechanisms is reflected in the formulation of new models. The present overview shows that recent modelling has been more oriented towards the stochastic fluctuations connected to radiation energy deposition. This implies that the traditional cell surivival models ought to be complemented by models of stochastic energy deposition processes and repair processes at the intracellular level. (orig.)

  17. Cell and Molecular Biology of Ataxia Telangiectasia Heterozygous Human Mammary Epithelial Cells Irradiated in Culture

    Science.gov (United States)

    Richmond, Robert C.

    2001-01-01

    Autologous isolates of cell types from obligate heterozygotes with the autosomal disorder ataxia-telangiectasia (A-T)were used to begin a tissue culture model for assessing pathways of radiation-induced cancer formation in this target tissue. This was done by establishing cultures of stromal fibroblasts and long-term growth human mammary epithelial cells (HMEC) in standard 2-dimensional tissue culture in order to establish expression of markers detailing early steps of carcinogenesis. The presumptive breast cancer susceptibility of A-T heterozygotes as a sequel to damage caused by ionizing radiation provided reason to study expression of markers in irradiated HMEC. Findings from our study with HMEC have included determination of differences in specific protein expression amongst growth phase (e.g., log vs stationary) and growth progression (e.g., pass 7 vs pass 9), as well as differences in morphologic markers within populations of irradiated HMEC (e.g., development of multinucleated cells).

  18. Establishment and characterization of American elm cell suspension cultures

    Science.gov (United States)

    Steven M. Eshita; Joseph C. Kamalay; Vicki M. Gingas; Daniel A. Yaussy

    2000-01-01

    Cell suspension cultures of Dutch elm disease (DED)-tolerant and DED-susceptible American elms clones have been established and characterized as prerequisites for contrasts of cellular responses to pathogen-derived elicitors. Characteristics of cultured elm cell growth were monitored by A700 and media conductivity. Combined cell growth data for all experiments within a...

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

    Science.gov (United States)

    Vendrame, Wagner A.; Pinares, Ania

    2013-06-01

    evaluated in this study provide the basic ground work and pre-flight assessment needed to justify a model for microgravity studies with jatropha in vitro cell cultures. Future studies should focus on results of experiments performed with jatropha in vitro cultures in microgravity.

  20. Electrospinning of microbial polyester for cell culture

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Oh Hyeong [Department of Polymer Science and Engineering, Kumoh National Institute of Technology, 1 Yangho-dong, Gumi, Gyeongbuk 730-701 (Korea, Republic of); Lee, Ik Sang [Department of Polymer Science and Engineering, Kumoh National Institute of Technology, 1 Yangho-dong, Gumi, Gyeongbuk 730-701 (Korea, Republic of); Ko, Young-Gwang [Department of Polymer Science and Engineering, Kumoh National Institute of Technology, 1 Yangho-dong, Gumi, Gyeongbuk 730-701 (Korea, Republic of); Meng, Wan [Department of Polymer Science, Kyungpook National University, 1370 Sankyuk-dong, Buk-gu, Daegu 702-701 (Korea, Republic of); Jung, Kyung-Hye [Department of Polymer Science, Kyungpook National University, 1370 Sankyuk-dong, Buk-gu, Daegu 702-701 (Korea, Republic of); Kang, Inn-Kyu [Department of Polymer Science, Kyungpook National University, 1370 Sankyuk-dong, Buk-gu, Daegu 702-701 (Korea, Republic of); Ito, Yoshihiro [Kanagawa Academy of Science and Technology, KSP East 309, Sakado 3-2-1, Takatsu-ku, Kawasaki 213-0012 (Japan)

    2007-03-01

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

  1. Alginate foam-based three-dimensional culture to investigate drug sensitivity in primary leukaemia cells.

    Science.gov (United States)

    Karimpoor, Mahroo; Yebra-Fernandez, Eva; Parhizkar, Maryam; Orlu, Mine; Craig, Duncan; Khorashad, Jamshid S; Edirisinghe, Mohan

    2018-04-01

    The development of assays for evaluating the sensitivity of leukaemia cells to anti-cancer agents is becoming an important aspect of personalized medicine. Conventional cell cultures lack the three-dimensional (3D) structure of the bone marrow (BM), the extracellular matrix and stromal components which are crucial for the growth and survival of leukaemia stem cells. To accurately predict the sensitivity of the leukaemia cells in an in vitro assay a culturing system containing the essential components of BM is required. In this study, we developed a porous calcium alginate foam-based scaffold to be used for 3D culture. The new 3D culture was shown to be cell compatible as it supported the proliferation of both normal haematopoietic and leukaemia cells. Our cell differential assay for myeloid markers showed that the porous foam-based 3D culture enhanced myeloid differentiation in both leukaemia and normal haematopoietic cells compared to two-dimensional culture. The foam-based scaffold reduced the sensitivity of the leukaemia cells to the tested antileukaemia agents in K562 and HL60 leukaemia cell line model and also primary myeloid leukaemia cells. This observation supports the application of calcium alginate foams as scaffold components of the 3D cultures for investigation of sensitivity to antileukaemia agents in primary myeloid cells. © 2018 The Author(s).

  2. Generation of primary cultures of bovine brain endothelial cells and setup of cocultures with rat astrocytes

    DEFF Research Database (Denmark)

    Helms, Hans C; Brodin, Birger

    2014-01-01

    -brain barrier. The present protocol describes the setup of an in vitro coculture model based on primary cultures of endothelial cells from bovine brain microvessels and primary cultures of rat astrocytes. The model displays a high electrical tightness and expresses blood-brain barrier marker proteins....

  3. IFN-γ alters the expression of diverse immunity related genes in a cell culture model designed to represent maturing neutrophils.

    Directory of Open Access Journals (Sweden)

    Michael A Ellison

    expression within these classes of genes could help explain the immune supportive effects of IFN-γ. Next we explored if the effect of IFN-γ on expression of these genes is dependent on whether the cells are undergoing maturation; to do this we compared the effects of IFN-γ on cells cultured with and without DMSO. For a subset of genes the expression level changes caused by IFN-γ were much greater in maturing cells than non-maturing cells. These findings indicate that developmental changes associated with cell maturation can modulate the effects of IFN-γ but that this is gene specific. Since the effects of IFN-γ depend on whether cells are maturing, the gene expression changes observed in this study must be due to more than just prolonged application of IFN-γ and are instead the result of interplay between cell maturation and changes caused by the chemokine. This supports our hypothesis that the effects of IFN-γ on developing neutrophils in the bone marrow may be very different from its effects on mature cells in the blood. Collectively the findings in this study enhance our understanding of the effects of IFN-γ on maturing myeloid cells and indicate possible mechanisms by which this cytokine could support immune function.

  4. Stimulation of the proliferation of hemopoietic stem cells in irradiated bone marrow cell culture

    International Nuclear Information System (INIS)

    Mori, K.J.; Izumi, H.; Seto, A.

    1981-01-01

    Long-term hemopoiesis was established in bone marrow cell culture in vitro. This culture was shown to support the recovery proliferation of hemopoietic stem cells completely in vitro after irradiation. Hemopoietic stem cells were stimulated into proliferation in culture when normal bone marrow cells were overlayed on top of the irradiated adherent cell colonies. These results indicate that proliferation and differentiation of hemopoietic stem cells in vitro are also supported by stromahemopoietic cell interactions

  5. Hydrolyzed fish proteins modulates both inflammatory and antioxidant gene expression as well as protein expression in a co culture model of liver and head kidney cells isolated from Atlantic salmon (Salmo salar).

    Science.gov (United States)

    Holen, Elisabeth; He, Juyun; Araujo, Pedro; Seliussen, Jørgen; Espe, Marit

    2016-07-01

    Hydrolyzed fish proteins (H-pro) contain high concentrations of free amino acids and low molecular peptides that potentially may benefit fish health. The following study aimed to test whether the water-soluble phase of H-pro could attenuate lipopolysaccharide (LPS) provoked inflammation in liver cells and head kidney cells isolated from Atlantic salmon. Cells were grown as mono cultures or co cultures to assess possible crosstalk between immune cells and metabolic cells during treatments. Cells were added media with or without H-pro for 2 days before LPS exposure and harvested 24 h post LPS exposure. Respective cells without H-pro and LPS were used as controls. H-pro alone could affect expression of proteins directly as H-pro increased catalase protein expression in head kidney- and liver cells, regardless of culturing methods and LPS treatment. Leukotriene B4 (LTB4) production was also increased by H-pro in head kidney cells co cultured with liver cells. H-pro increased LPS induced interleukin 1β (IL-1β) transcription in liver cells co cultured with head kidney cells. All cultures of head kidney cells showed a significant increase in IL-1β transcription when treated with H-pro + LPS. H-pro decreased caspase-3 transcription in liver cells cultured co cultured with head kidney cells. Peroxisome proliferator activated receptor α (PPAR α) was upregulated, regardless of treatment, in liver cells co cultured with head kidney cells clearly showing that culturing method alone affected gene transcription. H-pro alone and together with LPS as an inflammation inducer, affect both antioxidant and inflammatory responses. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2011-12-17

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

  7. Culture of human cells in experimental units for spaceflight impacts on their behavior.

    Science.gov (United States)

    Cazzaniga, Alessandra; Moscheni, Claudia; Maier, Jeanette Am; Castiglioni, Sara

    2017-05-01

    Because space missions produce pathophysiological alterations such as cardiovascular disorders and bone demineralization which are very common on Earth, biomedical research in space is a frontier that holds important promises not only to counterbalance space-associated disorders in astronauts but also to ameliorate the health of Earth-bound population. Experiments in space are complex to design. Cells must be cultured in closed cell culture systems (from now defined experimental units (EUs)), which are biocompatible, functional, safe to minimize any potential hazard to the crew, and with a high degree of automation. Therefore, to perform experiments in orbit, it is relevant to know how closely culture in the EUs reflects cellular behavior under normal growth conditions. We compared the performances in these units of three different human cell types, which were recently space flown, i.e. bone mesenchymal stem cells, micro- and macrovascular endothelial cells. Endothelial cells are only slightly and transiently affected by culture in the EUs, whereas these devices accelerate mesenchymal stem cell reprogramming toward osteogenic differentiation, in part by increasing the amounts of reactive oxygen species. We conclude that cell culture conditions in the EUs do not exactly mimic what happens in a culture dish and that more efforts are necessary to optimize these devices for biomedical experiments in space. Impact statement Cell cultures represent valuable preclinical models to decipher pathogenic circuitries. This is true also for biomedical research in space. A lot has been learnt about cell adaptation and reaction from the experiments performed on many different cell types flown to space. Obviously, cell culture in space has to meet specific requirements for the safety of the crew and to comply with the unique environmental challenges. For these reasons, specific devices for cell culture in space have been developed. It is important to clarify whether these

  8. Modeling the genealogy of a cultural trait.

    Science.gov (United States)

    Aguilar, Elliot; Ghirlanda, Stefano

    2015-05-01

    The mathematical study of genealogies has yielded important insights in population biology, such as the ability to estimate the time to the most recent common ancestor (MRCA) of a sample of genetic sequences or of a group of individuals. Here we introduce a model of cultural genealogies that is a step toward answering similar questions for cultural traits. In our model individuals can inherit from a variable, potentially large number of ancestors, rather than from a fixed, small number of ancestors (one or two) as is typical of genetic evolution. We first show that, given a sample of individuals, a cultural common ancestor does not necessarily exist. We then introduce a related concept: the most recent unique ancestor (MRUA), i.e., the most recent single individual who is the earliest cultural ancestor of the sample. We show that, under neutral evolution, the time to the MRUA can be staggeringly larger than the time to MRCA in a single ancestor model, except when the average number of learning opportunities per individuals is small. Our results point out that the properties of cultural genealogies may be very different from those of genetic genealogies, with potential implications for reconstructing the histories of cultural traits. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. The Role of Glucose, Serum, and Three-Dimensional Cell Culture on the Metabolism of Bone Marrow-Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Byron Deorosan

    2011-01-01

    factors in the metabolic response of the cells. However, cells cultured in low density collagen exhibited considerable cell death, likely because of physical contraction of the collagen hydrogel which was not observed in the higher density collagen. These findings will be useful to the development of in vitro cell culture models that properly mimic in vivo physiological processes.

  10. Particle Trajectories in Rotating Wall Cell Culture Devices

    Science.gov (United States)

    Ramachandran N.; Downey, J. P.

    1999-01-01

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

  11. Microfluidic engineered high cell density three-dimensional neural cultures

    Science.gov (United States)

    Cullen, D. Kacy; Vukasinovic, Jelena; Glezer, Ari; La Placa, Michelle C.

    2007-06-01

    Three-dimensional (3D) neural cultures with cells distributed throughout a thick, bioactive protein scaffold may better represent neurobiological phenomena than planar correlates lacking matrix support. Neural cells in vivo interact within a complex, multicellular environment with tightly coupled 3D cell-cell/cell-matrix interactions; however, thick 3D neural cultures at cell densities approaching that of brain rapidly decay, presumably due to diffusion limited interstitial mass transport. To address this issue, we have developed a novel perfusion platform that utilizes forced intercellular convection to enhance mass transport. First, we demonstrated that in thick (>500 µm) 3D neural cultures supported by passive diffusion, cell densities =104 cells mm-3), continuous medium perfusion at 2.0-11.0 µL min-1 improved viability compared to non-perfused cultures (p death and matrix degradation. In perfused cultures, survival was dependent on proximity to the perfusion source at 2.00-6.25 µL min-1 (p 90% viability in both neuronal cultures and neuronal-astrocytic co-cultures. This work demonstrates the utility of forced interstitial convection in improving the survival of high cell density 3D engineered neural constructs and may aid in the development of novel tissue-engineered systems reconstituting 3D cell-cell/cell-matrix interactions.

  12. Usability and Applicability of Microfluidic Cell Culture Systems

    DEFF Research Database (Denmark)

    Hemmingsen, Mette

    possibilities for, for example, precise control of the chemical environment, 3D cultures, controlled co-culture of different cell types or automated, individual control of up to 96 cell culture chambers in one integrated system. Despite the great new opportunities to perform novel experimental designs......Microfluidic cell culture has been a research area with great attention the last decade due to its potential to mimic the in vivo cellular environment more closely compared to what is possible by conventional cell culture methods. Many exciting and complex devices have been presented providing......, these devices still lack general implementation into biological research laboratories. In this project, the usability and applicability of microfluidic cell culture systems have been investigated. The tested systems display good properties regarding optics and compatibility with standard laboratory equipment...

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

    Science.gov (United States)

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

    2015-07-01

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

  14. THE ALKALOID CYTISINE IN THE CELL CULTURE

    Directory of Open Access Journals (Sweden)

    Gazaliev A.M.

    2012-08-01

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

  15. Isolation and culture of larval cells from C. elegans.

    Directory of Open Access Journals (Sweden)

    Sihui Zhang

    Full Text Available Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×10(4 cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81% of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology.

  16. Rat brain sagittal organotypic slice cultures as an ex vivo dopamine cell loss system.

    Science.gov (United States)

    McCaughey-Chapman, Amy; Connor, Bronwen

    2017-02-01

    Organotypic brain slice cultures are a useful tool to study neurological function as they provide a more complex, 3-dimensional system than standard 2-dimensional in vitro cell cultures. Building on a previously developed mouse brain slice culture protocol, we have developed a rat sagittal brain slice culture system as an ex vivo model of dopamine cell loss. We show that rat brain organotypic slice cultures remain viable for up to 6 weeks in culture. Using Fluoro-Gold axonal tracing, we demonstrate that the slice 3-dimensional cytoarchitecture is maintained over a 4 week culturing period, with particular focus on the nigrostriatal pathway. Treatment of the cultures with 6-hydroxydopamine and desipramine induces a progressive loss of Fluoro-Gold-positive nigral cells with a sustained loss of tyrosine hydroxylase-positive nigral cells. This recapitulates the pattern of dopaminergic degeneration observed in the rat partial 6-hydroxydopamine lesion model and, most importantly, the progressive pathology of Parkinson's disease. Our slice culture platform provides an advance over other systems, as we demonstrate for the first time 3-dimensional cytoarchitecture maintenance of rat nigrostriatal sagittal slices for up to 6 weeks. Our ex vivo organotypic slice culture system provides a long term cellular platform to model Parkinson's disease, allowing for the elucidation of mechanisms involved in dopaminergic neuron degeneration and the capability to study cellular integration and plasticity ex vivo. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Indole and synthetic derivative activate chaperone expression to reduce polyQ aggregation in SCA17 neuronal cell and slice culture models

    Directory of Open Access Journals (Sweden)

    Kung PJ

    2014-10-01

    Full Text Available Pin-Jui Kung,1,* Yu-Chen Tao,1,* Ho-Chiang Hsu,1 Wan-Ling Chen,1 Te-Hsien Lin,1 Donala Janreddy,2 Ching-Fa Yao,2 Kuo-Hsuan Chang,3 Jung-Yaw Lin,1 Ming-Tsan Su,1 Chung-Hsin Wu,1 Guey-Jen Lee-Chen,1 Hsiu-Mei Hsieh-Li1 1Department of Life Science, 2Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan; 3Department of Neurology, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taipei, Taiwan *These authors contributed equally to this work Abstract: In spinocerebellar ataxia type 17 (SCA17, the expansion of a translated CAG repeat in the TATA box binding protein (TBP gene results in a long polyglutamine (polyQ tract in the TBP protein, leading to intracellular accumulation of aggregated TBP and cell death. The molecular chaperones act in preventing protein aggregation to ameliorate downstream harmful events. In this study, we used Tet-On SH-SY5Y cells with inducible SCA17 TBP/Q79-green fluorescent protein (GFP expression to test indole and synthetic derivative NC001-8 for neuroprotection. We found that indole and NC001-8 up-regulated chaperone expression to reduce polyQ aggregation in neuronal differentiated TBP/Q79 cells. The effects on promoting neurite outgrowth and on reduction of aggregation on Purkinje cells were also confirmed with cerebellar primary and slice cultures of SCA17 transgenic mice. Our results demonstrate how indole and derivative NC001-8 reduce polyQ aggregation to support their therapeutic potentials in SCA17 treatment. Keywords: spinocerebellar ataxia type 17, TATA box binding protein, polyQ aggregation, indole and derivative, therapeutics

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

    Science.gov (United States)

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

    2011-11-15

    The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here, we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells, as predicted from a numerical model of cell migration, and results in significant increases in cell growth of undifferentiated cells. These chemically defined xeno-free substrates generate more than three times the number of cells than feeder-containing substrates per surface area. Further, reprogramming and typical gene-targeting protocols can be readily performed on these engineered surfaces. These substrates provide an attractive cell culture platform for the production of clinically relevant factor-free reprogrammed cells from patient tissue samples and facilitate the definition of standardized scale-up friendly methods for disease modeling and cell therapeutic applications.

  19. Investigating Effects of Gelatin-Chitosan Film on Culture of Bone Marrow Stromal Cells in Rat

    Directory of Open Access Journals (Sweden)

    A Karami joyani

    2015-02-01

    Conclusion: Results of proliferation,differentiation and apoptosis cultured BMSCs on a gelatin-chitosan film showed that gelatin-chitosan film can be used as a good model of a biodegradable scaffold in tissue engineering and cell therapy.

  20. Biogelx: Cell Culture on Self-Assembling Peptide Gels.

    Science.gov (United States)

    Harper, Mhairi M; Connolly, Michael L; Goldie, Laura; Irvine, Eleanore J; Shaw, Joshua E; Jayawarna, Vineetha; Richardson, Stephen M; Dalby, Matthew J; Lightbody, David; Ulijn, Rein V

    2018-01-01

    Aromatic peptide amphiphiles can form self-supporting nanostructured hydrogels with tunable mechanical properties and chemical compositions. These hydrogels are increasingly applied in two-dimensional (2D) and three-dimensional (3D) cell culture, where there is a rapidly growing need to store, grow, proliferate, and manipulate naturally derived cells within a hydrated, 3D matrix. Biogelx Limited is a biomaterials company, created to commercialize these bio-inspired hydrogels to cell biologists for a range of cell culture applications. This chapter describes methods of various characterization and cell culture techniques specifically optimized for compatibility with Biogelx products.

  1. Culture media from hypoxia conditioned endothelial cells protect human intestinal cells from hypoxia/reoxygenation injury.

    Science.gov (United States)

    Hummitzsch, Lars; Zitta, Karina; Bein, Berthold; Steinfath, Markus; Albrecht, Martin

    2014-03-10

    Remote ischemic preconditioning (RIPC) is a phenomenon, whereby short episodes of non-lethal ischemia to an organ or tissue exert protection against ischemia/reperfusion injury in a distant organ. However, there is still an apparent lack of knowledge concerning the RIPC-mediated mechanisms within the target organ and the released factors. Here we established a human cell culture model to investigate cellular and molecular effects of RIPC and to identify factors responsible for RIPC-mediated intestinal protection. Human umbilical vein cells (HUVEC) were exposed to repeated episodes of hypoxia (3 × 15 min) and conditioned culture media (CM) were collected after 24h. Human intestinal cells (CaCo-2) were cultured with or without CM and subjected to 90 min of hypoxia/reoxygenation injury. Reverse transcription-polymerase chain reaction, Western blotting, gelatin zymography, hydrogen peroxide measurements and lactate dehydrogenase (LDH) assays were performed. In HUVEC cultures hypoxic conditioning did not influence the profile of secreted proteins but led to an increased gelatinase activity (Pcultures 90 min of hypoxia/reoxygenation resulted in morphological signs of cell damage, increased LDH levels (Pculture model may help to unravel RIPC-mediated cellular events and to identify molecules released by RIPC. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Development of a microfluidic perfusion 3D cell culture system

    Science.gov (United States)

    Park, D. H.; Jeon, H. J.; Kim, M. J.; Nguyen, X. D.; Morten, K.; Go, J. S.

    2018-04-01

    Recently, 3-dimensional in vitro cell cultures have gained much attention in biomedical sciences because of the closer relevance between in vitro cell cultures and in vivo environments. This paper presents a microfluidic perfusion 3D cell culture system with consistent control of long-term culture conditions to mimic an in vivo microenvironment. It consists of two sudden expansion reservoirs to trap incoming air bubbles, gradient generators to provide a linear concentration, and microchannel mixers. Specifically, the air bubbles disturb a flow in the microfluidic channel resulting in the instability of the perfusion cell culture conditions. For long-term stable operation, the sudden expansion reservoir is designed to trap air bubbles by using buoyancy before they enter the culture system. The performance of the developed microfluidic perfusion 3D cell culture system was examined experimentally and compared with analytical results. Finally, it was applied to test the cytotoxicity of cells infected with Ewing’s sarcoma. Cell death was observed for different concentrations of H2O2. For future work, the developed microfluidic perfusion 3D cell culture system can be used to examine the behavior of cells treated with various drugs and concentrations for high-throughput drug screening.

  3. What is culture in «cultural economy»? Defining culture to create measurable models in cultural economy

    Directory of Open Access Journals (Sweden)

    Aníbal Monasterio Astobiza

    2017-07-01

    Full Text Available The idea of culture is somewhat vague and ambiguous for the formal goals of economics. The aim of this paper is to define the notion of culture better so as to help build economic explanations based on culture and therefore to measure its impact in every activity or beliefs associated with culture. To define culture according to the canonical evolutionary definition, it is any kind of ritualised behaviour that becomes meaningful for a group and that remains more or less constant and is transmitted down through the generations. Economic institutions are founded, implicitly or explicitly, on a worldview of how humans function; culture is an essential part of understanding us as humans, making it necessary to describe what we understand by culture correctly. In this paper we review the literature on evolutionary anthropology and psychology dealing with the concept of culture to warn that economic modelling ignores intangible benefits of culture rendering economics unable to measure certain cultural items in the digital consumer society.

  4. A method for culturing human hair follicle cells.

    Science.gov (United States)

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

    1981-01-01

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

  5. Growth Inhibition of Osteosarcoma Cell Lines in 3D Cultures: Role of Nitrosative and Oxidative Stress.

    Science.gov (United States)

    Gorska, Magdalena; Krzywiec, Pawel Bieniasz; Kuban-Jankowska, Alicja; Zmijewski, Michal; Wozniak, Michal; Wierzbicka, Justyna; Piotrowska, Anna; Siwicka, Karolina

    2016-01-01

    3D cell cultures have revolutionized the understanding of cell behavior, allowing culture of cells with the possibility of resembling in vivo intercellular signaling and cell-extracellular matrix interaction. The effect of limited oxygen penetration into 3D culture of highly metastatic osteosarcoma 143B cells in terms of expression of nitro-oxidative stress markers was investigated and compared to standard 2D cell culture. Human osteosarcoma (143B cell line) cells were cultured as monolayers, in collagen and Matrigel. Cell viability, gene expression of nitro-oxidative stress markers, and vascular endothelial growth factor were determined using Trypan blue assay, quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Three-dimensional environments modify nitro-oxidative stress and influence gene expression and cell proliferation of OS 143B cells. Commercial cell lines might not constitute a good model of 3D cultures for bone tissue engineering, as they are highly sensitive to hypoxia, and hypoxic conditions can induce oxidation of the cellular environment. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  6. Antiviral Effect of Ribavirin against HCV Associated with Increased Frequency of G-to-A and C-to-U Transitions in Infectious Cell Culture Model

    DEFF Research Database (Denmark)

    Galli, Andrea; Mens, Helene; Gottwein, Judith M

    2018-01-01

    -polymerase gene was amplified, cloned, and sequenced to estimate genetic distances. We confirm that the antiviral effect of all three RBV-drug forms on HCV relies on induction of specific transitions (G-to-A and C-to-U). These mutations lead to generation of non-infectious virions, reflected by decreased spread......Ribavirin (RBV) is a broad-spectrum antiviral active against a wide range of RNA viruses. Despite having been used for decades in the treatment of chronic hepatitis C virus (HCV) infection, the precise mechanism of action of RBV is unknown. In other viruses, it inhibits propagation by increasing...... the rate of G-to-A and C-to-U transitions. Here, we utilized the J6/JFH1 HCV cell-culture system to investigate whether RBV inhibits HCV through the same mechanism. Infected Huh7.5 cells were treated with increasing concentrations of RBV or its phosphorylated forms. A fragment of the HCV NS5B...

  7. Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture.

    Science.gov (United States)

    Barta, Tomas; Dolezalova, Dasa; Holubcova, Zuzana; Hampl, Ales

    2013-03-01

    Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.

  8. Molecular mechanisms underlying protective effects of quercetin against mitochondrial dysfunction and progressive dopaminergic neurodegeneration in cell culture and MitoPark transgenic mouse models of Parkinson's Disease.

    Science.gov (United States)

    Ay, Muhammet; Luo, Jie; Langley, Monica; Jin, Huajun; Anantharam, Vellareddy; Kanthasamy, Arthi; Kanthasamy, Anumantha G

    2017-06-01

    Quercetin, one of the major flavonoids in plants, has been recently reported to have neuroprotective effects against neurodegenerative processes. However, since the molecular signaling mechanisms governing these effects are not well clarified, we evaluated quercetin's effect on the neuroprotective signaling events in dopaminergic neuronal models and further tested its efficacy in the MitoPark transgenic mouse model of Parkinson's disease (PD). Western blot analysis revealed that quercetin significantly induced the activation of two major cell survival kinases, protein kinase D1 (PKD1) and Akt in MN9D dopaminergic neuronal cells. Furthermore, pharmacological inhibition or siRNA knockdown of PKD1 blocked the activation of Akt, suggesting that PKD1 acts as an upstream regulator of Akt in quercetin-mediated neuroprotective signaling. Quercetin also enhanced cAMP response-element binding protein phosphorylation and expression of the cAMP response-element binding protein target gene brain-derived neurotrophic factor. Results from qRT-PCR, Western blot analysis, mtDNA content analysis, and MitoTracker assay experiments revealed that quercetin augmented mitochondrial biogenesis. Quercetin also increased mitochondrial bioenergetics capacity and protected MN9D cells against 6-hydroxydopamine-induced neurotoxicity. To further evaluate the neuroprotective efficacy of quercetin against the mitochondrial dysfunction underlying PD, we used the progressive dopaminergic neurodegenerative MitoPark transgenic mouse model of PD. Oral administration of quercetin significantly reversed behavioral deficits, striatal dopamine depletion, and TH neuronal cell loss in MitoPark mice. Together, our findings demonstrate that quercetin activates the PKD1-Akt cell survival signaling axis and suggest that further exploration of quercetin as a promising neuroprotective agent for treating PD may offer clinical benefits. © 2017 International Society for Neurochemistry.

  9. Rabbit uterine epithelial cells: Co-culture with spermatozoa

    International Nuclear Information System (INIS)

    Boice, M.L.

    1988-01-01

    A primary culture of rabbit uterine epithelial cells was established and their effects on sperm function were examined in vitro. Epithelial cells were isolated from uteri of estrous rabbits and cultured on floating collagen gels in phenol red-free medium supplemented with 5% fetal bovine serum. Light microscopy and keratin staining showed that the epithelial cell population established in culture had morphological characteristics similar to that seen in the intact endometrium. Cells were cultured with 3 H-leucine and uptake of label by cells and its incorporation into cellular and secretory proteins determined. When compared to cells cultured for 24-48 h, incorporation of label into cellular protein was lower at 72-96 h, but secretion increased. Estradiol 17-β did not affect label uptake or incorporation, but did enhance proliferation of cells as judged by total DNA content of the cell population. Analysis of proteins in media by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography suggested that epithelial and stromal cells synthesis proteins that may be secretory in nature during 72-96 h culture. Twenty-nine to thirty-one h after initiation of epithelial cultures, 1-2 x 10 6 sperm were co-incubated with cells and sperm viability, motility, loss of acrosome and fertilizing ability determined

  10. Metabolic Activation of the Organic Fraction Coated Onto Air Pollution PM2.5 and its Genotoxicity in a Co Culture Model of Human Lung Cells

    International Nuclear Information System (INIS)

    Abbas, I; Garcon, G; Billet, S.; Verdin, A.; Escande, F.; Saint-Georges, F.; Mulliez, Ph.; Gosset, P.; Shirali, P.

    2011-01-01

    Air pollution Particulate Matter (PM 2 .5) is described as one of the major risk factors affecting human health. Hence, the objective of our research project was to evaluate the lung toxicity of PM 2 .5 collected in Dunkerque (France), through the study of the metabolic activation of its organic fraction (e.g. Polycyclic Aromatic Hydrocarbons, PAHs; Volatile Organic Compounds, VOCs) and its genotoxicity in two human cell models: embryonic lung epithelial L132 cells and Alveolar Macrophages (AM) isolated from bronchiolo-alveolar lavages of healthy outpatients, in mono- and/or coculture. The coculture system we used allowed the direct exposure of AM to PM 2 .5, and the interaction between the two cell types only through soluble factor diffusion. Exposure to Dunkerque City's PM 2 .5 induced the gene expression of phase I and phase II enzymes (e.g. CYP1A1, CYP2E1, CYP2F1, NQO1, GSTπ1, GSTμ3) involved in the metabolic activation of PAHS and/or VOCS, in AM, in mono- and coculture, and in L132 cells, only in monoculture. Taken together, these results reinforced the key role of AM in lung defenses, and indicated that particles, as physical vector of the penetration and retention of coated-PAHS and/or VOCS within cells, enabled them to exert a durable toxicity. DNA bulky adduct formation was also reported not only in Dunkerque City's PM 2 .5-exposed AM, in mono- and coculture, but also in L132 cells from PAH-exposed coculture. Loss of Heterozygosity (LOH) and/or MicroSatellite Instability (MSI) of some MicroSatellites (MS) located in multiple critical regions of chromosome 3 were reported in L132 cells from Dunkerque City's PM 2 .5-exposed mono- or cocultures. (author)

  11. Long-term maintenance of human induced pluripotent stem cells by automated cell culture system.

    Science.gov (United States)

    Konagaya, Shuhei; Ando, Takeshi; Yamauchi, Toshiaki; Suemori, Hirofumi; Iwata, Hiroo

    2015-11-17

    Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem (iPS) cells, are regarded as new sources for cell replacement therapy. These cells can unlimitedly expand under undifferentiated conditions and be differentiated into multiple cell types. Automated culture systems enable the large-scale production of cells. In addition to reducing the time and effort of researchers, an automated culture system improves the reproducibility of cell cultures. In the present study, we newly designed a fully automated cell culture system for human iPS maintenance. Using an automated culture system, hiPS cells maintained their undifferentiated state for 60 days. Automatically prepared hiPS cells had a potency of differentiation into three germ layer cells including dopaminergic neurons and pancreatic cells.

  12. Biona-C Cell Culture pH Monitoring System

    Science.gov (United States)

    Friedericks, C.

    1999-01-01

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

  13. Sequential cancer mutations in cultured human intestinal stem cells

    NARCIS (Netherlands)

    Drost, Jarno; van Jaarsveld, Richard H.; Ponsioen, Bas; Zimberlin, Cheryl; van Boxtel, Ruben; Buijs, Arjan; Sachs, Norman; Overmeer, René M.; Offerhaus, G. Johan; Begthel, Harry; Korving, Jeroen; van de Wetering, Marc; Schwank, Gerald; Logtenberg, Meike; Cuppen, Edwin; Snippert, Hugo J.; Medema, Jan Paul; Kops, Geert J. P. L.; Clevers, Hans

    2015-01-01

    Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-07-01

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

  16. PECULIARITIES OF SECONDARY METABOLITES BIOSYNTHESIS IN PLANT CELL CULTURES

    Directory of Open Access Journals (Sweden)

    A.M. NOSOV

    2014-06-01

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

  17. Inferring cultural models from corpus data

    DEFF Research Database (Denmark)

    Jensen, Kim Ebensgaard

    2015-01-01

    on three instantiations of the construction – namely, [too young to V], [too proud to V], and [too macho to V] – to see whether there are patterns in their ranges of coattracted verbs that are indicative of force-dynamic relations in cultural models of age, pride, and machismo respectively....

  18. Hexavalent chromium at low concentration alters Sertoli cell barrier and connexin 43 gap junction but not claudin-11 and N-cadherin in the rat seminiferous tubule culture model

    Energy Technology Data Exchange (ETDEWEB)

    Carette, Diane [INSERM U 1065, Team 5 “Physiopathology of Germ Cell Control: Genomic and Non Genomic Mechanisms” C3M, University of Nice Sophia Antipolis, Nice (France); UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris (France); Perrard, Marie-Hélène, E-mail: marie-helene.durand@ens-lyon.fr [Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon I, CNRS, INRA, Ecole Normale Supérieure de Lyon, Lyon (France); Prisant, Nadia [University of Versailles/St Quentin-en-Yvelines (France); UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris (France); Gilleron, Jérome; Pointis, Georges [INSERM U 1065, Team 5 “Physiopathology of Germ Cell Control: Genomic and Non Genomic Mechanisms” C3M, University of Nice Sophia Antipolis, Nice (France); Segretain, Dominique [University of Versailles/St Quentin-en-Yvelines (France); UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris (France); Durand, Philippe [Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon I, CNRS, INRA, Ecole Normale Supérieure de Lyon, Lyon (France); Kallistem SAS Ecole Normale Supérieure de Lyon, Lyon (France)

    2013-04-01

    Exposure to toxic metals, specifically those belonging to the nonessential group leads to human health defects and among them reprotoxic effects. The mechanisms by which these metals produce their negative effects on spermatogenesis have not been fully elucidated. By using the Durand's validated seminiferous tubule culture model, which mimics the in vivo situation, we recently reported that concentrations of hexavalent chromium, reported in the literature to be closed to that found in the blood circulation of men, increase the number of germ cell cytogenetic abnormalities. Since this metal is also known to affect cellular junctions, we investigated, in the present study, its potential influence on the Sertoli cell barrier and on junctional proteins present at this level such as connexin 43, claudin-11 and N-cadherin. Cultured seminiferous tubules in bicameral chambers expressed the three junctional proteins and ZO-1 for at least 12 days. Exposure to low concentrations of chromium (10 μg/l) increased the trans-epithelial resistance without major changes of claudin-11 and N-cadherin expressions but strongly delocalized the gap junction protein connexin 43 from the membrane to the cytoplasm of Sertoli cells. The possibility that the hexavalent chromium-induced alteration of connexin 43 indirectly mediates the effect of the toxic metal on the blood–testis barrier dynamic is postulated. - Highlights: ► Influence of Cr(VI) on the Sertoli cell barrier and on junctional proteins ► Use of cultured seminiferous tubules in bicameral chambers ► Low concentrations of Cr(VI) (10 μg/l) altered the trans-epithelial resistance. ► Cr(VI) did not alter claudin-11 and N-cadherin. ► Cr(VI) delocalized connexin 43 from the membrane to the cytoplasm of Sertoli cells.

  19. Ultrasonic three-dimensional on-chip cell culture for dynamic studies of tumor immune surveillance by natural killer cells.

    Science.gov (United States)

    Christakou, Athanasia E; Ohlin, Mathias; Önfelt, Björn; Wiklund, Martin

    2015-08-07

    We demonstrate a simple method for three-dimensional (3D) cell culture controlled by ultrasonic standing waves in a multi-well microplate. The method gently arranges cells in a suspension into a single aggregate in each well of the microplate and, by this, nucleates 3D tissue-like cell growth for culture times between two and seven days. The microplate device is compatible with both high-resolution optical microscopy and maintenance in a standard cell incubator. The result is a scaffold- and coating-free method for 3D cell culture that can be used for controlling the cellular architecture, as well as the cellular and molecular composition of the microenvironment in and around the formed cell structures. We demonstrate the parallel production of one hundred synthetic 3D solid tumors comprising up to thousands of human hepatocellular carcinoma (HCC) HepG2 cells, we characterize the tumor structure by high-resolution optical microscopy, and we monitor the functional behavior of natural killer (NK) cells migrating, docking and interacting with the tumor model during culture. Our results show that the method can be used for determining the collective ability of a given number of NK cells to defeat a solid tumor having a certain size, shape and composition. The ultrasound-based method itself is generic and can meet any demand from applications where it is advantageous to monitor cell culture from production to analysis of 3D tissue or tumor models using microscopy in one single microplate device.

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

    Science.gov (United States)

    Gebhard, C; Gabriel, C; Walter, I

    2016-06-01

    Spheroid cell culture emerges as powerful in vitro tool for experimental tumour research. In this study, we established a scaffold-free three-dimensional spheroid system built from canine osteosarcoma (OS) cells (D17). Spheroids (7, 14 and 19 days of cultivation) and monolayer cultures (2 and 7 days of cultivation) were evaluated and compared on light and electron microscopy. Monolayer and spheroid cultures were tested for vimentin, cytokeratin, alkaline phosphatase, osteocalcin and collagen I by means of immunohistochemistry. The spheroid cell culture exhibited a distinct network of collagen I in particular after 19-day cultivation, whereas in monolayer cultures, collagen I was arranged as a lamellar basal structure. Necrotic centres of large spheroids, as observed in 14- and 19-day cultures, were characterized by significant amounts of osteocalcin. Proliferative activity as determined by Ki-67 immunoreactivity showed an even distribution in two-dimensional cultures. In spheroids, proliferation was predominating in the peripheral areas. Metastasis-associated markers ezrin and S100A4 were shown to be continuously expressed in monolayer and spheroid cultures. We conclude that the scaffold-free spheroid system from canine OS cells has the ability to mimic the architecture of the in vivo tumour, in particular cell-cell and cell-matrix interactions. © 2015 The Authors. Anatomia, Histologia, Embryologia Published by Blackwell Verlag GmbH.

  1. Cell cultures in uterine leiomyomas: rapid disappearance of cells carrying MED12 mutations.

    Science.gov (United States)

    Nadine Markowski, Dominique; Tadayyon, Mahboobeh; Bartnitzke, Sabine; Belge, Gazanfer; Maria Helmke, Burkhard; Bullerdiek, Jörn

    2014-04-01

    Uterine leiomyomas (UL) are the most frequent symptomatic human tumors. Nevertheless, their molecular pathogenesis is not yet fully understood. To learn more about the biology of these common neoplasms and their response to treatment, cell cultures derived from UL are a frequently used model system, but until recently appropriate genetic markers confirming their origin from the tumor cell population were lacking for most UL, i.e., those not displaying karyotypic abnormalities. The identification of MED12 mutations in the majority of UL makes it possible to trace the tumor cell population during in vitro passaging in the absence of cytogenetic abnormalities. The present study is addressing the in vitro survival of cells carrying MED12 mutations and its association with karyotypic alterations. The results challenge numerous in vitro studies into the biology and behavior of leiomyomas. Cells of one genetic subtype of UL, i.e., those with rearrangements of the high mobility AT-hook 2 protein gene (HMGA2), seem to be able to proliferate in vitro for many passages whereas tumor cells from the much more frequent MED12-mutated lesions barely survive even the first passages. Apparently, for the most frequent type of human UL no good in vitro model seems to exist because cells do not survive culturing. On the other hand, this inability may point to an Achilles' heel of this type of UL. Copyright © 2014 Wiley Periodicals, Inc.

  2. Cultural models, socialization goals, and parenting ethnotheories: A multicultural analysis

    OpenAIRE

    Keller, H; Lamm, B; Abels, M; Yovsi, R; Borke, J; Jensen, H; Papaligoura, Z; Holub, C; Lo, W; Tomiyama, AJ; Su, Y; Wang, Y; Chaudhary, N

    2006-01-01

    This study conceptualizes a cultural model of parenting. It is argued that cultural models are expressed in the degree of familism, which informs socialization goals that are embodied in parenting ethnotheories. Three cultural models were differentiated a priori: independent, interdependent, and autonomous-related. Samples were recruited that were expected to represent these cultural models: German, Euro-American, and Greek middle-class women representing the independent cultural model; Camer...

  3. Role of differential physical properties in emergent behavior of 3D cell co-cultures

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Babst Benjamin A

    2009-12-01

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

  5. Cell cycle phase of nondividing cells in aging human cell cultures determined by DNA content and chromosomal constitution

    International Nuclear Information System (INIS)

    Yanishevsky, R.M.

    1975-01-01

    Human diploid cell cultures, strain WI-38, have a finite proliferative capacity and have been proposed as a model of biological aging. To identify the cell cycle phase of the nondividing cells, cultures of various ages were exposed to 3 Hdt for 48 hours to label dividing cells, then the cycle phase was identified for individual cells by one of two methods, and finally, the proliferative status of the same cells was scored by autoradiographic evidence of 3 HdT uptake. The methods to identify the cycle phase were: determination of DNA strain content by Feulgen scanning cytophotometry, and determination of chromosome constitution by the technique of premature chromosome condensation (PCC). Preliminary experiments showed the effect of continuous exposure to various levels of 3 HdT on cell growth. High levels of 3 HdT inhibited cell cycle traverse: the cell number and labeling index curves reached a plateau; the cell volume increased; the cells accumulated with 4C DNA contents and it appeared that they blocked in G 2 phase. This pattern is consistent with a radiation effect. (U.S.)

  6. Assessment of long-term effects of nanoparticles in a microcarrier cell culture system.

    Directory of Open Access Journals (Sweden)

    Maria Mrakovcic

    Full Text Available 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 (BioLevitator(TM. 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.

  7. Morphology of primary human venous endothelial cell cultures before and after culture medium exchange.

    Science.gov (United States)

    Krüger-Genge, A; Fuhrmann, R; Jung, F; Franke, R P

    2015-01-01

    The evaluation of the interaction of human, venous endothelial cells (HUVEC) with body foreign materials on the cellular level cannot be performed in vivo, but is investigated in vitro under standard culture conditions. To maintain the vitality, proliferation and morphology of HUVEC seeded on body foreign substrates over days, the cell culture medium is usually exchanged every second day. It is well known, that alterations in the microenvironment of cells bear the risk of influencing cell morphology and function. In the current study the influence of cell culture medium exchange on HUVEC cytoskeletal microfilament structure and function was investigated. HUVEC in the third passage were seeded on extracellular matrix (ECM) - which was secreted from bovine corneal endothelial cells on glass- until functional confluence was reached. The experiment started 11 days after HUVEC seeding with an exchange of the cell culture medium followed by a staining of the actin microfilaments with phalloidin-rhodamin 1.5 and 5 minutes after medium exchange. The microfilaments were documented by use of an Olympus microscope (IMT-2) equipped with a UV lamp and online connected to a TV chain (Sony XC 50 ST/monochrome) implying an OPTIMAS - Image analysis system. Prostacyclin was analysed in the cell culture supernatant. 1.5 min after culture medium exchange in the functionally confluent cultures a slight disturbance of the actin microfilament structure with a broadening of the marginal filament band, a partial disconnection of cell-cell contacts and the appearance of intercellular fenestrations were observed. 5 minutes after medium exchange a redevelopment of the slightly disturbed microfilament structure with a condensation and narrowing of the marginal filament band was seen. 12 h later a further consolidation of the microfilament structure occurred. In addition, a perturbation of the cultured HUVEC occurred after cell culture medium exchange. The prostacyclin concentration in the

  8. Neuroprotective effects of a brain permeant 6-aminoquinoxaline derivative in cell culture conditions that model the loss of dopaminergic neurons in Parkinson disease.

    Science.gov (United States)

    Le Douaron, Gael; Schmidt, Fanny; Amar, Majid; Kadar, Hanane; Debortoli, Lucila; Latini, Alexandra; Séon-Méniel, Blandine; Ferrié, Laurent; Michel, Patrick Pierre; Touboul, David; Brunelle, Alain; Raisman-Vozari, Rita; Figadère, Bruno

    2015-01-07

    Parkinson disease is a neurodegenerative disorder of aging, characterized by disabling motor symptoms resulting from the loss of midbrain dopaminergic neurons and the decrease of dopamine in the striatum. Current therapies are directed at treating the symptoms but there is presently no cure for the disease. In order to discover neuroprotective compounds with a therapeutical potential, our research team has established original and highly regioselective methods for the synthesis of 2,3-disubstituted 6-aminoquinoxalines. To evaluate the neuroprotective activity of these molecules, we used midbrain cultures and various experimental conditions that promote dopaminergic cell loss. Among a series of 11 molecules, only compound MPAQ (2-methyl-3-phenyl-6-aminoquinoxaline) afforded substantial protection in a paradigm where dopaminergic neurons die spontaneously and progressively as they mature. Prediction of blood-brain barrier permeation by Quantitative Structure-Activity Relationship studies (QSARs) suggested that MPAQ was able to reach the brain parenchyma with sufficient efficacy. HPLC-MS/MS quantification in brain homogenates and MALDI-TOF mass spectrometry imaging on brain tissue sections performed in MPAQ-treated mice allowed us to confirm this prediction and to demonstrate, by MALDI-TOF mass spectrometry imaging, that MPAQ was localized in areas containing vulnerable neurons and/or their terminals. Of interest, MPAQ also rescued dopaminergic neurons, which (i) acquired dependency on the trophic peptide GDNF for their survival or (ii) underwent oxidative stress-mediated insults mediated by catalytically active iron. In summary, MPAQ possesses an interesting pharmacological profile as it penetrates the brain parenchyma and counteracts mechanisms possibly contributive to dopaminergic cell death in Parkinson disease. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  9. Radiosensitivity of primary cultured fish cells with different ploidy

    International Nuclear Information System (INIS)

    Mitani, Hiroshi; Egami, Nobuo; Kobayashi, Hiromu.

    1986-01-01

    The radiosensitivity of primary cultured goldfish cells (Carassius auratus) was investigated by colony formation assay. The radiosensitivity of cells from two varieties of goldfish, which show different sensitivity to lethal effect of ionizing radiation in vivo, was almost identical. Primary cultured cells from diploid, triploid and tetraploid fish retained their DNA content as measured by microfluorometry, and the nuclear size increases as ploidy increases. However, radiosensitivity was not related to ploidy. (author)

  10. Study of the cell activity in three-dimensional cell culture by using Raman spectroscopy

    Science.gov (United States)

    Arunngam, Pakajiraporn; Mahardika, Anggara; Hiroko, Matsuyoshi; Andriana, Bibin Bintang; Tabata, Yasuhiko; Sato, Hidetoshi

    2018-02-01

    The purpose of this study is to develop a estimation technique of local cell activity in cultured 3D cell aggregate with gelatin hydrogel microspheres by using Raman spectroscopy. It is an invaluable technique allowing real-time, nondestructive, and invasive measurement. Cells in body generally exist in 3D structure, which physiological cell-cell interaction enhances cell survival and biological functions. Although a 3D cell aggregate is a good model of the cells in living tissues, it was difficult to estimate their physiological conditions because there is no effective technique to make observation of intact cells in the 3D structure. In this study, cell aggregates were formed by MC3T-E1 (pre-osteoblast) cells and gelatin hydrogel microspheres. In appropriate condition MC3T-E1 cells can differentiate into osteoblast. We assume that the activity of the cell would be different according to the location in the aggregate because the cells near the surface of the aggregate have more access to oxygen and nutrient. Raman imaging technique was applied to measure 3D image of the aggregate. The concentration of the hydroxyapatite (HA) is generated by osteoblast was estimated with a strong band at 950-970 cm-1 which assigned to PO43- in HA. It reflects an activity of the specific site in the cell aggregate. The cell density in this specific site was analyzed by multivariate analysis of the 3D Raman image. Hence, the ratio between intensity and cell density in the site represents the cell activity.

  11. Comparative expression profile of NOD1/2 and certain acute inflammatory cytokines in thermal-stressed cell culture model of native and crossbred cattle

    Science.gov (United States)

    Bhanuprakash, V.; Singh, Umesh; Sengar, Gyanendra Singh; Raja, T. V.; Sajjanar, Basavraj; Alex, Rani; Kumar, Sushil; Alyethodi, R. R.; Kumar, Ashish; Sharma, Ankur; Kumar, Suresh; Bhusan, Bharat; Deb, Rajib

    2017-05-01

    Thermotolerance depends mainly on the health and immune status of the animals. The variation in the immune status of the animals may alter the level of tolerance of animals exposed to heat or cold stress. The present study was conducted to investigate the expression profile of two important nucleotide binding and oligomerization domain receptors (NLRs) (NOD1 and NOD2) and their central signalling molecule RIP2 gene during in vitro thermal-stressed bovine peripheral blood mononuclear cells (PBMCs) of native (Sahiwal) and crossbred (Sahiwal X HF) cattle. We also examined the differential expression profile of certain acute inflammatory cytokines in in vitro thermal-stressed PBMC culture among native and its crossbred counterparts. Results revealed that the expression profile of NOD1/2 positively correlates with the thermal stress, signalling molecule and cytokines. Present findings also highlighted that the expression patterns during thermal stress were comparatively superior among indigenous compared to crossbred cattle which may add references regarding the better immune adaptability of Zebu cattle.

  12. A cultural model of household energy consumption

    International Nuclear Information System (INIS)

    Lutzenhiser, Loren

    1992-01-01

    In this paper, we consider the development of demand-side research, from an early interest in conservation behavior to a later focus on physical, economic, psychological and social models of energy consumption. Unfortunately, none of these models account satisfactorily for measured energy consumption in the residential sector. Growing interest in the end-uses of energy (e.g. in support of load forecasting, demand-side management and least-cost utility planning), increasing international studies of energy use, and continuing work in the energy and lifestyles research tradition now support an emerging cultural perspective on household energy use. The ecological foundations of the cultural model and its applications in energy research are discussed, along with some of the analytic consequences of this approach. (author)

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

    Science.gov (United States)

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

    1996-01-01

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

  14. Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells.

    Science.gov (United States)

    Skottman, H; Muranen, J; Lähdekorpi, H; Pajula, E; Mäkelä, K; Koivusalo, L; Koistinen, A; Uusitalo, H; Kaarniranta, K; Juuti-Uusitalo, K

    2017-10-01

    Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Radiation adaptive response for the growth of cultured glial cells

    International Nuclear Information System (INIS)

    Suzuki, S.; Miura, Y.; Kano, M.; Toda, T.; Urano, S.

    2003-01-01

    Full text: To examine the molecular mechanism of radiation adaptive response (RAR) for the growth of cultured glial cells and to investigate the influence of aging on the response, glial cells were cultured from young and aged rats (1 month and 24 months old). RAR for the growth of glial cells conditioned with a low dose of X-rays and subsequently exposed to a high dose of X-rays was examined for cell number and BrdU incorporation. Involvement of the subcellular signaling pathway factors in RAR was investigated using their inhibitors, activators and mutated glial cells. RAR was observed in cells cultured from young rats, but was not in cells from aged rats. The inhibitors of protein kinase C (PKC) and DNA-dependent protein kinase (DNA-PK) or phosphatidylinositol 3-kinase (PI3K) suppressed RAR. The activators of PKC instead of low dose irradiation also caused RAR. Moreover, glial cells cultured from severe combined immunodeficiency (scid) mice (CB-17 scid) and ataxia-telangiectasia (AT) cells from AT patients showed no RAR. These results indicated that PKC, ATM, DNAPK and/or PI3K were involved in RAR for growth and BrdU incorporation of cultured glial cells and RAR decreased with aging. Proteomics data of glial cells exposed to severe stress of H 2 O 2 or X-rays also will be presented in the conference since little or no difference has not been observed with slight stress yet

  16. A human thymic epithelial cell culture system for the promotion of lymphopoiesis from hematopoietic stem cells.

    Science.gov (United States)

    Beaudette-Zlatanova, Britte C; Knight, Katherine L; Zhang, Shubin; Stiff, Patrick J; Zúñiga-Pflücker, Juan Carlos; Le, Phong T

    2011-05-01

    A human thymic epithelial cell (TEC) line expressing human leukocyte antigen-ABC and human leukocyte antigen-DR was engineered to overexpress murine Delta-like 1 (TEC-Dl1) for the purpose of establishing a human culture system that supports T lymphopoiesis from hematopoietic progenitor cells (HPCs). Cord blood or bone marrow HPCs were co-cultured with either the parental TEC line expressing low levels of the Notch ligands, Delta-like 1 and Delta-like 4, or with TEC-Dl1 to determine if these cell lines support human lymphopoiesis. In co-cultures with cord blood or bone marrow HPCs, TEC-Dl1 cells promote de novo generation of CD7(pos)CD1a(pos) T-lineage committed cells. Most CD7(pos)CD1a(hi) cells are CD4(pos)CD8(pos) double-positive (DP). We found that TEC-Dl1 cells are insufficient to generate mature CD3(hi) CD4(pos) or CD3(hi) CD8(pos) single-positive (SP) T cells from the CD4(pos)CD8(pos) DP T cells; however, we detected CD3(lo) cells within the DP and SP CD4 and CD8 populations. The CD3(lo) SP cells expressed lower levels of interleukin-2Rα and interleukin-7Rα compared to CD3(lo) DP cells. In contrast to the TEC-Dl1 line, the parental TEC-84 line expressing low levels of human Notch ligands permits HPC differentiation to the B-cell lineage. We report for the first time a human TEC line that supports lymphopoiesis from cord blood and bone marrow HPC. The TEC cell lines described herein provide a novel human thymic stroma model to study the contribution of human leukocyte antigen molecules and Notch ligands to T-cell commitment and maturation and could be utilized to promote lymphopoiesis for immune cell therapy. Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

  17. Electrospinning PCL Scaffolds Manufacture for Three-Dimensional Breast Cancer Cell Culture

    Directory of Open Access Journals (Sweden)

    Marc Rabionet

    2017-08-01

    Full Text Available In vitro cell culture is traditionally performed within two-dimensional (2D environments, providing a quick and cheap way to study cell properties in a laboratory. However, 2D systems differ from the in vivo environment and may not mimic the physiological cell behavior realistically. For instance, 2D culture models are thought to induce cancer stem cells (CSCs differentiation, a rare cancer cell subpopulation responsible for tumor initiation and relapse. This fact hinders the development of therapeutic strategies for tumors with a high relapse percentage, such as triple negative breast cancer (TNBC. Thus, three-dimensional (3D scaffolds have emerged as an attractive alternative to monolayer culture, simulating the extracellular matrix structure and maintaining the differentiation state of cells. In this work, scaffolds were fabricated through electrospinning different poly(ε-caprolactone-acetone solutions. Poly(ε-caprolactone (PCL meshes were seeded with triple negative breast cancer (TNBC cells and 15% PCL scaffolds displayed significantly (p < 0.05 higher cell proliferation and elongation than the other culture systems. Moreover, cells cultured on PCL scaffolds exhibited higher mammosphere forming capacity and aldehyde dehydrogenase activity than 2D-cultured cells, indicating a breast CSCs enrichment. These results prove the powerful capability of electrospinning technology in terms of poly(ε-caprolactone nanofibers fabrication. In addition, this study has demonstrated that electrospun 15% PCL scaffolds are suitable tools to culture breast cancer cells in a more physiological way and to expand the niche of breast CSCs. In conclusion, three-dimensional cell culture using PCL scaffolds could be useful to study cancer stem cell behavior and may also trigger the development of new specific targets against such malignant subpopulation.

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

    NARCIS (Netherlands)

    Heo, Inha; Clevers, Hans

    2015-01-01

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

  19. Novel culturing platform for brain slices and neuronal cells

    DEFF Research Database (Denmark)

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

    2015-01-01

    In this paper we demonstrate a novel culturing system for brain slices and neuronal cells, which can control the concentration of nutrients and the waste removal from the culture by adjusting the fluid flow within the device. The entire system can be placed in an incubator. The system has been...... tested successfully with brain slices and PC12 cells. The culture substrate can be modified using metal electrodes and/or nanostructures for conducting electrical measurements while culturing and for better mimicking the in vivo conditions....

  20. Metabolic activity of tree saps of different origin towards cultured human cells in the light of grade correspondence analysis and multiple regression modeling

    Directory of Open Access Journals (Sweden)

    Artur Wnorowski

    2017-06-01

    Full Text Available Tree saps are nourishing biological media commonly used for beverage and syrup production. Although the nutritional aspect of tree saps is widely acknowledged, the exact relationship between the sap composition, origin, and effect on the metabolic rate of human cells is still elusive. Thus, we collected saps from seven different tree species and conducted composition-activity analysis. Saps from trees of Betulaceae, but not from Salicaceae, Sapindaceae, nor Juglandaceae families, were increasing the metabolic rate of HepG2 cells, as measured using tetrazolium-based assay. Content of glucose, fructose, sucrose, chlorides, nitrates, sulphates, fumarates, malates, and succinates in sap samples varied across different tree species. Grade correspondence analysis clustered trees based on the saps’ chemical footprint indicating its usability in chemotaxonomy. Multiple regression modeling showed that glucose and fumarate present in saps from silver birch (Betula pendula Roth., black alder (Alnus glutinosa Gaertn., and European hornbeam (Carpinus betulus L. are positively affecting the metabolic activity of HepG2 cells.

  1. Stimulation and support of haemopoietic stem cell proliferation by irradiated stroma cell colonies in bone marrow cell culture in vitro

    International Nuclear Information System (INIS)

    Mori, K.J.; Izumi, Hiroko; Seto, Akira

    1981-01-01

    A culture system was established in which haemopoietic stem cells can undergo a recovery proliferation after a depletion of the stem cells, completely in vitro. To elucidate the source of the stimulatory factors, normal bone marrow cells were overlayed on top of the irradiated adherent 'stromal' cell colonies in the bone marrow cell culture. This stimulated the proliferation of haemopoietic stem cells in the cultured cells in suspension. The present results indicate that the stromal cells produce factors which stimulate stem cell proliferation. Whether the stimulation is evoked by direct cell-cell interactions or by humoral factors is as yet to be studied. (author)

  2. Human skeletal muscle-derived stem cells retain stem cell properties after expansion in myosphere culture

    International Nuclear Information System (INIS)

    Wei, Yan; Li, Yuan; Chen, Chao; Stoelzel, Katharina; Kaufmann, Andreas M.; Albers, Andreas E.

    2011-01-01

    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.

  3. Quantitative volumetric Raman imaging of three dimensional cell cultures

    Science.gov (United States)

    Kallepitis, Charalambos; Bergholt, Mads S.; Mazo, Manuel M.; Leonardo, Vincent; Skaalure, Stacey C.; Maynard, Stephanie A.; Stevens, Molly M.

    2017-03-01

    The ability to simultaneously image multiple biomolecules in biologically relevant three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell-material interactions. Here, we present a computational framework for label-free quantitative volumetric Raman imaging (qVRI). We apply qVRI to a selection of biological systems: human pluripotent stem cells with their cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy.

  4. Cell-cycle research with synchronous cultures: an evaluation

    Science.gov (United States)

    Helmstetter, C. E.; Thornton, M.; Grover, N. B.

    2001-01-01

    The baby-machine system, which produces new-born Escherichia coli cells from cultures immobilized on a membrane, was developed many years ago in an attempt to attain optimal synchrony with minimal disturbance of steady-state growth. In the present article, we put forward a model to describe the behaviour of cells produced by this method, and provide quantitative evaluation of the parameters involved, at each of four different growth rates. Considering the high level of selection achievable with this technique and the natural dispersion in interdivision times, we believe that the output of the baby machine is probably close to optimal in terms of both quality and persistence of synchrony. We show that considerable information on events in the cell cycle can be obtained from populations with age distributions very much broader than those achieved with the baby machine and differing only modestly from steady state. The data presented here, together with the long and fruitful history of findings employing the baby-machine technique, suggest that minimisation of stress on cells is the single most important factor for successful cell-cycle analysis.

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

    International Nuclear Information System (INIS)

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

    1984-01-01

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

  6. Reparative properties of the traditional Chinese medicine Cordyceps sinensis (Chinese caterpillar mushroom) using HT29 cell culture and rat gastric damage models of injury.

    Science.gov (United States)

    Marchbank, Tania; Ojobo, Ehighale; Playford, Christopher J; Playford, Raymond J

    2011-05-01

    Cordyceps sinensis (CS) is a traditional Chinese medicine and health food used to support many organ systems. It is commercially produced by cultivation in a liquid medium or on a solid (grain/potato) phase. We tested the effects of hot water extracts of liquid-phase and solid-phase commercially grown CS on its ability to influence proliferation (using Alamar blue, an oxidation/reduction indicator), migration (serial-wounded monolayer photomicroscopy), invasion through collagen gel (fluorometric assay) and indomethacin-induced apoptosis (active caspase-3 colorimetric assay) of human colon cancer HT29 cells. An in vivo study used a rat gastric damage model (indomethacin 20 mg/kg and 4 h restraint with oral administration). The CS extract stimulated cell proliferation threefold when added at 10 μg/ml (P < 0·01). Cell migration increased by 69 % and invasion by 17 % when CS was added at 5 mg/ml (P < 0·01). The results also showed that 93 % of the pro-proliferative activity was soluble in ethanol, whereas pro-migratory activity was divided (61:49) into both ethanol-soluble and ethanol-insoluble sub-fractions. Indomethacin-induced apoptosis was not affected by the presence of CS. CS reduced the amount of gastric injury by 63 % when administered orally at 20 mg/ml (P < 0·01), the results being similar to using the potent cytoprotective agent epidermal growth factor at 25 μg/ml (83 % reduction). We conclude that both methods of cultivated CS possess biological activity when analysed using a variety of gut models of injury and repair. Functional foods, such as CS, could provide a novel approach for the prevention and treatment of injury to the bowel.

  7. Growth of melanocytes in human epidermal cell cultures

    International Nuclear Information System (INIS)

    Staiano-Coico, L.; Hefton, J.M.; Amadeo, C.; Pagan-Charry, I.; Madden, M.R.; Cardon-Cardo, C.

    1990-01-01

    Epidermal cell cultures were grown in keratinocyte-conditioned medium for use as burn wound grafts; the melanocyte composition of the grafts was studied under a variety of conditions. Melanocytes were identified by immunohistochemistry based on a monoclonal antibody (MEL-5) that has previously been shown to react specifically with melanocytes. During the first 7 days of growth in primary culture, the total number of melanocytes in the epidermal cultures decreased to 10% of the number present in normal skin. Beginning on day 2 of culture, bipolar melanocytes were present at a mean cell density of 116 +/- 2/mm2; the keratinocyte to melanocyte ratio was preserved during further primary culture and through three subpassages. Moreover, exposure of cultures to mild UVB irradiation stimulated the melanocytes to proliferate, suggesting that the melanocytes growing in culture maintained their responsiveness to external stimuli. When the sheets of cultured cells were enzymatically detached from the plastic culture flasks before grafting, melanocytes remained in the basal layer of cells as part of the graft applied to the patient

  8. 3D cell culture to determine in vitro biocompatibility of bioactive glass in association with chitosan.

    Science.gov (United States)

    Bédouin, Y; Pellen Mussi, P; Tricot-Doleux, S; Chauvel-Lebret, D; Auroy, P; Ravalec, X; Oudadesse, H; Perez, F

    2015-01-01

    This study reports the in vitro biocompatibility of a composite biomaterial composed of 46S6 bioactive glass in association with chitosan (CH) by using 3D osteoblast culture of SaOS2. The 46S6 and CH composite (46S6-CH) forms small hydroxyapatite crystals on its surface after only three days immersion in the simulated body fluid. For 2D osteoblast culture, a significant increase in cell proliferation was observed after three days of contact with 46S6 or 46S6-CH-immersed media. After six days, 46S6-CH led to a significant increase in cell proliferation (128%) compared with pure 46S6 (113%) and pure CH (122%). For 3D osteoblast culture, after six days of culture, there was an increase in gene expression of markers of the early osteoblastic differentiation (RUNX2, ALP, COL1A1). Geometric structures corresponding to small apatite clusters were observed by SEM on the surface of the spheroids cultivated with 46S6 or 46S6-CH-immersed media. We showed different cellular responses depending on the 2D and 3D cell culture model. The induction of osteoblast differentiation in the 3D cell culture explained the differences of cell proliferation in contact with 46S6, CH or 46S6-CH-immersed media. This study confirmed that the 3D cell culture model is a very promising tool for in vitro biological evaluation of bone substitutes' properties.

  9. Reassembly of anterior pituitary organization by hanging drop three-dimensional cell culture.

    Science.gov (United States)

    Tsukada, Takehiro; Kouki, Tom; Fujiwara, Ken; Ramadhani, Dini; Horiguchi, Kotaro; Kikuchi, Motoshi; Yashiro, Takashi

    2013-08-29

    The anterior pituitary gland comprises 5 types of hormone-producing cells and non-endocrine cells, such as folliculostellate (FS) cells. The cells form a lobular structure surrounded by extracellular matrix (ECM) but are not randomly distributed in each lobule; hormone-producing cells have affinities for specific cell types (topographic affinity), and FS cells form a homotypic meshwork. To determine whether this cell and ECM organization can be reproduced in vitro, we developed a 3-dimensional (3D) model that utilizes hanging drop cell culture. We found that the topographic affinities of hormone-producing cells were indeed maintained (ie, GH to ACTH cells, GH to TSH cells, PRL to LH/FSH cells). Fine structures in hormone-producing cells retained their normal appearance. In addition, FS cells displayed well-developed cytoplasmic protrusions, which interconnected with adjacent FS cells to form a 3D meshwork. In addition, reassembly of gap junctions and pseudofollicles among FS cells was observed in cell aggregates. Major ECM components-collagens and laminin-were deposited and distributed around the cells. In sum, the dissociated anterior pituitary cells largely maintained their in vivo anterior pituitary architectures. This culture system appears to be a powerful experimental tool for detailed analysis of anterior pituitary cell organization.

  10. SAHA-induced TRAIL-sensitisation of Multiple Myeloma cells is enhanced in 3D cell culture.

    Science.gov (United States)

    Arhoma, A; Chantry, A D; Haywood-Small, S L; Cross, N A

    2017-11-15

    Multiple Myeloma (MM) is currently incurable despite many novel therapies. Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) is a potential anti-tumour agent although effects as a single agent are limited. In this study, we investigated whether the Histone Deacetylase (HDAC) inhibitor SAHA can enhance TRAIL-induced apoptosis and target TRAIL resistance in both suspension culture, and 3D cell culture as a model of disseminated MM lesions that form in bone. The effects of SAHA and/or TRAIL in 6 Multiple Myeloma cell lines were assessed in both suspension cultures and in an Alginate-based 3D cell culture model. The effect of SAHA and/or TRAIL was assessed on apoptosis by assessment of nuclear morphology using Hoechst 33342/Propidium Iodide staining. Viable cell number was assessed by CellTiter-Glo luminescence assay, Caspase-8 and -9 activities were measured by Caspase-Glo™ assay kit. TRAIL-resistant cells were generated by culture of RPMI 8226 and NCI-H929 by acute exposure to TRAIL followed by selection of TRAIL-resistant cells. TRAIL significantly induced apoptosis in a dose-dependent manner in OPM-2, RPMI 8226, NCI-H929, U266, JJN-3 MM cell lines and ADC-1 plasma cell leukaemia cells. SAHA amplified TRAIL responses in all lines except OPM-2, and enhanced TRAIL responses were both via Caspase-8 and -9. SAHA treatment induced growth inhibition that further increased in the combination treatment with TRAIL in MM cells. The co-treatment of TRAIL and SAHA reduced viable cell numbers all cell lines. TRAIL responses were further potentiated by SAHA in 3D cell culture in NCI-H929, RPMI 8226 and U266 at lower TRAIL + SAHA doses than in suspension culture. However TRAIL responses in cells that had been selected for TRAIL resistance were not further enhanced by SAHA treatment. SAHA is a potent sensitizer of TRAIL responses in both TRAIL sensitive and resistant cell lines, in both suspension and 3D culture, however SAHA did not sensitise TRAIL-sensitive cell

  11. Cell chirality: emergence of asymmetry from cell culture.

    Science.gov (United States)

    Wan, Leo Q; Chin, Amanda S; Worley, Kathryn E; Ray, Poulomi

    2016-12-19

    Increasing evidence suggests that intrinsic cell chirality significantly contributes to the left-right (LR) asymmetry in embryonic development, which is a well-conserved characteristic of living organisms. With animal embryos, several theories have been established, but there are still controversies regarding mechanisms associated with embryonic LR symmetry breaking and the formation of asymmetric internal organs. Recently, in vitro systems have been developed to determine cell chirality and to recapitulate multicellular chiral morphogenesis on a chip. These studies demonstrate that chirality is indeed a universal property of the cell that can be observed with well-controlled experiments such as micropatterning. In this paper, we discuss the possible benefits of these in vitro systems to research in LR asymmetry, categorize available platforms for single-cell chirality and multicellular chiral morphogenesis, and review mathematical models used for in vitro cell chirality and its applications in in vivo embryonic development. These recent developments enable the interrogation of the intracellular machinery in LR axis establishment and accelerate research in birth defects in laterality.This article is part of the themed issue 'Provocative questions in left-right asymmetry'. © 2016 The Author(s).

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

    DEFF Research Database (Denmark)

    Ziolkowska, K.; Jedrych, E.; Kwapiszewski, R.

    2010-01-01

    In this paper, hybrid (PDMS/glass) microfluidic cell culture system (MCCS) integrated with the concentration gradient generator (CGG) is presented. PDMS gas permeability enabled cells' respiration in the fabricated microdevices and excellent glass hydrophilicity allowed successful cells' seeding...

  13. Organoids as Model for Infectious Diseases : Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

    NARCIS (Netherlands)

    Bartfeld, Sina; Clevers, Hans

    2015-01-01

    Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of

  14. Microfluidic bioreactors for culture of non-adherent cells

    DEFF Research Database (Denmark)

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

    2011-01-01

    Microfluidic bioreactors (μBR) are becoming increasingly popular for cell culture, sample preparation and analysis in case of routine genetic and clinical diagnostics. We present a novel μBR for non-adherent cells designed to mimic in vivo perfusion of cells based on diffusion of media through...

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

    DEFF Research Database (Denmark)

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

    1984-01-01

    Casein kinase II (CKII) activity is enhanced as much as 2-3 fold in established and 4-5-fold in transformed human cell lines when compared to that of fibroblasts and primary human tumour cell cultures where CKII activity never exceeded a basic level. The high activity of CKII in transformed cells...

  16. Protein biosynthesis in cultured human hair follicle cells.

    Science.gov (United States)

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

    1980-10-31

    A new technique has been used for culturing human keratinocytes. The cells grow on the basement membrane-like capsules of bovine lenses. Lens cells were removed from the capsules by rigid trypsinization. In order to exclude any contamination with remaining living cells the isolated capsules were irradiated with X-rays at a dose of 10,000 rad. In this way human epithelial cells can be brought in culture from individual hair follicles. Since feeder cells are not used in this culture technique, the biosynthesis of keratinocyte proteins can be studied in these cultures. The newly synthesized proteins can be separated into a water-soluble, a urea-soluble, and a urea-insoluble fraction. Product analysis has been performed on the first two fractions revealing protein patterns identical to those of intact hair follicles. Product analysis of the urea-soluble fractions of microdissected hair follicles shows that the protein pattern of the cultured keratinocytes resembles the protein pattern of the hair follicle sheath. Studies on the metabolism of benzo(a)pyrene revealed that the enzyme aryl hydrocarbon hydroxylase (AHH) is present in cultured hair follicle cells. A possible use of our culture system for eventual detection of inherited predisposition for smoking-dependent lung cancer is discussed.

  17. 3D hepatic cultures simultaneously maintain primary hepatocyte and liver sinusoidal endothelial cell phenotypes.

    Directory of Open Access Journals (Sweden)

    Yeonhee Kim

    Full Text Available 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

  18. Primary culture of human Schwann and schwannoma cells: improved and simplified protocol.

    Science.gov (United States)

    Dilwali, Sonam; Patel, Pratik B; Roberts, Daniel S; Basinsky, Gina M; Harris, Gordon J; Emerick, Kevin S; Stankovic, Konstantina M

    2014-09-01

    Primary culture of human Schwann cells (SCs) and vestibular schwannoma (VS) cells are invaluable tools to investigate SC physiology and VS pathobiology, and to devise effective pharmacotherapies against VS, which are sorely needed. However, existing culture protocols, in aiming to create robust, pure cultures, employ methods that can lead to loss of biological characteristics of the original cells, potentially resulting in misleading biological findings. We have developed a minimally manipulative method to culture primary human SC and VS cells, without the use of selective mitogens, toxins, or time-consuming and potentially transformative laboratory techniques. Schwann cell purity was quantified longitudinally using S100 staining in SC cultures derived from the great auricular nerve and VS cultures followed for 7 and 12 weeks, respectively. SC cultures retained approximately ≥85% purity for 2 weeks. VS cultures retained approximately ≥80% purity for the majority of the span of 12 weeks, with maximal purity of 87% at 2 weeks. The VS cultures showed high level of biological similarity (68% on average) to their respective parent tumors, as assessed using a protein array featuring 41 growth factors and receptors. Apoptosis rate in vitro negatively correlated with tumor volume. Our results, obtained using a faster, simplified culturing method than previously utilized, indicate that highly pure, primary human SC and VS cultures can be established with minimal manipulation, reaching maximal purity at 2 weeks of culture. The VS cultures recapitulate the parent tumors' biology to a great degree, making them relevant models to investigate VS pathobiology. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Cystine uptake by cultured cells originating from dog proximal tubule segments

    International Nuclear Information System (INIS)

    States, B.; Reynolds, R.; Lee, J.; Segal, S.

    1990-01-01

    Large numbers of kidney epithelial cells were cultured successfully from isolated dog proximal tubule segments. Cells in primary culture and in first passage retained the cystine-dibasic amino acid co-transporter system which is found in vivo and in freshly isolated proximal tubule segments. In contrast to other cultured cells, the cystine-glutamate anti-porter was absent in primary cultures. However, this anti-porter system seemed to be developing in cells in first passage. The intracellular ratio of cysteine:reduced glutathione (CSH:GSH) was maintained at 1:36 in both primary cultures and in low passage cells. Incubation of cells in primary culture for 5 min at 37 degrees C with 0.025 mM [ 35 S]L-cystine resulted in incorporation of approximately 36 and 8.5% of the label into intracellular CSH and GSH, respectively. These cultured cells, therefore, seem to be an excellent model system for the eventual elucidation of (a) the inticacies of cystine metabolism and (b) regulation of (1) the cystine-dibasic amino acid co-transporter system and (2) the development of the cysteine-glutamate anti-porter system

  20. Direct Conversion of Equine Adipose-Derived Stem Cells into Induced Neuronal Cells Is Enhanced in Three-Dimensional Culture.

    Science.gov (United States)

    Petersen, Gayle F; Hilbert, Bryan J; Trope, Gareth D; Kalle, Wouter H J; Strappe, Padraig M

    2015-12-01

    The ability to culture neurons from horses may allow further investigation into equine neurological disorders. In this study, we demonstrate the generation of induced neuronal cells from equine adipose-derived stem cells (EADSCs) using a combination of lentiviral vector expression of the neuronal transcription factors Brn2, Ascl1, Myt1l (BAM) and NeuroD1 and a defined chemical induction medium, with βIII-tubulin-positive induced neuronal cells displaying a distinct neuronal morphology of rounded and compact cell bodies, extensive neurite outgrowth, and branching of processes. Furthermore, we investigated the effects of dimensionality on neuronal transdifferentiation, comparing conventional two-dimensional (2D) monolayer culture against three-dimensional (3D) culture on a porous polystyrene scaffold. Neuronal transdifferentiation was enhanced in 3D culture, with evenly distributed cells located on the surface and throughout the scaffold. Transdifferentiation efficiency was increased in 3D culture, with an increase in mean percent conversion of more than 100% compared to 2D culture. Additionally, induced neuronal cells were shown to transit through a Nestin-positive precursor state, with MAP2 and Synapsin 2 expression significantly increased in 3D culture. These findings will help to increase our understanding of equine neuropathogenesis, with prospective roles in disease modeling, drug screening, and cellular replacement for treatment of equine neurological disorders.

  1. Control of fibronectin synthesis by rat granulosa cells in culture

    International Nuclear Information System (INIS)

    Skinner, M.K.; Dorrington, J.H.

    1984-01-01

    The secreted and cellular [ 35 S]methionine-radiolabeled proteins of cultured rat granulosa cells were separated by electrophoresis on sodium dodecylsulfate (SDS) polyacrylamide gradient slab gels. From 24 to 72 h of culture FSH increased the intensity of labeling of most of the secreted proteins. A 220,000-dalton protein, however, increased in intensity only in control cultures and became the major secreted protein after 72 h, comprising 20% of the total radiolabeled proteins. This protein was identified as fibronectin by immunoprecipitation. There was no increase in the secreted or cellular fibronectin in FSH- or testosterone- and insulin-treated cultures. These studies indicate that a component of extracellular matrix is a major secretory product of unstimulated immature granulosa cells. As hormones induce the differentiated functions of granulosa cells in culture, the secretion of fibronectin is inhibited

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

    Directory of Open Access Journals (Sweden)

    Song-I Chun

    Full Text Available 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.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.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 (0-10 mM.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.

  3. Irradiated murine fibroblasts as feeder layer used in human cell culture

    International Nuclear Information System (INIS)

    Almeida, Tiago L.; Klingbeil, Fatima G.; Yoshito, Daniele; Caproni, Priscila; Mathor, Monica B.; Herson, Marisa R.

    2007-01-01

    In 1975, Rheinwald and Green published an in vitro model for keratinocyte cell cultures in which the use of murine fibroblasts, as a feeder layer was introduced. These cells are modified fibroblasts, which presence render keratinocyte cells to remain proliferative for longer periods of time. This optimization of culture outputs has allowed for several clinical applications of confluent keratinocyte cultures as skin substitutes or wound dressings in situations such as post burn extensive skin loss, loss of oral mucosa, and other skin disorders. Nevertheless, proliferation of fibroblast in co-culture with keratinocytes must be controlled by anti-proliferative measures such as irradiation; at the same time, keratinocytes require specific nutrients in the culture medium, which may interfere with the fibroblast feeder layer viability. Therefore, the thorough understanding of the impact of different issues such as culture media composition, irradiation dose and pre-plating storage conditions of irradiated fibroblast to be used as feeder layer in these co-culture systems is important. In this work, changes as far as viability and proliferative rates of irradiated fibroblasts in culture were evaluated in relation to the type of culture medium used, dose of gamma radiation exposure, storage and timing of cell plating post irradiation. Results indicate that the type of culture medium used and time-lag between irradiation, refrigeration and plating of irradiated cells do not have significant impact in culture outcomes. However, the dose of gamma radiation administered to the cells may influence the final quality of these cells if to be used as a feeder layer. (author)

  4. Slow conduction in mixed cultured strands of primary ventricular cells and stem cell-derived cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Jan Pavel Kucera

    2015-09-01

    Full Text Available Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs. However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands.CV was significantly lower in strands composed purely of SCMs (5.5±1.5 cm/s, n=11 as compared to PCMs (34.9±2.9 cm/s, n=21 at similar refractoriness (100% SCMs: 122±25 ms, n=9; 100% PCMs: 139±67 ms, n=14. In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV.These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias.

  5. Microfluidic perfusion culture of human induced pluripotent stem cells under fully defined culture conditions.

    Science.gov (United States)

    Yoshimitsu, Ryosuke; Hattori, Koji; Sugiura, Shinji; Kondo, Yuki; Yamada, Rotaro; Tachikawa, Saoko; Satoh, Taku; Kurisaki, Akira; Ohnuma, Kiyoshi; Asashima, Makoto; Kanamori, Toshiyuki

    2014-05-01

    Human induced pluripotent stem cells (hiPSCs) are a promising cell source for drug screening. For this application, self-renewal or differentiation of the cells is required, and undefined factors in the culture conditions are not desirable. Microfluidic perfusion culture allows the production of small volume cultures with precisely controlled microenvironments, and is applicable to high-throughput cellular environment screening. Here, we developed a microfluidic perfusion culture system for hiPSCs that uses a microchamber array chip under defined extracellular matrix (ECM) and culture medium conditions. By screening various ECMs we determined that fibronectin and laminin are appropriate for microfluidic devices made out of the most popular material, polydimethylsiloxane (PDMS). We found that the growth rate of hiPSCs under pressure-driven perfusion culture conditions was higher than under static culture conditions in the microchamber array. We applied our new system to self-renewal and differentiation cultures of hiPSCs, and immunocytochemical analysis showed that the state of the hiPSCs was successfully controlled. The effects of three antitumor drugs on hiPSCs were comparable between microchamber array and 96-well plates. We believe that our system will be a platform technology for future large-scale screening of fully defined conditions for differentiation cultures on integrated microfluidic devices. © 2013 Wiley Periodicals, Inc.

  6. Quantitative volumetric Raman imaging of three dimensional cell cultures

    KAUST Repository

    Kallepitis, Charalambos

    2017-03-22

    The ability to simultaneously image multiple biomolecules in biologically relevant three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell–material interactions. Here, we present a computational framework for label-free quantitative volumetric Raman imaging (qVRI). We apply qVRI to a selection of biological systems: human pluripotent stem cells with their cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy.

  7. Adherence of Moraxella bovis to cell cultures of bovine origin.

    Science.gov (United States)

    Annuar, B O; Wilcox, G E

    1985-09-01

    The adherence of five strains of Moraxella bovis to cell cultures was investigated. M bovis adhered to cultures of bovine corneal epithelial and Madin-Darby bovine kidney cells but not to cell types of non-bovine origin. Both piliated and unpiliated strains adhered but piliated strains adhered to a greater extent than unpiliated strains. Antiserum against pili of one strain inhibited adherence of piliated strains but caused only slight inhibition of adherence to the unpiliated strains. Treatment of bacteria with magnesium chloride caused detachment of pili from the bacterial cell and markedly inhibited adherence of piliated strains but caused only slight inhibition of adherence by the unpiliated strains. The results suggested that adhesion of piliated strains to cell cultures was mediated via pili but that adhesins other than pili may be involved in the attachment of unpiliated strains of M bovis to cells.

  8. Self-assembling Fmoc dipeptide hydrogel for in situ 3D cell culturing

    Directory of Open Access Journals (Sweden)

    Akpe Victor

    2007-12-01

    Full Text Available Abstract Background Conventional cell culture studies have been performed on 2D surfaces, resulting in flat, extended cell growth. More relevant studies are desired to better mimic 3D in vivo tissue growth. Such realistic environments should be the aim of any cell growth study, requiring new methods for culturing cells in vitro. Cell biology is also tending toward miniaturization for increased efficiency and specificity. This paper discusses the application of a self-assembling peptide-derived hydrogel for use as a 3D cell culture scaffold at the microscale. Results Phenylalanine derivative hydrogel formation was seen to occur in multiple dispersion media. Cells were immobilized in situ within microchambers designed for cell analysis. Use of the highly biocompatible hydrogel components and simplistic procedures significantly reduced the cytotoxic effects seen with alternate 3D culture materials and microstructure loading methods. Cells were easily immobilized, sustained and removed from microchambers. Differences in growth morphology were seen in the cultured cells, owing to the 3-dimentional character of the gel structure. Degradation improved the removal of hydrogel from the microstructures, permitting reuse of the analysis platforms. Conclusion Self-assembling diphenylalanine derivative hydrogel provided a method to dramatically reduce the typical difficulties of microculture formation. Effective generation of patterned 3D cultures will lead to improved cell study results by better modeling in vivo growth environments and increasing efficiency and specificity of cell studies. Use of simplified growth scaffolds such as peptide-derived hydrogel should be seen as highly advantageous and will likely become more commonplace in cell culture methodology.

  9. Defining process design space for monoclonal antibody cell culture.

    Science.gov (United States)

    Abu-Absi, Susan Fugett; Yang, LiYing; Thompson, Patrick; Jiang, Canping; Kandula, Sunitha; Schilling, Bernhard; Shukla, Abhinav A

    2010-08-15

    The concept of design space has been taking root as a foundation of in-process control strategies for biopharmaceutical manufacturing processes. During mapping of the process design space, the multidimensional combination of operational variables is studied to quantify the impact on process performance in terms of productivity and product quality. An efficient methodology to map the design space for a monoclonal antibody cell culture process is described. A failure modes and effects analysis (FMEA) was used as the basis for the process characterization exercise. This was followed by an integrated study of the inoculum stage of the process which includes progressive shake flask and seed bioreactor steps. The operating conditions for the seed bioreactor were studied in an integrated fashion with the production bioreactor using a two stage design of experiments (DOE) methodology to enable optimization of operating conditions. A two level Resolution IV design was followed by a central composite design (CCD). These experiments enabled identification of the edge of failure and classification of the operational parameters as non-key, key or critical. In addition, the models generated from the data provide further insight into balancing productivity of the cell culture process with product quality considerations. Finally, process and product-related impurity clearance was evaluated by studies linking the upstream process with downstream purification. Production bioreactor parameters that directly influence antibody charge variants and glycosylation in CHO systems were identified.

  10. Cell-cycle distributions and radiation responses of Chinese hamster cells cultured continuously under hypoxic conditions

    International Nuclear Information System (INIS)

    Tokita, N.; Carpenter, S.G.; Raju, M.R.

    1984-01-01

    Cell-cycle distributions were measured by flow cytometry for Chinese hamster (CHO) cells cultured continuously under hypoxic conditions. DNA histograms showed an accumulation of cells in the early S phase followed by a traverse delay through the S phase, and a G 2 block. During hypoxic culturing, cell viability decreased rapidly to less than 0.1% at 120 h. Radiation responses for cells cultured under these conditions showed an extreme radioresistance at 72 h. Results suggest that hypoxia induces a condition similar to cell synchrony which itself changes the radioresistance of hypoxic cells. (author)

  11. In vitro production of azadirachtin from cell suspension cultures of ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR G

    proven effective in the control of agricultural pests in an environmentally ..... Prakash G and Srivastava A K 2005 Statistical media optimization for cell growth and ... Juss. suspension cultures; Process Biochemistry 40 3795–3800. Prakash G ...

  12. Establishment of the callus and cell suspension culture of ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-05

    Oct 5, 2009 ... Full Length Research Paper. Establishment of the callus ... study provided an efficient way for E. angustifolia cell suspension culture to produce secondary metabolite. .... was also observed that in these treatments the stem.

  13. Enhancement of Diosgenin Production in Plantlet and Cell Cultures ...

    African Journals Online (AJOL)

    Enhancement of Diosgenin Production in Plantlet and Cell Cultures of Dioscorea zingiberensis by Palmarumycin C13 from the Endophytic fungus, Berkleasmium sp. Dzf12. Y Mou, K Zhou, D Xu, R Yu, J Li, C Yin, L Zhou ...

  14. Establishment of sorghum cell suspension culture system for ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-03-18

    Mar 18, 2008 ... Additionally, sorghum cell suspension cultures have been initiated from the friable ... proteomics technologies. The field of proteomics is .... air dried at room temperature and resuspended in 2 ml of urea buffer [9 M urea, 2 M ...

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

    NARCIS (Netherlands)

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

    1995-01-01

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

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

    NARCIS (Netherlands)

    Huang, L.; van Loveren, C.; Ling, J.; Wei, X.; Crielaard, W.; Deng, D.M.

    2016-01-01

    Porphyromonas gingivalis is present as a biofilm at the sites of periodontal infections. The detachment of gingival epithelial cells induced by P. gingivalis biofilms was examined using planktonic cultures as a comparison. Exponentially grown planktonic cultures or 40-h biofilms were co-incubated

  17. Free-energy carriers in human cultured muscle cells

    NARCIS (Netherlands)

    Bolhuis, P. A.; de Zwart, H. J.; Ponne, N. J.; de Jong, J. M.

    1985-01-01

    Creatine phosphate (CrP), adenosine triphosphate (ATP), creatine kinase (CK), adenylate kinase (AK), protein, and DNA were quantified in human muscle cell cultures undergoing transition from dividing myoblasts to multinucleate myotubes. CrP is negligible in cultures grown in commonly applied media

  18. Radiation effects on cultured human lymphoid cells

    International Nuclear Information System (INIS)

    Johansson, L.; Nilsson, K.; Carlsson, J.; Larsson, B.; Jakobsson, P.

    1981-01-01

    The cloning efficiency of human normal and malignant lymphoid cells is usually low. Radiation effects in vitro on such cells can therefore not be analysed with conventional cloning. However, this problem can be circumscribed by using the growth extrapolation method. A panel of human leukemia-lymphoma cell-lines representing Epstein-Barr virus carrying lymphoblastoid cells of presumed non-neoplastic derivation and neoplastic T- and B-lymphocytes was used to test the efficiency of this method. The sensitivity to radiation could be determined for all these cell types. The growth extrapolation method gave generally the same result as conventional cloning demonstrated by comparison with one exceptional cell-line with capacity for cloning in agar. The sensitivity varied largely between the different cell types. A common feature was that none of the cell lines had a good capacity to accumulate sublethal radiation injury. (Auth.)

  19. Radiosensitivity of cultured insect cells: II. Diptera

    International Nuclear Information System (INIS)

    Koval, T.M.

    1983-01-01

    The radiosensitivity of five dipteran cell lines representing three mosquito genera and one fruit fly genus were examined. These lines are: (1) ATC-10, Aedes aegypti; (2) RU-TAE-14, Toxorhynchites amboinensis; (3) RU-ASE-2A, Anopheles stephensi; (4) WR69-DM-1, Drosophila melanogaster; and (5) WR69-DM-2, Drosophila melanogaster. Population doubling times for these lines range from approximately 16 to 48 hr. Diploid chromosome numbers are six for the mosquito cells and eight for the fruit fly cells D 0 values are 5.1 and 6.5 Gy for the Drosophila cell lines and 3.6, 6.2, and 10.2 Gy for the mosquito cell lines. The results of this study demonstrate that dipteran insect cells are a few times more resistant to radiation than mammalian cells, but not nearly as radioresistant as lepidopteran cells

  20. Impact of cell culture process changes on endogenous retrovirus expression.

    Science.gov (United States)

    Brorson, Kurt; De Wit, Christina; Hamilton, Elizabeth; Mustafa, Mehnaz; Swann, Patrick G; Kiss, Robert; Taticek, Ron; Polastri, Gian; Stein, Kathryn E; Xu, Yuan

    2002-11-05

    Cell culture process changes (e.g., changes in scale, medium formulation, operational conditions) and cell line changes are common during the development life cycle of a therapeutic protein. To ensure that the impact of such process changes on product quality and safety is minimal, it is standard practice to compare critical product quality and safety attributes before and after the changes. One potential concern introduced by cell culture process improvements is the possibility of increased endogenous retrovirus expression to a level above the clearance capability of the subsequent purification process. To address this, retrovirus expression was measured in scaled down and full production scaled Chinese hamster ovary (CHO) cell cultures of four monoclonal antibodies and one recombinant protein before and after process changes. Two highly sensitive, quantitative (Q)-PCR-based assays were used to measure endogenous retroviruses. It is shown that cell culture process changes that primarily alter media components, nutrient feed volume, seed density, cell bank source (i.e., master cell bank vs. working cell bank), and vial size, or culture scale, singly or in combination, do not impact the rate of retrovirus expression to an extent greater than the variability of the Q-PCR assays (0.2-0.5 log(10)). Cell culture changes that significantly alter the metabolic state of the cells and/or rates of protein expression (e.g., pH and temperature shifts, NaButyrate addition) measurably impact the rate of retrovirus synthesis (up to 2 log(10)). The greatest degree of variation in endogenous retrovirus expression was observed between individual cell lines (up to 3 log(10)). These data support the practice of measuring endogenous retrovirus output for each new cell line introduced into manufacturing or after process changes that significantly increase product-specific productivity or alter the metabolic state, but suggest that reassessment of retrovirus expression after other

  1. Polymer microfilters with nanostructured surfaces for the culture of circulating cancer cells

    International Nuclear Information System (INIS)

    Makarova, Olga V.; Adams, Daniel L.; Divan, Ralu; Rosenmann, Daniel; Zhu, Peixuan; Li, Shuhong; Amstutz, Platte; Tang, Cha-Mei

    2016-01-01

    There is a critical need to improve the accuracy of drug screening and testing through the development of in vitro culture systems that more effectively mimic the in vivo environment. Surface topographical features on the nanoscale level, in short nanotopography, effect the cell growth patterns, and hence affect cell function in culture. We report the preliminary results on the fabrication, and subsequent cellular growth, of nanoscale surface topography on polymer microfilters using cell lines as a precursor to circulating tumor cells (CTCs). To create various nanoscale features on the microfilter surface, we used reactive ion etching (RIE) with and without an etching mask. An anodized aluminum oxide (AAO) membrane fabricated directly on the polymer surface served as an etching mask. Polymer filters with a variety of modified surfaces were used to compare the effects on the culture of cancer cell lines in blank culture wells, with untreated microfilters or with RIE-treated microfilters. We then report the differences of cell shape, phenotype and growth patterns of bladder and glioblastoma cancer cell lines after isolation on the various types of material modifications. Our data suggest that RIE modified polymer filters can isolate model cell lines while retaining ell viability, and that the RIE filter modification allows T24 monolayering cells to proliferate as a structured cluster. - Highlights: • Surface topographical effects the growth patterns and cell function of cancer cells • Nanoscale surface topography on polymer filters for circulating tumor cell culture • Membrane fabricated directly on polymer surfaces utilized for polymer etching • Nanotopography alters cell shape, phenotype and growth patterns of cancer cells • Nanoscale surface topography dictates monolayering or 3D structured cell culture

  2. Polymer microfilters with nanostructured surfaces for the culture of circulating cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Makarova, Olga V. [Creatv MicroTech, Inc., 2242 West Harrison St., Chicago 60612, IL (United States); Adams, Daniel L., E-mail: dan@creatvmicrotech.com [Creatv MicroTech, Inc., 1 Deer Park Drive, Monmouth Junction, NJ 08852 (United States); Divan, Ralu; Rosenmann, Daniel [Center for Nanoscale Materials, Argonne National Laboratory, 9700 South Cass Ave., Argonne 60439, IL (United States); Zhu, Peixuan; Li, Shuhong; Amstutz, Platte; Tang, Cha-Mei [Creatv MicroTech, Inc., 11609 Lake Potomac Drive, Potomac 20854, MD (United States)

    2016-09-01

    There is a critical need to improve the accuracy of drug screening and testing through the development of in vitro culture systems that more effectively mimic the in vivo environment. Surface topographical features on the nanoscale level, in short nanotopography, effect the cell growth patterns, and hence affect cell function in culture. We report the preliminary results on the fabrication, and subsequent cellular growth, of nanoscale surface topography on polymer microfilters using cell lines as a precursor to circulating tumor cells (CTCs). To create various nanoscale features on the microfilter surface, we used reactive ion etching (RIE) with and without an etching mask. An anodized aluminum oxide (AAO) membrane fabricated directly on the polymer surface served as an etching mask. Polymer filters with a variety of modified surfaces were used to compare the effects on the culture of cancer cell lines in blank culture wells, with untreated microfilters or with RIE-treated microfilters. We then report the differences of cell shape, phenotype and growth patterns of bladder and glioblastoma c