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1

Studying cell-cell communication in co-culture  

PubMed Central

Heterotypic and homotypic cellular interactions are essential for biological function, and co-culture models are versatile tools for investigating these cellular interactions in vitro. Physiologically relevant co-culture models have been used to elucidate the effects of cell-cell physical contact and/or secreted factors, as well as the influence of substrate geometry and interaction scale on cell response. Identifying the relative contribution of each cell population to co-culture is often experimentally challenging for these cellular interactions studies. In this issue of Biotechnology Journal, Hamilton et al. [1] report on a hydrogel-based co-culture system, that enables paracrine interactions. A simple and elegant method for enzymatic separation of cell populations post co-culture is introduced, thereby enhancing the ease for post-culture analysis of the effects of co-culture on individual cell populations. PMID:23554248

Bogdanowicz, Danielle R.; Lu, Helen H.

2014-01-01

2

Characterizing parameters of Jatropha curcas cell cultures for microgravity studies  

NASA Astrophysics Data System (ADS)

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

Vendrame, Wagner A.; Pinares, Ania

2013-06-01

3

Morphological study of the TK cholangiocarcinoma cell line with three-dimensional cell culture.  

PubMed

Cholangiocarcinoma is an intractable carcinoma originating from the bile duct epithelium. To gain an understanding of the cell biology of cholangiocarcinoma, in vitro cell culture is valuable. However, well?characterized cell lines are limited. In the present study, the morphology of the TK cholangiocarcinoma cell line was analyzed by three?dimensional culture. Dispersed TK cells were injected into a gelatin mesh scaffold and cultivated for 3?20 days. The morphology of the TK cells was investigated by phase?contrast microscopy, optical microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). TK cells were observed to proliferate three-dimensionally in the scaffold. The cells exhibited a globoid structure and attached to the scaffold. The SEM observation demonstrated typical microvilli and plicae on the surface of the structure. Light microscopy and TEM confirmed intercellular and cell?to?scaffold attachment in the three?dimensional mesh. The culture also exhibited the formation of a duct-like structure covered by structured microvilli. In conclusion, three?dimensional culture of TK cells demonstrated the morphological characteristics of cholangiocarcinoma in vitro. Production of high levels of carbohydrate antigen (CA)19?9, CA50 and carcinoembryonic antigen was previously confirmed in the TK cell line. As a characteristic morphology was demonstrated in the present study, the TK cholangiocarcinoma cell line may be useful as an experimental model for further study of cholangiocarcinoma. PMID:24535710

Akiyoshi, Kohei; Kamada, Minori; Akiyama, Nobutake; Suzuki, Masafumi; Watanabe, Michiko; Fujioka, Kouki; Ikeda, Keiichi; Mizuno, Shuichi; Manome, Yoshinobu

2014-04-01

4

Studies on cultured rat Schwann cells. I. Establishment of purified populations from cultures of peripheral nerve.  

PubMed

We have previously reported that in dissociated cultures of neonatal rat sciatic nerve, all of the cells could be identified by indirect immunofluorescence with two antisera to cell surface antigens. The Schwann cells, but not the fibroblasts, expressed the Ran-1 antigen, while the fibroblasts, but not the Schwann cells, expressed the Thy-1 antigen. We have exploited this difference to derive pure populations of Schwann cells. A combination of [3H]thymidine autoradiography and immunofluorescence marking showed that in Modified Eagle's Medium with 10% foetal calf serum, the Schwann cells divided slowly while the fibroblasts divided rapidly. Accordingly, two day old cultures were exposed to cytosine arabinoside to select against the fibroblasts, followed by growth in medium containing an extract of bovine pituitary which stimulated division of the Schwann cells. After 7 days the confluent cultures, which contained 80-90% Schwann cells, were passaged after treatment in suspension with antiserum to Thy-1 and rabbit complement. After continued growth in medium with pituitary extract, the secondary cultures contained greater than 99.5% Schwann cells. These purified populations have been maintained in culture for as long as 150 days (6 passages) and retained the Ran-1 marker. The cultured Schwann cells expressed the S100 antigen, as shown by indirect immunofluorescence and complement fixation, and receptors for cholera toxin. They did not express the large external transformation sensitive protein, the glial fibrillary acidic protein, or receptors for tetanus toxin. PMID:371755

Brockes, J P; Fields, K L; Raff, M C

1979-04-01

5

Advances in cell culture  

SciTech Connect

This book presents papers on advances in cell culture. Topics covered include: Genetic changes in the influenza viruses during growth in cultured cells; The biochemistry and genetics of mosquito cells in culture; and Tree tissue culture applications.

Maramorosch, K. (Dept. of Entomology, Rutgers Univ., New Brunswick, NJ (US))

1987-01-01

6

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

PubMed Central

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

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

2011-01-01

7

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

PubMed Central

Background. Mucociliary transport (MCT) is a defense mechanism of the airway. To study the underlying mechanisms of MCT, we have both developed an experimental model of cultures, from human adenoid tissue of ciliated and secretory cells, and characterized the response to local chemical signals that control ciliary activity and the secretion of respiratory mucins in vitro. Materials and Methods. In ciliated cell cultures, ciliary beat frequency (CBF) and intracellular Ca2+ levels were measured in response to ATP, UTP, and adenosine. In secretory cultures, mucin synthesis and secretion were identified by using immunodetection. Mucin content was taken from conditioned medium and analyzed in the presence or absence of UTP. Results. Enriched ciliated cell monolayers and secretory cells were obtained. Ciliated cells showed a basal CBF of 10.7?Hz that increased significantly after exposure to ATP, UTP, or adenosine. Mature secretory cells showed active secretion of granules containing different glycoproteins, including MUC5AC. Conclusion. Culture of ciliated and secretory cells grown from adenoid epithelium is a reproducible and feasible experimental model, in which it is possible to observe ciliary and secretory activities, with a potential use as a model to understand mucociliary transport control mechanisms. PMID:23484122

Gonzalez, Claudia; Espinosa, Marisol; Sanchez, Maria Trinidad; Droguett, Karla; Rios, Mariana; Fonseca, Ximena; Villalon, Manuel

2013-01-01

8

Cell isolation and culture.  

PubMed

Cell isolation and culture are essential tools for the study of cell function. Isolated cells grown under controlled conditions can be manipulated and imaged at a level of resolution that is not possible in whole animals or even tissue explants. Recent advances have allowed for large-scale isolation and culture of primary C. elegans cells from both embryos and all four larval stages. Isolated cells can be used for single-cell profiling, electrophysiology, and high-resolution microscopy to assay cell autonomous development and behavior. This chapter describes protocols for the isolation and culture of C. elegans embryonic and larval stage cells. Our protocols describe isolation of embryonic and L1 stage cells from nematodes grown on high-density NA22 bacterial plates and isolation of L2 through L4 stage cells from nematodes grown in axenic liquid culture. Both embryonic and larval cells can be isolated from nematode populations within 3 hours and can be cultured for several days. A primer on sterile cell culture techniques is given in the appendices. PMID:23430760

Zhang, Sihui; Kuhn, Jeffrey R

2013-01-01

9

Introduction Removal of endotoxin from solutions for animal studies, cell culture, transplanta-  

E-print Network

as a defense mechanism against gram-negative bacteria [1,2]. This degranulation re- leases a series of enzymesIntroduction Removal of endotoxin from solutions for animal studies, cell culture, transplanta,7]. To make protein samples suitable for animal studies and cell culture, contaminat- ing pyrogens

Lebendiker, Mario

10

Hepatic parenchymal cell glutamylation of methotrexate studied in monolayer culture.  

PubMed

A detailed analysis of the gamma-glutamylation of methotrexate has been conducted in primary cultures of rat hepatic parenchymal cells in monolayer culture. The rates of glutamylation are concentration dependent and saturable when measured over a 6-h period at concentrations between 2 and 50 microM. The removal of folate and inclusion of insulin, dexamethasone, and tocopherol enhance glutamylation. Omission of methionine from the medium increases glutamylation, whereas the 6-h period of syntheses, methotrexate the reaction. During the 6-h period of syntheses, methotrexate diglutamate is the primary product, whereas the di- and triglutamates are the major cellular species when the incubation is extended to 24 h. Lower extracellular methotrexate concentrations result in the formation of relatively greater amounts of longer chain length derivatives. The accumulation of methotrexate polyglutamates at steady state is saturable and occurs by 24 h. The predominant species contain two to four glutamate residues, and the distribution depends upon the culture conditions and the extracellular methotrexate concentration. The turnover of cellular polyglutamates at saturation occurs at 30 to 40% of the total cell pool over a 6-h period. Placement of hepatocytes with saturating levels of methotrexate polyglutamates in medium lacking methotrexate results in the slow loss of all derivatives, and the rate of loss is inversely related to polyglutamate chain length. Following a pulse dose of methotrexate, hepatocytes continue to increase the chain length of the cellular pool of polyglutamates, and this process is impaired by addition of folinic acid to the medium. In the pulse experiments as in the longer term incubations, the primary species are the di- and triglutamates. The results demonstrate limited capacity of the hepatocytes to glutamylate longer chain length polyglutamate derivatives. PMID:2416430

Galivan, J; Pupons, A; Rhee, M S

1986-02-01

11

Molluscan cells in culture: primary cell cultures and cell lines  

PubMed Central

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

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

2013-01-01

12

Assessment of cell death studies by monitoring hydrogen peroxide in cell culture.  

PubMed

Hydrogen peroxide (H2O2) has been widely used to study the oxidative stress response. However, H2O2 is unstable and easily decomposes into H2O and O2. Consequently, a wide range of exposure times and treatment concentrations has been described in the literature. In the present study, we established a ferrous oxidation-xylenol orange (FOX) assay, which was originally described for food and body liquids, as a method for the precise quantification of H2O2 concentrations in cell culture media. We observed that the presence of FCS and high cell densities significantly accelerate the decomposition of H2O2, therefore acting as a protection against cell death by accidental necrosis. PMID:24747006

Hirsch, Irina; Prell, Erik; Weiwad, Matthias

2014-07-01

13

Microspore-derived cell suspension cultures of oilseed rape as a system for studying gene expression  

Microsoft Academic Search

Abiotic stress, such as extreme temperature, drought, or excessive salinity, is one of the leading causes of crop loss worldwide.\\u000a Microspore-derived (MD) cell suspension cultures of Brassica napus L. cv. Jet Neuf have been shown to be a useful system for studying the biochemistry of developing oilseeds. In the present\\u000a study, we describe the application of MD cell suspension cultures

Yongzhong Shi; Gangbiao Xu; Timothy B. Warrington; Gordon K. Murdoch; E. Chris Kazala; Crystal L. Snyder; Randall J. Weselake

2008-01-01

14

Reporting of sex as a variable in cardiovascular studies using cultured cells  

PubMed Central

Background Chromosomal complement, including that provided by the sex chromosomes, influences expression of proteins and molecular signaling in every cell. However, less than 50% of the scientific studies published in 2009 using experimental animals reported sex as a biological variable. Because every cell has a sex, we conducted a literature review to determine the extent to which sex is reported as a variable in cardiovascular studies on cultured cells. Methods Articles from 10 cardiovascular journals with high impact factors (Circulation, J Am Coll Cardiol, Eur Heart J, Circ Res, Arterioscler Thromb Vasc Biol, Cardiovasc Res, J Mol Cell Cardiol, Am J Physiol Heart Circ Physiol, J Heart Lung Transplant and J Cardiovasc Pharmacol) and published in 2010 were searched using terms 'cultured' and 'cells' in any order to determine if the sex of those cells was reported. Studies using established cell lines were excluded. Results Using two separate search strategies, we found that only 25 of 90 articles (28%) and 20 of 101 articles (19.8%) reported the sex of cells. Of those reporting the sex of cells, most (68.9%; n = 31) used only male cells and none used exclusively female cells. In studies reporting the sex of cells of cardiovascular origin, 40% used vascular smooth-muscle cells, and 30% used stem/progenitor cells. In studies using cells of human origin, 35% did not report the sex of those cells. None of the studies using neonatal cardiac myocytes reported the sex of those cells. Conclusions The complement of sex chromosomes in cells studied in culture has the potential to affect expression of proteins and 'mechanistic' signaling pathways. Therefore, consistent with scientific excellence, editorial policies should require reporting sex of cells used in in vitro experiments. PMID:22060014

2011-01-01

15

A system for culturing iris pigment epithelial cells to study lens regeneration in newt.  

PubMed

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw¹. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month²(,)³ . This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2)?(,)?. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration. PMID:21730940

Bhavsar, Rital B; Nakamura, Kenta; Tsonis, Panagiotis A

2011-01-01

16

Mechanically and chemically tunable cell culture system for studying the myofibroblast phenotype.  

PubMed

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

Saums, Michele K; Wang, Weifeng; Han, Biao; Madhavan, Lakshmi; Han, Lin; Lee, Daeyeon; Wells, Rebecca G

2014-05-20

17

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

E-print Network

. Cells (1 × 105) were stained with annexin V and propidium iodide using the Apoptosis Detection Kit and following the manufacturer’s instructions (R&D Systems, Abingdon, Oxford, UK). Cells were analyzed by flow cytometry using a Coulter EPICS XL flow... Primary research A novel cell culture model for studying differentiation and apoptosis in the mouse mammary gland Katrina E Gordon, Bert Binas*, Rachel S Chapman†, Kathreena M Kurian†, Richard W E Clarkson‡, A John Clark, E Birgitte Lane...

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

2000-03-07

18

Culturing of cerebellar granule cells to study neuronal migration: gradient and local perfusion assays.  

PubMed

Cultures of cerebellar granule cells are a suitable model to analyze the mechanisms governing neuronal migration. In this unit, we describe a protocol to obtain cultures of dissociated granule cells at a low density, where individual cells can be easily observed. In addition, we include a protocol for studying neuronal migration in these cultures, using single, actively migrating cerebellar granule cells. Following this protocol, a factor of interest can be applied either in a gradient concentration by means of a micropipet located near the neuron, or in a homogeneous concentration by locally perfusing a certain region of the neuron. Time-lapse images are taken to analyze changes in the speed and/or directionality of the observed neuron. Overall, the two protocols take more or less a day and a half to perform, and are a useful way to evaluate a certain factor/drug for its chemotactic activity or its capacity to alter migration speed. PMID:22752893

Guijarro, Patricia; Jiang, Jian; Yuan, Xiao-bing

2012-07-01

19

A Novel Three-Dimensional Culture System of Polarized Epithelial Cells to Study Endometrial Carcinogenesis  

PubMed Central

Development of three-dimensional (3D) cultures that mimic in vivo tissue organization has a pivotal role in the investigation of the involvement of cell adhesion and polarity genes in the pathogenesis of epithelial cancers. Here we describe a novel 3D culture model with primary mouse endometrial epithelial cells. In this model, isolated endometrial epithelial cells develop single-lumened, polarized glandular structures resembling those observed in endometrial tissue. Our in vitro 3D culture model of endometrial glands requires the use of serum-free defined medium with only epidermal growth factor and insulin as growth supplements and 3% Matrigel as reconstituted extracellular matrix. Under these culture conditions, glands of epithelial cells displaying typical apicobasal polarity and proper positioning of tight and adherent junctions are formed by hollowing as early as 7 to 8 days in culture. Addition of the phosphatidylinositol 3-kinase inhibitor LY294002 completely inhibits bromodeoxyuridine incorporation and cyclinD1 expression, confirming that in vitro growth of endometrial glands depends on phosphatidylinositol 3-kinase/Akt signaling. To prove that our culture method is a good model to study endometrial carcinogenesis, we knocked down E-cadherin or phosphatase and tensin homolog expression by lentivirus-delivered short hairpin RNAs. Down-regulation of E-cadherin resulted in complete loss of epithelial cell polarity and glandular formation, whereas phosphatase and tensin homolog down-regulation resulted in increased proliferation of glandular epithelial cells. These properties indicate that our 3D culture model is suitable to study the effect of growth factors, drugs, and gene alterations in endometrial carcinogenesis and to study normal endometrial biology/physiology. PMID:20395448

Eritja, Nuria; Llobet, David; Domingo, Monica; Santacana, Maria; Yeramian, Andree; Matias-Guiu, Xavier; Dolcet, Xavi

2010-01-01

20

Stem cell culture engineering  

Microsoft Academic Search

Stem cells have the capacity for self renewal and undergo multilineage differentiation. Stem cells isolated from both blastocysts and adult tissues represent valuable sources of cells for applications in cell therapy, drug screening and tissue engineering. While expanding stem cells in culture, it is critical to maintain their self?renewal and differentiation capacity. In generating particular cell types for specific applications,

Gargi Seth; Catherine M. Verfaillie

2005-01-01

21

Collagen-based co-culture for invasive study on cancer cells-fibroblasts interaction.  

PubMed

The roles of tumor stroma in carcinogenesis are still unclear. This study was aimed at designing an in vitro model for investigating the effects of stromal fibroblasts in the invasive growth of squamous cell carcinoma. Using two cancer cell lines, we performed three-dimensional co-culture with dermal equivalents to evaluate the effects of fibroblasts in cancer invasion. In vitro models for cellular interaction study were designed as follows: a collagen gel-based direct co-culture model (C-Dr) and a collagen gel-based indirect co-culture model (C-In). The invasive growth was found only in the dermal equivalents with fibroblasts. MMP-2 activity could be induced by direct contact between cancer cells and stromal fibroblasts. Cathepsin D was also highly expressed when co-cultured with cancer cells and fibroblasts. The present study demonstrated that the presence of fibroblasts is essential in cancer invasion and that collagen gel-based co-culture models might be useful for invasive study. PMID:16756953

Che, Zhong Min; Jung, Tae Heob; Choi, Jung Hee; Yoon, Do Jun; Jeong, Hyun Jeong; Lee, Eun Ju; Kim, Jin

2006-07-21

22

Cell Culture Made Easy.  

ERIC Educational Resources Information Center

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…

Dye, Frank J.

1985-01-01

23

Basics of Cell Culture  

NSDL National Science Digital Library

These manuals are used in the Stem Cell Culture Course at City College of San Francisco. This course is about general mammalian cell culture techniques but includes a laboratory exercise using stem cells (takes 3 weeks to complete). The course is taught to high school students but the materials are also used for college students. Laboratory exercises provide instruction in basic techniques of routine cell culture using common cell lines before progressing to differentiation of mouse embryonic stem cells. Photographs and explanations of common equipment (laminar flow hood, inverted microscope, etc.) and reagents are provided. Laboratory exercises include the following: Basic Aseptic Technique; Media Preparation; Plating cells from frozen stock; Cell counting and plating; Survival assay (UV); Live Cell Identification; Transfection; Freezing cells; Stem cell differentiation. A student lab manual and an instructor manual are provided.

Afshar, Golnar

2012-03-12

24

Progeria: A cell culture study and clinical report of familial incidence  

Microsoft Academic Search

This report relates the case histories of two sisters who demonstrated the typical symptoms of progeria at birth. One of these children had died previous to this study. The familial occurrence underlines the thesis that progeria is an autosomal-recessive disorder. The examination of the cultured skin fibroblasts from the younger child showed a clear decrease in cell growth. On the

Thomas Rautenstrauch; Friedemann Snigula; Thomas Krieg; Steffen Gay; P. K. Müller

1977-01-01

25

Magnetic approaches to study collective three-dimensional cell mechanics in long-term cultures (invited)  

NASA Astrophysics Data System (ADS)

Contractile forces generated by cells and the stiffness of the surrounding extracellular matrix are two central mechanical factors that regulate cell function. To characterize the dynamic evolution of these two mechanical parameters during tissue morphogenesis, we developed a magnetically actuated micro-mechanical testing system in which fibroblast-populated collagen microtissues formed spontaneously in arrays of microwells that each contains a pair of elastomeric microcantilevers. We characterized the magnetic actuation performance of this system and evaluated its capacity to support long-term cell culture. We showed that cells in the microtissues remained viable during prolonged culture periods of up to 15 days, and that the mechanical properties of the microtissues reached and maintained at a stable state after a fast initial increase stage. Together, these findings demonstrate the utility of this microfabricated bio-magneto-mechanical system in extended mechanobiological studies in a physiologically relevant 3D environment.

Zhao, Ruogang; Boudou, Thomas; Wang, Wei-Gang; Chen, Christopher S.; Reich, Daniel H.

2014-05-01

26

A comparative review of cell culture systems for the study of microglial biology in Alzheimer's disease.  

PubMed

Over the past two decades, it has become increasingly apparent that Alzheimer's disease neuropathology is characterized by activated microglia (brain resident macrophages) as well as the classic features of amyloid plaques and neurofibrillary tangles. The intricacy of microglial biology has also become apparent, leading to a heightened research interest in this particular cell type. Over the years a number of different microglial cell culturing techniques have been developed to study either primary mammalian microglia, or immortalized cell lines. Each microglial system has advantages and disadvantages and should be selected for its appropriateness in a particular research context. This review summarizes several of the most common microglial cell culture systems currently being employed in Alzheimer's research including primary microglia; BV2 and N9 retroviral immortalized microglia; human immortalized microglia (HMO6); and spontaneously immortalized rodent microglial lines (EOC lines and HAPI cells). Particularities of cell culture requirements and characteristics of microglial behavior, especially in response to applied inflammogen stimuli, are compared and discussed across these cell types. PMID:22651808

Stansley, Branden; Post, Jan; Hensley, Kenneth

2012-01-01

27

Multigenerational study of chemically induced cytotoxicity and proliferation in cultures of human proximal tubular cells.  

PubMed

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

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

2014-01-01

28

Pumps for microfluidic cell culture.  

PubMed

In comparison to traditional in vitro cell culture in Petri dishes or well plates, cell culture in microfluidic-based devices enables better control over chemical and physical environments, higher levels of experimental automation, and a reduction in experimental materials. Over the past decade, the advantages associated with cell culturing in microfluidic-based platforms have garnered significant interest and have led to a plethora of studies for high throughput cell assays, organs-on-a-chip applications, temporal signaling studies, and cell sorting. A clear concern for performing cell culture in microfluidic-based devices is deciding on a technique to deliver and pump media to cells that are encased in a microfluidic device. In this review, we summarize recent advances in pumping techniques for microfluidic cell culture and discuss their advantages and possible drawbacks. The ultimate goal of our review is to distill the large body of information available related to pumps for microfluidic cell culture in an effort to assist current and potential users of microfluidic-based devices for advanced in vitro cellular studies. PMID:23893649

Byun, Chang Kyu; Abi-Samra, Kameel; Cho, Yoon-Kyoung; Takayama, Shuichi

2014-02-01

29

Mammalian Cell Culture  

NSDL National Science Digital Library

This "Course-in-a-Box" from Bio-Link is a good starting point for instructors to develop a course on how to maintain mammalian cells in culture. Students will learn "basic techniques of routine cell culture using common cell lines before progressing to differentiation of mouse embryonic stem cells." Laboratories include Basic Aseptic Technique, Media Preparation, and Plating Cells from Frozen Stock. Materials include an Instructor Laboratory Manual, Student Laboratory Manual, Problem Sets, and Quizzes. A free login is required to access the materials.

2014-08-21

30

Neurotoxic effects of indocyanine green -cerebellar granule cell culture viability study  

PubMed Central

The aim of this study was to examine neurotoxicity indocyanine green (ICG). We assessed viability of primary cerebellar granule cell culture (CGC) exposed to ICG to test two mechanisms that could be the first triggers causing neuronal toxicity: imbalance in calcium homeostasis and the degree of oligomerization of ICG molecules. We have observed this imbalance in CGC after exposure to 75-125?? ICG and dose and application sequence dependent protective effect of Gadovist on surviving neurons in vitro when used with ICG. Spectroscopic studies suggest the major cause of toxicity of the ICG is connected with oligomers formation. ICG at concentration of 25 ?M (which is about 4 times higher than the highest concentration of ICG in the brain applied in in-vivo human studies) is not neurotoxic in the cell culture. PMID:24688815

Toczylowska, Beata; Zieminska, Elzbieta; Goch, Grazyna; Milej, Daniel; Gerega, Anna; Liebert, Adam

2014-01-01

31

Development of oligodendrocytes. Studies of rat glial cells cultured in chemically-defined medium.  

PubMed

Oligodendrocytes are macroglial cells that synthesize and maintain myelin in the central nervous system. Oligodendrocytes in rodent brain are formed postnatally from glial progenitor cells. These progenitors cells are bipotential and differentiate in a later stage of development into type-2 astrocytes. Recent studies with cultured cells indicate that growth factors such as platelet-derived growth factor and ciliary neurotrophic factor are instrumental in the control of these events. This paper discusses various methods for the isolation of oligodendrocytes and for their maintenance in culture. We use cerebra or spinal cords from one-week old rat pups to prepare glial cultures that are enriched in oligodendrocytes (60-80% or greater than or equal to 90%, respectively). After one day in serum-containing medium the cells are kept in chemically-defined medium, supplemented with the hormones insulin, T3 and hydrocortisone. The activities of astrocyte-and oligodendrocyte-specific marker enzymes were measured to evaluate the influence of these hormones on the differentiation of the oligodendrocytes. Finally, glial energy metabolism and the utilization of ketone bodies and of fatty acids are discussed briefly. PMID:2696749

Lopes-Cardozo, M; Sykes, J E; Van der Pal, R H; van Golde, L M

1989-09-01

32

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

PubMed

Grooved substrates are commonly used to guide cell alignment and produce in vitro tissues that mimic certain aspects of in vivo cellular organization. These more sophisticated tissues provide valuable in vitro models for testing drugs and for dissecting out molecular mechanisms that direct tissue organization. To increase the accessibility of these tissue models we describe a simple and yet reproducible strategy to produce 1?µm-spaced grooved well plates suitable for conducting automated analysis of cellular responses. We characterize the alignment of four human cell types: retinal epithelial cells, umbilical vein endothelial cells, foreskin fibroblasts, and human pluripotent stem-cell-derived cardiac cells on grooves. We find all cells align along the grooves to differing extents at both sparse and confluent densities. To increase the sophistication of in vitro tissue organization possible, we also created hybrid substrates with controlled patterns of microgrooved and flat regions that can be identified in real-time using optical microscopy. Using our hybrid patterned surfaces we explore: (i) the ability of neighboring cells to provide a template to organize surrounding cells that are not directly exposed to grooved topographic cues, and (ii) the distance over which this template effect can operate in confluent cell sheets. We find that in fibroblast sheets, but not epithelial sheets, cells aligned on grooves can direct alignment of neighboring cells in flat regions over a limited distance of approximately 200??m. Our hybrid surface plate provides a novel tool for studying the collective response of groups of cells exposed to differential topographical cues. Biotechnol. Bioeng. 2014;111: 2537-2548. © 2014 Wiley Periodicals, Inc. PMID:24889796

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

2014-12-01

33

Use of plant cell cultures to study the metabolism of environmental chemicals  

SciTech Connect

The metabolism of the following environmental chemicals has been studied in cell suspension cultures of wheat (Triticum aestivum L.) and soybean (Glycine max L.):2, 4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), hexachlorobenzene, pentachlorophenol, diethylhexylphthalate , benzo (alpha) pyrene, and DDT. All chemicals tested, including the persistent ones, were partially metabolized. Polar conjugates predominated in all cases. A covalent incorporation into lignin could be demonstrated for 2,4-D and pentachlorophenol. A specific deposition in the cellular vacuole could be demonstrated for the beta-D-glucopyranoside conjugates derived from 2,4-D. A rapid assay procedure to evaluate the metabolism of a given /sup 14/C-labeled chemical in plant cell suspension cultures is described. This procedure requires about 1 week, and the reproducibility of the results obtained has been assessed.

Sandermann, H. Jr.; Scheel, D.; v.d.Trenck, T.

1984-04-01

34

Poly(dimethylsiloxane) thin films as biocompatible coatings for microfluidic devices : cell culture and flow studies with glial cells.  

SciTech Connect

Oxygen plasma treatment of poly(dimethylsiloxane) (PDMS) thin films produced a hydrophilic surface that was biocompatible and resistant to biofouling in microfluidic studies. Thin film coatings of PDMS were previously developed to provide protection for semiconductor-based microoptical devices from rapid degradation by biofluids. However, the hydrophobic surface of native PDMS induced rapid clogging of microfluidic channels with glial cells. To evaluate the various issues of surface hydrophobicity and chemistry on material biocompatibility, we tested both native and oxidized PDMS (ox-PDMS) coatings as well as bare silicon and hydrophobic alkane and hydrophilic oligoethylene glycol silane monolayer coated under both cell culture and microfluidic studies. For the culture studies, the observed trend was that the hydrophilic surfaces supported cell adhesion and growth, whereas the hydrophobic ones were inhibitive. However, for the fluidic studies, a glass-silicon microfluidic device coated with the hydrophilic ox-PDMS had an unperturbed flow rate over 14 min of operation, whereas the uncoated device suffered a loss in rate of 12%, and the native PDMS coating showed a loss of nearly 40%. Possible protein modification of the surfaces from the culture medium also were examined with adsorbed films of albumin, collagen, and fibrinogen to evaluate their effect on cell adhesion.

Peterson, Sophie Louise; Sasaki, Darryl Yoshio; Gourley, Paul Lee; McDonald, Anthony Eugene

2004-06-01

35

Electron microscopic study of the African strain of malignant catarrhal fever virus in bovine cell cultures.  

PubMed

Malignant catarrhal fever (African strain) is a viral disease of ruminants which is considered an exotic disease in the United States. Viral isolates obtained from one clinically ill gaur (Bos gaurus) and a greater kudu (Tragelaphus strepsiceros) located in a zoologic park in Oklahoma, and from one heifer (Bos taurus) and a domestic white-tailed deer (Odocoileus virginianus) experimentally inoculated with the isolated and identified African strain of malignant catarrhal fever virus (MCFV), were each studied in bovine cell cultures by electron microscopy. Certain of the viral isolated were previously characterized as MCFV by serologic and morphologic examinations, their cytopathic effect in cell cultures, and their ability to reproduce disease in a ruminant host. The virions of MCFV (African) examined by electron microscopy were icosahedral similar to herpes-virus, were between 98 and 194 nm, developed in the nucleus and matured in the cytoplasm of the cell, and exhibited budding. The virus in infected cells passed through the nuclear and plasma membranes and also into cytoplasmic vesicles from which it acquired one or more envelopes. Virions of malignant catarrhal fever were closely associated with the cellular endoplasmic reticulum, and aberrant morphologic forms of MCFV were observed in virus-infected cells. PMID:7073076

Castro, A E; Daley, G G

1982-04-01

36

Cytogenetic studies of Haplopappus gracilis in both callus and suspension cell cultures.  

PubMed

Investigations have been carried out on karyotype change in both callus and suspension cell cultures of Haplopappus gracilis (2n=4). It has been found that polyploidization arises directly in culture to give up to six times the normal diploid chromosome number in some cultures. In polyploid cultures, both chromosome loss and chromosome rearrangements occur to give rise to aneuploid karyotypes displaying chromosomes which differ in morphology from the diploid set. Whole or partial chromosome loss has been observed in the form of lagging chromosomes and chromosome bridges at anaphase, and micronuclei, ring chromosomes and chromosome fragments at other stages in mitosis. C-banded preparations have confirmed the occurrence of chromosomal rearrangements. Comparative investigations suggest that (i) more polyploidy occurs in callus cultures than in suspension cell cultures, and (ii) the presence of cytokinin (kinetin) in the culture medium may reduce the extent of karyotype change. PMID:24227152

Ashmore, S E; Shapcott, A S

1989-08-01

37

Cell Culturing of Cytoskeleton  

NASA Technical Reports Server (NTRS)

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.

2004-01-01

38

Cell Culturing of Cytoskeleton  

NASA Technical Reports Server (NTRS)

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.

2004-01-01

39

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

NASA Astrophysics Data System (ADS)

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

Kudo, Nobuki; Kinoshita, Yuto

2012-11-01

40

Hyaluronan synthesis in cultured tobacco cells (BY-2) expressing a chlorovirus enzyme: cytological studies.  

PubMed

Extraction of hyaluronan from animals or microbial fermentation has risks including contamination with pathogens and microbial toxins. In this work, tobacco cultured-cells (BY-2) were successfully transformed with a chloroviral hyaluronan synthase (cvHAS) gene to produce hyaluronan. Cytological studies revealed accumulation of HA on the cells, and also in subcellular fractions (protoplasts, miniplasts, vacuoplasts, and vacuoles). Transgenic BY-2 cells harboring a vSPO-cvHAS construct containing the vacuolar targeting signal of sporamin connected to the N-terminus of cvHAS accumulated significant amounts of HA in vacuoles. These results suggested that cvHAS successfully functions on the vacuolar membrane and synthesizes/transports HA into vacuoles. Efficient synthesis of HA using this system provides a new method for practical production of HA. PMID:23404209

Rakkhumkaew, Numfon; Shibatani, Shigeo; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

2013-04-01

41

CYSTIC FIBROSIS OF THE PANCREAS A STUDY rN CELL CULTUR~.*,~:  

E-print Network

Cystic fibrosis of the pancreas is probably the most frequent inherited disease affecting Caucasian populations. The classic clinical features of this grave childhood disorder include chronic bronchiolar obstruction, repeated pulmonary infections, steatorrhea, azotorrhea, and elevation of electrolytes in the sweat. The clinical and pathological aspects of cystic fibrosis have been comprehensively reviewed by di Sant'Agnese and Talamo (1). Several studies have emphasized that the clinical expression of the disease may be extremely variable (2, 3). Although formal genetic analysis (4-7) indicates that the disease is inherited in a simple autosomal recessive fashion, the primary molecular abnormality in cystic fibrosis is presently unknown. Recent advances in cell culture make it possible to grow human fibroblasts and to establish diploid cell lines for detailed morphological and chemical studies (8). Preliminary experiments undertaken in this laboratory revealed a consistent morphological abnormality of cytoplasmic metachromasia in skin fibroblasts derived from patients with cystic fibrosis and known heterozygous

B. Shannon Danes; Am Alexander; G. Bearn

1968-01-01

42

Ultrafast-Laser Interactions with Soft Biological Tissues a Study with Viable 3-D Hydrogel Cell Cultures  

E-print Network

Ultrafast-Laser Interactions with Soft Biological Tissues ­ a Study with Viable 3-D Hydrogel Cell., Toronto, Ontario M5G 2M9 Canada Abstract: We've developed a 3-dimensional hydrogel cell culture-type differentiation. As a simpler proxy for in vivo studies, we have developed an agar-based hydrogel as a 3D tissue

Marjoribanks, Robin S.

43

[Human myoblast culture as muscle stem cells in medical and biological studies].  

PubMed

The method for obtaining human myoblast culture has been modified to consider the specific histological localization of the satellite cells as well as their growth properties; the cultivation conditions have been selected to grow up to 150000 cells/cm2. At high densities, the cells remain mononuclear and preserve their typical myoblast morphology as well as the capacity for fusion and the formation of myotubes. By contrast to fibroblasts, up to 80% of the cells in the myoblast culture were positive in the acid phosphatase test, which indicates their stem nature. The obtained myoblast cultures were used in the clinical tests of cell-mediated gene therapy of Duchenne's muscular dystrophy as well as in the bioassay for the effects of biologically active compounds. PMID:15926341

Terekhov, S M; Krokhina, T B; Shishkin, S S; Krakhmaleva, I N; Zakharov, S F; Ershova, E S

2001-01-01

44

A microfluidic system for automatic cell culture  

NASA Astrophysics Data System (ADS)

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

Huang, Chun-Wei; Lee, Gwo-Bin

2007-07-01

45

J Cell Biochem . Author manuscript Optimizing stem cell culture  

E-print Network

cell culture is widely used in basic research for studying stem cell biology, but also owingJ Cell Biochem . Author manuscript Page /1 7 Optimizing stem cell culture Boudewijn Van Der Sanden * Correspondence should be adressed to: Didier Wion Abstract Stem cells always

Paris-Sud XI, Université de

46

Effect of pulse duration on KrF laser treatment of a polyethersulfone film: cell culture study  

NASA Astrophysics Data System (ADS)

KrF laser processing of polyethersulfone (PES) films at three different pulse durations regarding to the change in biocompatibility is presented with cell culture study. Various microstructure and chemistry results were obtained on the surface upon laser irradiation. It is shown that nanoscale hydrophilic ripples significantly affect the adhesion of L929 cells on the surface.

Pazokian, Hedieh; Mollabashi, Mahmoud; Selimis, Alexandros; Stratakis, Emmanuel; Barzin, Jalal; Jelvani, Saeid

2013-03-01

47

Urothelial cell culture.  

PubMed

This chapter reviews the use of urothelial cells as a means to enhance tissue regeneration and wound healing in urinary tract system. It addresses the properties of urothelial cells, including their role as a permeability barrier to protect underlying muscle tissue from the caustic effects of urine and as one of the main cell types, along with smooth muscle cells, that are used in urethral or bladder tissue engineering today. This description includes a general overview of various isolation techniques and culture methods that have been developed to improve urinary tract reconstruction in vivo and aid the characterization of growth factor expression in vitro. The chapter then describes various applications using urothelial cells, including production of multilayer urothelial sheets, tissue engineered bladder mucosa, tissue engineered urethra, and tissue engineered bladder. It also outlines the advantages of sandwich and layered coculture of these cells and the effects of epithelial-stromal cell interactions during tissue regeneration or wound healing processes in the urinary tract. PMID:24029928

Zhang, Yuanyuan; Atala, Anthony

2013-01-01

48

Are quantum dots toxic? Exploring the discrepancy between cell culture and animal studies.  

PubMed

Despite significant interest in developing quantum dots (QDs) for biomedical applications, many researchers are convinced that QDs will never be used for treating patients because of their potential toxicity. The perception that QDs are toxic is rooted in two assumptions. Cadmium-containing QDs can kill cells in culture. Many researchers then assume that because QDs are toxic to cells, they must be toxic to humans. In addition, many researchers classify QDs as a homogeneous group of materials. Therefore, if CdSe QDs are harmful, they extrapolate this result to all QDs. Though unsubstantiated, these assumptions continue to drive QD research. When dosing is physiologically appropriate, QD toxicity has not been demonstrated in animal models. In addition, QDs are not uniform: each design is a unique combination of physicochemical properties that influence biological activity and toxicity. In this Account, we summarize key findings from in vitro and in vivo studies, explore the causes of the discrepancy in QD toxicological data, and provide our view of the future direction of the field. In vitro and in vivo QD studies have advanced our knowledge of cellular transport kinetics, mechanisms of QD toxicity, and biodistribution following animal injection. Cell culture experiments have shown that QDs undergo design-dependent intracellular localization and they can cause cytotoxicity by releasing free cadmium into solution and by generating free radical species. In animal experiments, QDs preferentially enter the liver and spleen following intravascular injection, undergo minimal excretion if larger than 6 nm, and appear to be safe to the animal. In vitro and in vivo studies show an apparent discrepancy with regard to toxicity. Dosing provides one explanation for these findings. Under culture conditions, a cell experiences a constant QD dose, but the in vivo QD concentration can vary, and the organ-specific dose may not be high enough to induce detectable toxicity. Because QDs are retained within animals, long-term toxicity may be a problem but has not been established. Future QD toxicity studies should be standardized and systematized because methodological variability in the current body of literature makes it difficult to compare and contrast results. We advocate the following steps for consistent, comparable toxicology data: (a) standardize dose metrics, (b) characterize QD uptake concentration, (c) identify in vitro models that reflect the cells QDs interact with in vivo, and (d) use multiple assays to determine sublethal toxicity and biocompatibility. Finally, we should ask more specific toxicological questions. For example: "At what dose are 5 nm CdSe QDs that are stabilized with mercaptoacetic acid and conjugated to the antibody herceptin toxic to HeLa cells?" rather than "Are QDs toxic?" QDs are still a long way from realizing their potential as a medical technology. Modifying the current QD toxicological research paradigm, investigating toxicity in a case-by-case manner, and improving study quality are important steps in identifying a QD formulation that is safe for human use. PMID:22853558

Tsoi, Kim M; Dai, Qin; Alman, Benjamin A; Chan, Warren C W

2013-03-19

49

Epitheliomesenchymal Transdifferentiation of Cultured RPE Cells  

Microsoft Academic Search

Retinal pigment epithelium (RPE) cells of the proliferative vitreoretinopathy (PVR) membrane take on the shape of fibroblasts and participate in fibrosis, thus deviating from the character of epithelial cells. This study was undertaken to evaluate RPE cell transdifferentiation in vitro. During the culture of porcine RPE cells, primary and 10th-passaged RPE cells were investigated for cell growth in response to

Sung-Chul Lee; Oh-Woong Kwon; Gong-Je Seong; Soon-Hyun Kim; Jae-Eun Ahn; Eun-Duck P. Kay

2001-01-01

50

Ex vivo electroporation of retinal cells: a novel, high efficiency method for functional studies in primary retinal cultures.  

PubMed

Primary retinal cultures constitute valuable tools not only for basic research on retinal cell development and physiology, but also for the identification of factors or drugs that promote cell survival and differentiation. In order to take full advantage of the benefits of this system it is imperative to develop efficient and reliable techniques for the manipulation of gene expression. However, achieving appropriate transfection efficiencies in these cultures has remained challenging. The purpose of this work was to develop and optimize a technique that would allow the transfection of chick retinal cells with high efficiency and reproducibility for multiple applications. We developed an ex vivo electroporation method applied to dissociated retinal cell cultures that offers a significant improvement over other currently available transfection techniques, increasing efficiency by five-fold. In this method, eyes were enucleated, devoid of RPE, and electroporated with GFP-encoding plasmids using custom-made electrodes. Electroporated retinas were then dissociated into single cells and plated in low density conditions, to be analyzed after 4 days of incubation. Parameters such as voltage and number of electric pulses, as well as plasmid concentration and developmental stage of the animal were optimized for efficiency. The characteristics of the cultures were assessed by morphology and immunocytochemistry, and cell viability was determined by ethidium homodimer staining. Cell imaging and counting was performed using an automated high-throughput system. This procedure resulted in transfection efficiencies in the order of 22-25% of cultured cells, encompassing both photoreceptors and non-photoreceptor neurons, and without affecting normal cell survival and differentiation. Finally, the feasibility of the technique for cell-autonomous studies of gene function in a biologically relevant context was tested by carrying out gain and loss-of-function experiments for the transcription factor PAX6. Electroporation of a plasmid construct expressing PAX6 resulted in a marked upregulation in the expression levels of this protein that could be measured in the whole culture as well as cell-intrinsically. This was accompanied by a significant decrease in the percentage of cells differentiating as photoreceptors among the transfected population. Conversely, electroporation of an RNAi construct targeting PAX6 resulted in a significant decrease in the levels of this protein, with a concomitant increase in the proportion of photoreceptors. Taken together these results provide strong proof-of-principle of the suitability of this technique for genetic studies in retinal cultures. The combination of the high transfection efficiency obtained by this method with automated high-throughput cell analysis supplies the scientific community with a powerful system for performing functional studies in a cell-autonomous manner. PMID:23370269

Vergara, M Natalia; Gutierrez, Christian; O'Brien, David R; Canto-Soler, M Valeria

2013-04-01

51

Three Dimensional Cultures: A Tool to Study Normal Acinar Architecture vs. Malignant Transformation of Breast Cells  

PubMed Central

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex-vivo system2–3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6–7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling. PMID:24797513

Pal, Anupama; Kleer, Celina G.

2014-01-01

52

Studies on the Nature of Receptors Involved in Attachment of Tissue Culture Cells to Mycoplasmas  

PubMed Central

Several mycoplasmas, from avian and mammalian sources, growing in the form of colonies on agar and sheets attached to plastic dishes, were tested for their ability to adsorb tissue culture cells in suspension. HeLa cells adsorbed to the majority of mycoplasmas tested; adsorption occurred to the sheets and not to the colonies of some mycoplasmas. Other tissue cells, in primary culture and of diploid origin, adsorbed also. The mechanism of adsorption of HeLa cells to 4 mycoplasmas was examined by treating the cells and mycoplasmas in various ways and then testing for adsorption. N-acetyl neuraminic acid residues on the tissue cells were responsible for adsorption to M. gallisepticum and M. pneumoniae. The receptors for M. hominis and M. salivarium were probably not of this kind since treatment of the cells with purified neuraminidase did not influence adsorption. However, the cell receptors for these mycoplasmas were associated with protein because they were inactivated by proteolytic enzymes and by formalin. The cell receptors for M. hominis were more heat stable than those for the other mycoplasmas. From the aspect of the mycoplasma membrane, in no instance did neuraminidase treatment affect adsorption. On the other hand, various experiments suggested that protein components of the mycoplasma membrane were involved. The significance of these findings is discussed. PMID:5773147

Manchee, R. J.; Taylor-Robinson, D.

1969-01-01

53

Cell culture's spider silk road.  

PubMed

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

Perkel, Jeffrey

2014-06-01

54

Cell culture purity issues and DFAT cells  

SciTech Connect

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

Wei, Shengjuan [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China) [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China); Department of Animal Sciences, Washington State University, Pullman, WA 99164 (United States); Bergen, Werner G. [Program in Cellular and Molecular Biosciences/Department of Animal Sciences, Auburn University, Auburn, AL 36849 (United States)] [Program in Cellular and Molecular Biosciences/Department of Animal Sciences, Auburn University, Auburn, AL 36849 (United States); Hausman, Gary J. [Animal Science Department, University of Georgia, Athens, GA 30602-2771 (United States)] [Animal Science Department, University of Georgia, Athens, GA 30602-2771 (United States); Zan, Linsen, E-mail: zanls@yahoo.com.cn [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China)] [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China); Dodson, Michael V., E-mail: dodson@wsu.edu [Department of Animal Sciences, Washington State University, Pullman, WA 99164 (United States)

2013-04-12

55

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

PubMed Central

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

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

2014-01-01

56

A scanning electron microscopic study of phenotypic plasticity and surface structural changes of aortal smooth muscle cells in primary culture.  

PubMed

Phenotypic modulation of smooth muscle cells (SMCs) from a contractile to a synthetic state characterised by active proliferation appears to be an early event in the pathogenesis of atherosclerosis. A similar transition occurs when SMCs are established in culture. In this study the phenotypic plasticity and surface structural changes of aortal smooth muscle cells during the transition from the contractile to the synthetic state and during maturation have been structurally assessed by scanning electron microscope (SEM). The experiments were performed on SMCs obtained from aorta of neonatal rats after enzymatic digestion and then cultured on glass coverslips. SEM observations revealed a three-dimensional appearance characteristic for different stages of SMCs. Intensively proliferating cells from monolayer region were large, polygonal in shape with lamellipodia and well spread. Long, uniform in diameter, finger-like microvilli were densely arranged on the surface of these cells. In the thickened region of culture, the cells were rather small, generally spindle-shaped, not well spread, with low density of short, bubble-like microvilli on the surface. Numerous plasma membrane structural alterations in apoptotic cells were observed by SEM: loss of cellular adhesion, smoothing, shrinkage and outpouching of membrane segments have been recognised as markers associated with the cell injury and death. It was concluded that scanning microscopy observations would allow a more complete understanding of SMCs and their changes in culture and atherosclerotic disease. PMID:12725484

Tukaj, Cecylia; Bohdanowicz, Jerzy; Kubasik-Juraniec, Jolanta

2002-01-01

57

Poetry After Cultural Studies  

Microsoft Academic Search

Poetry after Cultural Studies elucidates the potential of poetry scholarship when joined with cultural studies. In eight searching essays covering an astonishing range of poetic practices, geographical regions, and methodological approaches, this volume reflects on what poetry can accomplish in the broadest social and cultural contexts. From Depression-era Iowa to the postcolonial landscape of French-speaking Martinique, whether appearing in newspapers,

Heidi R. Bean; Michael Chasar

2011-01-01

58

Comparative genomic study of gastric epithelial cells co-cultured with Helicobacter pylori  

PubMed Central

AIM: To identify genes potentially involved in Helicobacter pylori (H. pylori)-induced gastric carcinogenesis. METHODS: GES-1 cells were co-cultured with H. pylori strains isolated from patients with gastric carcinoma (GC, n = 10) or chronic gastritis (CG, n = 10) for in vitro proliferation and apoptosis assays to identify the most and least virulent strains. These two strains were cagA-genotyped and used for further in vivo carcinogenic virulence assays by infecting Mongolian gerbils for 52 wk, respectively; a broth free of H. pylori was lavaged as control. Genomic profiles of GES-1 cells co-cultured with the most and least virulent strains were determined by microarray analysis. The most differentially expressed genes were further verified using quantitative real-time polymerase chain reaction in GES-1 cells infected with the most and least virulent strains, and by immunohistochemistry in H. pylori positive CG, precancerous diseases, and GC biopsy specimens in an independent experiment. RESULTS: GC-derived H. pylori strains induced a potent proliferative effect in GES-1 cells in co-culture, whereas CG-derived strains did not. The most (from a GC patient) and least (from a CG patient) virulent strains were cagA-positive and negative, respectively. At week 52, CG, atrophy, metaplasia, dysplasia, and GC were observed in 90.0%, 80.0%, 80.0%, 90%, and 60.0%, respectively, of the animals lavaged with the most virulent strain. However, only mild CG was observed in 90% of the animals lavaged with the least virulent strain. On microarray analysis, 800 differentially expressed genes (49 up- and 751 down-regulated), involving those associated with cell cycle regulation, cell apoptosis, cytoskeleton, immune response, and substance and energy metabolisms, were identified in cells co-cultured with the most virulent strain as compared with those co-cultured with the least virulent strain. The six most differentially expressed genes (with a betweenness centrality of 0.1-0.2) were identified among the significant differential gene profile network, including JUN, KRAS, BRCA1, SMAD2, TRAF1, and HDAC6. Quantitative real-time polymerase chain reaction analyses verified that HDAC6 and TRFA1 mRNA expressions were significantly more up-regulated in GES-1 cells co-cultured with the most virulent strain than in those co-cultured with the least virulent strain. Immunohistochemistry of gastric mucosal specimens from H. pylori-positive patients with CG, intestinal metaplasia (IM), dysplasia, and GC showed that moderately positive and strongly positive HDAC6 expression was detected in 21.7% of CG patients, 30.0% of IM patients, 54.5% of dysplasia patients, and 77.8% of GC patients (P < 0.001). The up-regulation of TRAF1 expressions was detected in 34.8%, 53.3%, 72.7%, and 88.9% specimens of CG, IM, dysplasia, and GC, respectively (P < 0.001). CONCLUSION: The overexpression of HDAC6 and TRAF1 in GES-1 cells co-cultured with the GC-derived strain and in H. pylori-positive dysplasia and GC suggests that HDAC6 and TRAF1 may be involved in H. pylori-induced gastric carcinogenesis. PMID:23326126

Wang, Fen; Luo, Li-Dan; Pan, Jian-Hua; Huang, Li-Hua; Lv, Hong-Wei; Guo, Qin; Xu, Can-Xia; Shen, Shou-Rong

2012-01-01

59

A multifunctional 3D co-culture system for studies of mammary tissue morphogenesis and stem cell biology.  

PubMed

Studies on the stem cell niche and the efficacy of cancer therapeutics require complex multicellular structures and interactions between different cell types and extracellular matrix (ECM) in three dimensional (3D) space. We have engineered a 3D in vitro model of mammary gland that encompasses a defined, porous collagen/hyaluronic acid (HA) scaffold forming a physiologically relevant foundation for epithelial and adipocyte co-culture. Polarized ductal and acinar structures form within this scaffold recapitulating normal tissue morphology in the absence of reconstituted basement membrane (rBM) hydrogel. Furthermore, organoid developmental outcome can be controlled by the ratio of collagen to HA, with a higher HA concentration favouring acinar morphological development. Importantly, this culture system recapitulates the stem cell niche as primary mammary stem cells form complex organoids, emphasising the utility of this approach for developmental and tumorigenic studies using genetically altered animals or human biopsy material, and for screening cancer therapeutics for personalised medicine. PMID:21984937

Campbell, Jonathan J; Davidenko, Natalia; Caffarel, Maria M; Cameron, Ruth E; Watson, Christine J

2011-01-01

60

Electron microscopic studies of bovine viral diarrhea virus in tissues of diseased calves and in cell cultures  

Microsoft Academic Search

Summary Pathomorfological studies by electron microscopy (EM) were carried out on the intestines and lymphoid tissues, the buffy coat cells and cultured lymphocytes from calves suffering from mucosal disease (MD). This led to the detection of particles, 45–55 nm in diameter, within characteristic vesicular structures. As these findings coincided with the isolation of bovine viral diarrhea virus (BVDV) from the

H. Bielefeldt Ohmann; B. Bloch

1982-01-01

61

Marine Invertebrate Cell Cultures: New Millennium Trends  

Microsoft Academic Search

This review analyzes activities in the field of marine invertebrate cell culture during the years 1999 to 2004 and compares the outcomes with those of the preceding decade (1988 to 1998). During the last 5 years, 90 reports of primary cell culture studies of marine organisms belonging to only 6 taxa (Porifera, Cnidaria, Crustacea, Mollusca, Echinodermata, and Urochordata) have been

Baruch Rinkevich

2005-01-01

62

Cell culture experiments planned for the space bioreactor  

NASA Technical Reports Server (NTRS)

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.

Morrison, Dennis R.; Cross, John H.

1987-01-01

63

Regulation of cardiac myosin synthesis: Studies of RNA content in cultured heart cells  

SciTech Connect

Contraction regulates the myosin content and the rate of myosin synthesis in cultured neonatal rat heart cells. To further explore the mechanism for this regulation the authors examined various parameters of RNA content and RNA synthesis in contracting versus noncontracting myocytes. While contraction stimulated myosin heavy chain (MHC) synthesis by 72% compared to that of KCl-arrested cells, simultaneous analyses of polysome profiles were no different under the two culture conditions. Incorporation of ({sup 3}H) uridine monophosphate into cellular RNA revealed no change in the rate of total RNA or ribosomal subunits synthesis. In vitro translation of cellular RNA yielded similar incorporation of ({sup 35}S) methionine not trichloroacetic acid precipitable protein. Specific transcription of the MHC gene was examined by dot-blot analysis and was unaltered by contraction. Northern blot analysis of the MHC sequences detected by a cDNA probe revealed an mRNA sequence corresponding to a molecular weight of approximately 30 S. These data suggest that RNA synthesis and RNA content are unaltered by contraction in cultured heart cells and therefore the changes in myosin synthesis may be mediated at a post-transcriptional control level.

McDermott, P.; Whitaker-Dowling, P.; Klein, I. (Univ. of Pittsburgh, PA (United States) Cornell Univ., New York, NY (United States))

1987-11-01

64

Ultrastructural studies of surface features of human normal and tumor cells in tissue culture by scanning and transmission electron microscopy.  

PubMed

Human tumors of a variety of histopathologic types have been established in tissue culture. The surface features of these cell lines were investigated by scanning electron microscopy (SEM) with the use of new techniques for specimen preparation. Tumor cells demonstrated striking degrees of surface activity with numerous microvilli, filopodia, blebs, and ruffles. Intercellular contacts were also prominent in cultures of most solid tumors observed by SEM. At low cell density, normal human fibroblasts exhibited some surface features such as microvilli and blebs, but at higher cell density they lacked extensive surface modifications. By transmission electron microscopy (TEM), the cytoskeleton of normal fibroblasts was shown to be well organized, with parallel orientation of microfilaments, filaments, and microtubules. These structures were also in tumor cells, but they lacked the degree of organization of fibroblasts. Desmosomes were readily demonstrated in normal fibroblasts and carcinoma cells in culture but not in sarcomas, melanomas, or tumors of neural origin. These studies have provided the first correlative SEM and TEM analyses of solid human tumor cells of diverse pathologic types in vitro. PMID:1255758

Gonda, M A; Aaronson, S A; Ellmore, N; Zeve, V H; Nagashima, K

1976-02-01

65

AlgiMatrix(TM) Based 3D Cell Culture System as an In-Vitro Tumor Model for Anticancer Studies  

PubMed Central

Background 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. Methods 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. Results IC50 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. Conclusion 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. PMID:23349734

Godugu, Chandraiah; Patel, Apurva R.; Desai, Utkarsh; Andey, Terrick; Sams, Alexandria; Singh, Mandip

2013-01-01

66

A polydimethylsiloxane-polycarbonate hybrid microfluidic device capable of generating perpendicular chemical and oxygen gradients for cell culture studies.  

PubMed

This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) hybrid microfluidic device capable of performing cell culture under combinations of chemical and oxygen gradients. The microfluidic device is constructed of two PDMS layers with microfluidic channel patterns separated by a thin PDMS membrane. The top layer contains an embedded PC film and a serpentine channel for a spatially confined oxygen scavenging chemical reaction to generate an oxygen gradient in the bottom layer for cell culture. Using the chemical reaction method, the device can be operated with a small amount of chemicals, without bulky gas cylinders and sophisticated flow control schemes. Furthermore, it can be directly used in conventional incubators with syringe pumps to simplify the system setup. The bottom layer contains arrangements of serpentine channels for chemical gradient generation and a cell culture chamber in the downstream. The generated chemical and oxygen gradients are experimentally characterized using a fluorescein solution and an oxygen-sensitive fluorescent dye, respectively. For demonstration, a 48 hour cell-based drug test and a cell migration assay using human lung adenocarcinoma epithelial cells (A549) are conducted under various combinations of the chemical and oxygen gradients in the experiments. The drug testing results show an increase in A549 cell apoptosis due to the hypoxia-activated cytotoxicity of tirapazamine (TPZ) and also suggest great cell compatibility and gradient controllability of the device. In addition, the A549 cell migration assay results demonstrate an aerotactic behavior of the A549 cells and suggest that the oxygen gradient plays an essential role in guiding cell migration. The migration results, under combinations of chemokine and oxygen gradients, cannot be simply superposed with single gradient results. The device is promising to advance the control of in vitro microenvironments, to better study cellular responses under various physiological conditions for biomedical applications. PMID:25096368

Chang, Chia-Wen; Cheng, Yung-Ju; Tu, Melissa; Chen, Ying-Hua; Peng, Chien-Chung; Liao, Wei-Hao; Tung, Yi-Chung

2014-10-01

67

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

PubMed

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

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

2014-09-01

68

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

PubMed Central

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

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

2014-01-01

69

Some relationships among germ, satellite and interstitial cells during chick gonad differentiation : A tissue culture study  

E-print Network

collaborators carried out remarkable experiments on gonadal differentiation, achieving feminization of the male and 14 and in the testis between ED 14 and 17 (Swift, 1915, 1916 ; Brode, 1928). This study was carried Leghorn or White Plymouth Rock x Cornish chick embryos on ED 8 to 16. The tissue culture technique has

Boyer, Edmond

70

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

EPA Science Inventory

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

71

Biochemical studies on PT523, a potent nonpolyglutamatable antifolate, in cultured cells.  

PubMed

Studies on the mode of action of PT523 [N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine], a potent nonpolyglutamatable antifolate, were carried out in sensitive and resistant H35 rat hepatoma cell lines in culture, to compare it with other antifolates, including three dihydrofolate reductase (DHFR) inhibitors, i.e., methotrexate (MTX), gamma-fluoro-MTX, and trimetrexate (TMQ), two thymidylate synthase inhibitors, i.e., N10-propargyl-5,8- dideazafolate (PDDF) and 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolate (dmPDDF), and the glycinamide ribonucleotide formyltransferase inhibitor 5,10-dideaza-5,6,7,8-tetrahydrofolate. PT523 was the most active compound in this group against the parental H35 cells, with an IC50 ranging from 2.5 nM for 72 hr of treatment to 0.21 microM for 2 hr of treatment. Sublines resistant to MTX by virtue of a transport defect or a combination of defective transport and increased DHFR activity were resistant to PT523 and MTX but not to PDDF, whereas sublines resistant to fluoropyrimidines by virtue of increased thymidylate synthase activity were resistant to PDDF but not to PT523, TMQ, or MTX. Inhibition of H35 cell growth by PT523 was associated with a concentration- and time-related decrease in de novo dTMP and purine biosynthesis. Growth inhibition by PT523, MTX, and TMQ was prevented by leucovorin or a combination of thymidine (dThd) and hypoxanthine but not by dThd or hypoxanthine alone; in contrast, growth inhibition by dmPDDF was prevented by dThd alone. Intracellular reduced folate polyglutamate pools were markedly altered by PT523 treatment, with the most pronounced effect being an increase in 7,8-dihydrofolate mono- and polyglutamates and a decrease in 5,10-methylene-5,6,7,8-tetrahydrofolate mono- and polyglutamates, 5,6,7,8-tetrahydrofolate mono- and polyglutamates, and 10-formyl-5,6,7,8-tetrahydrofolate mono- and polyglutamates. This pattern was qualitatively similar to that observed with MTX and TMQ but different from that observed with dmPDDF or 5,10-dideaza-5,6,7,8-tetrahydrofolate, which resulted in little or no change in the folate species. Uptake of [3H]MTX and [3H]folinic acid, but not [3H]folic acid, by H35 cells was inhibited in a dose-related manner by PT523, suggesting that penetration of the cell probably involves, at least in part, active transport by the MTX/reduced folate carrier. To determine whether the potent cellular effects of PT523 might be due to chemical or enzymic clevage to N'-(4-amino-4-deoxypteroyl)-L-ornithine, a potent inhibitor of folylpolyglutamate synthetase, the formation of [3H]MTX polyglutamates in CCRF-CEM lymphoblasts pulsed with [3H]MTX after preincubation with PT523 was examined.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7514264

Rhee, M S; Galivan, J; Wright, J E; Rosowsky, A

1994-04-01

72

Plant cell suspension culture rheology.  

PubMed

The results of rheological measurements on 10 different plant cell suspension cultures are presented. Nicotiana tabacum (tobacco) suspension cultures grown in serial batch subculture display high viscosity and power law rheology. This "undesirable" rheology is shown to be a result of elongated cell morphology. The rheology of Papaver somniferum (poppy) cell suspensions is quite different; poppy suspensions behave as Newtonian fluids and have relatively low viscosity (less than 15 cP) at fresh cell densities up to 250 g/L. This flow behavior can be attributed to a lack of elongation in batch-grown poppy cells. A simple correlation for the viscosity as a function of cell density is developed for poppy suspensions up to 300 g fresh weight (FW)/L. It is shown that tobacco cells do not elongate when grown in semicontinuous culture (daily media replacement). These semicontinuously cultured cells have rheological behavior that is indistinguishable from that of poppy, further confirming the dependence of rheology on plant cell morphology. The rheology of a wide variety of other plant suspensions at 200 g FW/L is presented. Most cell suspensions, including soybean, cotton, bindweed, and potato, display low viscosities similar to poppy suspensions. Only carrot and atriplex exhibit slight pseudoplastic behavior which corresponded to a slight degree of cellular elongation for these cultures. This demonstrates that complex rheology associated with elongated cell morphology is much less common than low-viscosity Newtonian behavior. High viscosity in plant cell culture is therefore not an intrinsic characteristic of plant cells but, instead, is a result of the ability to grow cultures to extremely high cell densities due to low biological oxygen demand. PMID:18613057

Curtis, W R; Emery, A H

1993-08-01

73

Adaptation of culture methods for NMR studies of anchorage-dependent cells.  

PubMed

Two methods for growing anchorage-dependent cells were adapted for nuclear magnetic resonance (NMR) measurements: growing cells on agarose polyacrolein microsphere beads and on "filters" made of nonwoven polyester fabric. Both were found to be convenient and most suitable for NMR studies in any conventional spectrometer without probe modification. These methods were employed in studies of human breast cancer T47D-A11 cells, using scanning electron microscopy and 31P NMR spectroscopy. The results show that the contents per cell of phosphorylcholine, phosphorylethanolamine, and their glycerol derivatives depend on the mode of cell assembly and decrease gradually with the increase in cell-cell interaction along the growth curve. PMID:3398771

Neeman, M; Rushkin, E; Kadouri, A; Degani, H

1988-06-01

74

High density cell culture system  

NASA Technical Reports Server (NTRS)

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

Spaulding, Glenn F. (inventor)

1994-01-01

75

Dynamized Preparations in Cell Culture  

PubMed Central

Although reports on the efficacy of homeopathic medicines in animal models are limited, there are even fewer reports on the in vitro action of these dynamized preparations. We have evaluated the cytotoxic activity of 30C and 200C potencies of ten dynamized medicines against Dalton's Lymphoma Ascites, Ehrlich's Ascites Carcinoma, lung fibroblast (L929) and Chinese Hamster Ovary (CHO) cell lines and compared activity with their mother tinctures during short-term and long-term cell culture. The effect of dynamized medicines to induce apoptosis was also evaluated and we studied how dynamized medicines affected genes expressed during apoptosis. Mother tinctures as well as some dynamized medicines showed significant cytotoxicity to cells during short and long-term incubation. Potentiated alcohol control did not produce any cytotoxicity at concentrations studied. The dynamized medicines were found to inhibit CHO cell colony formation and thymidine uptake in L929 cells and those of Thuja, Hydrastis and Carcinosinum were found to induce apoptosis in DLA cells. Moreover, dynamized Carcinosinum was found to induce the expression of p53 while dynamized Thuja produced characteristic laddering pattern in agarose gel electrophoresis of DNA. These results indicate that dynamized medicines possess cytotoxic as well as apoptosis-inducing properties. PMID:18955237

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

2009-01-01

76

LANTHANUM IN HEART CELL CULTURE  

PubMed Central

Correlation of the localization of La+++ with its effects on Ca++ exchange in cultured rat heart cells is examined with the use of a recently developed technique. 75% of cellular Ca++ is exchangeable and is completely accounted for by two kinetically defined phases. The rapidly exchangeable phase has a t ½ = 1.15 min and accounts for 1 1 mmoles Ca++/kg wet cells or 43% of the exchangeable Ca++ (cells perfused with [Ca++]o = 1 mM) Phase 2 has a t ½ = 19.2 min and accounts for 1.5 mmoles Ca++/kg wet cells or 57% of the exchangeable Ca++. 0.5 mM [La+++]o displaces 0 52 mmoles Ca++/kg wet cells—all from phase 1—and almost completely abolishes subsequent Ca++ influx and efflux The presence of La+++ in the washout converts the washout pattern to a single phase system with a t ½ = 124 min. The effects upon Ca++ exchange are coincident with abolition of contractile tension but regenerative depolarization of the tissue is maintained Electron microscope localization of the La+++ places it exclusively in the external lamina or basement membrane of the cells. The study indicates that negatively charged sites in the basement membrane play a crucial role in the E-C coupling process in heart muscle PMID:5044754

Langer, G. A.; Frank, J. S.

1972-01-01

77

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

EPA Science Inventory

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

78

Cultural Environmental Studies  

NSDL National Science Digital Library

The American Studies Program of Washington State University offers this online directory to Websites and resources in cultural environmental studies. The directory presents a subject overview followed by a dozen or more subtopic headings which lead to annotated listings further broken down by subheadings. The site is frequently updated and provides a wealth of links for studying the last two centuries from a cultural studies viewpoint.

79

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

Microsoft Academic Search

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

Ulf T. Brunk; Jan L. E. Ericsson

1972-01-01

80

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

NASA Technical Reports Server (NTRS)

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

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

1990-01-01

81

156 Literary and Cultural Studies Literary and Cultural Studies  

E-print Network

156 · Literary and Cultural Studies Literary and Cultural Studies Advisory Committee: Knight). The program in Literary and Cultural Studies brings an interdis- ciplinary perspective to the study of culture of their Literary and Cultural Studies major). Students are equally encouraged to take courses in related

Lewis, Robert Michael

82

Studies on the purification of thrombopoietin from kidney cell culture medium.  

PubMed

A thrombocytopoiesis-stimulating factor (TSF) has been purified from human embryonic kidney (HEK) cell culture medium. In the initial purification step, crude HEK cell culture medium was fractionated with saturated ammonium sulfate (step I). The proteins precipitated by 40% to 60% and 60% to 80% ammonium sulfate saturation increased the percent of sulfur 35 incorporation into platelets of assay mice (P less than 0.01). The ammonium sulfate-precipitated proteins that contained significant TSF activity were further refined on Sephadex G-75 columns (step II). The fraction containing the highest specific activity (greatest 35S incorporation into platelets of assay mice per milligram of protein) was further purified by diethylaminoethyl (DEAE)-cellulose column chromatography (step III). TSF activity was eluted from the columns between 0.3 and 1.0 mol/L NaCl. Additional Sephadex chromatography of post-DEAE-chromatographic preparations further increased the purity of the TSF (step IV). TSF from this four-step procedure was further processed on a DEAE-high-performance liquid chromatography (HPLC) column (step Va) or size exclusion (SE)-HPLC columns (step Vb). After HPLC, the activity was localized in a region corresponding to a retention time of 6 to 8 minutes for the DEAE-HPLC, but longer times were found after SE-HPLC. TSF was further purified by additional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and SE-HPLC (step VI). The final product had significant TSF activity and represented a purification of approximately 500,000-fold. It was also shown that the isoelectric pH of partially purified TSF was 4.7 and the molecular weight of the more highly purified preparation was approximately 32,000. After extraction by a combination of chromatographic procedures, a single homogeneous product was obtained. PMID:4020244

McDonald, T P; Cottrell, M; Clift, R; Khouri, J A; Long, M D

1985-08-01

83

Electrophysiological studies of new-born rat nodose neurones in cell culture.  

PubMed Central

1. Neurones of the nodose ganglion of the vagus nerve were dissociated from new-born rats and grown in the virtual absence of non-neuronal cells and in the presence of nerve growth factor. 2. The resting potentials of the neurones ranged from -40 to -80 mV. Action potentials were of short duration, with no inflexion on the falling phase; others were of longer duration with a hump on the falling phase. 3. The inward current of the action potential was carried either predominantly by Na+ or by Na+ and Ca2+. 4. Tetrodotoxin (1 microM) blocked the Na+ channels of some neurones but in other neurones the Na+ channels were partially or completely resistant to tetrodotoxin (1-10 microM). 5. Many neurones formed excitatory synapses on neighbouring neurones which were blocked or greatly reduced by conventional ganglionic nicotinic antagonists. This indicates that these neurones secreted ACh and expressed ACh receptors at these synapses. 6. The accompanying paper (Baccaglini & Cooper, 1981) reports the effect of co-culturing nodose neurones with non-neuronal cells on the expression of functional nicotinic receptors. Images Plate 1 PMID:6284921

Baccaglini, P I; Cooper, E

1982-01-01

84

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

PubMed

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

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

2014-01-01

85

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

PubMed Central

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

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

2014-01-01

86

Cytochemical study of developing neurotransmitter properties of dissociated sympathetic neurons grown in co-culture with dissociated pineal cells.  

PubMed

We studied the development of neurotransmitter phenotype in sympathetic neurons grown in the presence of pinealocytes, a target tissue having adrenergic but not cholinergic receptors. Neurons, dissociated from neonatal rat superior cervical ganglia, were grown in co-culture with dissociated pineal cells. Both ganglionic and pineal non-neuronal background cells were allowed to grow nearly to confluency. Electron microscopic cytochemical techniques were used to examine sets of co-cultures at weekly intervals over 5 weeks. Adrenergic vesicles were identified by their dense granular precipitate following potassium permanganate fixation. We found that the percentage of small granular vesicles, both in synaptic boutons onto other neurons and in axonal varicosities, declined very little over 5 weeks. After an initial drop from 75 to 65%, the percentage of small granular vesicles remained remarkably constant. Throughout the 5 weeks, more than 70% of the boutons and varicosities contained a predominance of small granular vesicles; fewer than 20% contained a predominance of clear vesicles. Although both somal synaptic boutons and axonal varicosities retained a predominantly adrenergic ultrastructure, at certain weeks there was a statistically significant shift in the percent distribution of adrenergic vesicles in somal boutons compared with the distribution in axonal varicosities. Because these cultures were grown under conditions known to favor an induction of acetylcholine metabolism and a suppression of catecholamine metabolism, we conclude that the maintenance of adrenergic ultrastructure over 5 weeks may be due to the presence of the pineal cells. PMID:2872617

Phillips, C E; Freschi, J E

1986-04-01

87

Substrate Micropatterning as a New in Vitro Cell Culture System to Study Myelination  

PubMed Central

Myelination is a highly regulated developmental process whereby oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system ensheathe axons with a multilayered concentric membrane. Axonal myelination increases the velocity of nerve impulse propagation. In this work, we present a novel in vitro system for coculturing primary dorsal root ganglia neurons along with myelinating cells on a highly restrictive and micropatterned substrate. In this new coculture system, neurons survive for several weeks, extending long axons on defined Matrigel tracks. On these axons, myelinating cells can achieve robust myelination, as demonstrated by the distribution of compact myelin and nodal markers. Under these conditions, neurites and associated myelinating cells are easily accessible for studies on the mechanisms of myelin formation and on the effects of axonal damage on the myelin sheath. PMID:22348182

2011-01-01

88

Study of the Effects of Ultrasonic Waves on the Reproductive Integrity of Mammalian Cells Cultured in Vitro  

NASA Technical Reports Server (NTRS)

The effects of monochromatic ultrasonic waves of 0.1, 0.5, 1.0, 2.0 and, 3.3 MHz frequency on the colony-forming ability of mammalian cells (M3-1,V79, Chang's and T-1) cultured in vitro have been studied to determine the nature of the action of ultrasonic energy on biological systems at the cellular level. The combined effect of ultrasound and X-rays has also been studied. It is concluded: (1) Ultrasonic irradiation causes both lethal and sublethal damage. (2) There is a threshold dose rate for lethal effects. (3) The effectiveness of ultrasonic waves in causing cell death probably depends on the frequency and the amplitude of the waves for a given cell line, indicating a possible resonance phenomenon.

Martins, B. I.

1971-01-01

89

The culture of limbal epithelial cells.  

PubMed

The transplantation of cultured limbal epithelial cells (LEC) has since its first application in 1997 emerged as a promising technique for treating limbal stem cell deficiency. The culture methods hitherto used vary with respect to preparation of the harvested tissue, choice of culture medium, culture time, culture substrates, and supplementary techniques. In this chapter, we describe a procedure for establishing human LEC cultures using a feeder-free explant culture technique with human amniotic membrane (AM) as the culture substrate. PMID:23690008

Utheim, Tor Paaske; Lyberg, Torstein; Ræder, Sten

2013-01-01

90

Evaluation of Silicon Nitride as a Substrate for Culture of PC12 Cells: An Interfacial Model for Functional Studies in Neurons  

PubMed Central

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. PMID:24587271

Medina Benavente, Johan Jaime; Mogami, Hideo; Sakurai, Takashi; Sawada, Kazuaki

2014-01-01

91

Comparison of Coconut Water and Jordanian Propolis on Survival of Bench-dried Periodontal Ligament Cells: An in vitro Cell Culture Study  

PubMed Central

ABSTRACT Aim: The aim of this study is to assess and compare the efficacy of Jordanian propolis and full concentration mature coconut water in their ability to preserve periodontal ligament (PDL) cell viability after exposure of PDL cells to up to 120 minutes dry storage. Materials and methods: PDL cells were obtained from sound permanent first molars which were cultured in Dulbecco's Modified Eagles Medium (DMEM). Cultures were subjected to 0, 30, 45, 60, 90 and 120 minutes dry storage times then incubated with 100% mature coconut water, Jordanian propolis and DMEM for 45 minutes at room temperature (18-26°C). Untreated cells served as controls at each dry storage time tested. PDL cell viability was assessed by MTT assay. Statistical analysis of data was accomplished by using one-way analysis of variance complemented by Tukey test and the level of significance was 5% ( p < 0.05). Results: Up to 60 minutes dry storage, no significant improvement on the percentage of viable cells was found from soaking in all tested media. On the other hand, soaking in mature coconut water only resulted in higher percentages of viable cells at >60 minutes dry storage. However, this improvement was not significant (p > 0.05). Conclusion: Avulsed teeth which have been left dry for <45 minutes should be replanted immediately, whereas avulsed teeth which have been left dry for >45 minutes may benefit from soaking for 45 minutes in mature coconut water. How to cite this article: Al-Haj Ali SN, Al-Jundi S, Mhaidat N. Comparison of Coconut Water and Jordanian Propolis on Survival of Bench-dried Periodontal Ligament Cells: An in vitro Cell Culture Study. Int J Clin Pediatr Dent 2013;6(3):161-165.

Al-Jundi, Suhad; Mhaidat, Nizar

2013-01-01

92

Development of an Innovative 3D Cell Culture System to Study Tumour - Stroma Interactions in Non-Small Cell Lung Cancer Cells  

PubMed Central

Introduction We describe a novel 3D co-culture model using non-small cell lung cancer (NSCLC) cell lines in combination with lung fibroblasts. This model allows the investigation of tumour-stroma interactions and addresses the importance of having a more in vivo like cell culture model. Methods Automation-compatible multi-well hanging drop microtiter plates were used for the production of 3D mono- and co-cultures. In these hanging drops the two NSCLC cell lines A549 and Colo699 were cultivated either alone or co-cultured with lung fibroblasts. The viability of tumour spheroids was confirmed after five and ten days by using Annexin V/Propidium Iodide staining for flow-cytometry. Tumour fibroblast spheroid formation was characterized by scanning electron microscope (SEM), semi-thin sections, fluorescence microscope and immunohistochemistry (IHC). In addition to conventional histology, protein expression of E-Cadherin, vimentin, Ki67, fibronectin, cytokeratin 7 and ?-smooth muscle actin (?-SMA) was investigated by IHC. Results Lower viability was observed in A549 monocultures compared to co-cultures, whereas Colo699 monocultures showed better viability compared to co-cultures. Ki67 expression varied significantly between mono- and co-cultures in both tumour cell lines. An increase of vimentin and decreased E-Cadherin expression could be detected during the course of the cultivation suggesting a transition to a more mesenchymal phenotype. Furthermore, the fibroblast cell line showed an expression of ?-SMA only in co-culture with the cancer cell line A549, thereby indicating a mesenchymal to mesenchymal shift to an even more myofibroblast phenotype. Conclusion We demonstrate that our method is a promising tool for the generation of tumour spheroid co-cultures. Furthermore, these spheroids allow the investigation of tumour-stroma interactions and a better reflection of in vivo conditions of cancer cells in their microenvironment. Our method holds potential to contribute to the development of anti-cancer agents and support the search for biomarkers. PMID:24663399

Amann, Arno; Zwierzina, Marit; Gamerith, Gabriele; Bitsche, Mario; Huber, Julia M.; Vogel, Georg F.; Blumer, Michael; Koeck, Stefan; Pechriggl, Elisabeth J.; Kelm, Jens M.; Hilbe, Wolfgang; Zwierzina, Heinz

2014-01-01

93

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model  

PubMed Central

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS. The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13. We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS. PMID:22297395

Machado, Heather L.; Morris, Cindy A.; Höner zu Bentrup, Kerstin

2012-01-01

94

Basic Laboratory Techniques in Cell Culture.  

National Technical Information Service (NTIS)

This is a reference and instructional manual for the Centers for Disease Control (CDC) Course Number 8270-C, 'Basic Laboratory Techniques in Cell Culture.' It covers pertinent cell biology, cell culture terminology and definitions, the principles and meth...

B. R. Bird, F. T. Forrester

1981-01-01

95

Studies of immunomodulating actions of carotenoids. I. Effects of ??carotene and astaxanthin on murine lymphocyte functions and cell surface marker expression in in vitro culture system  

Microsoft Academic Search

The immunomodulating effects of carotenoids (??carotene and astaxanthin) on mouse lymphocytes were studied in in vitro culture system by use of assay for mitogen responses of spleen cells, thymocyte proliferation, interleukin 2 production, and antibody (Ab) production in vitro in response to sheep red blood cells. Changes of cell surface markers on spleen lymphocytes including la antigen (Ag), surface immunoglobulin,

Harumi Jyonouchi; Roberta J. Hill; Yoshifumi Tomita; Robert A. Good

1991-01-01

96

Versatile, Fully Automated, Microfluidic Cell Culture System  

E-print Network

on a microfluidic chip that creates arbitrary culture media formulations in 96 independent culture chambersVersatile, Fully Automated, Microfluidic Cell Culture System Rafael Go´mez-Sjo1berg, Anne A. Leyrat and quantita- tive cell culture technology, driven both by the intense activity in stem cell biology

Chen, Christopher S.

97

Fidelity of micropatterned cell cultures.  

PubMed

Methods that enable the culture of micropatterned cells may help advance our fundamental understanding of cell-cell and cell-surface interactions, while facilitating the development and implementation of cell-based biological assays. However, the long-term stability of the cell patterns can limit the time scales over which such methods can be informative. Here we used self-assembling monolayers (SAMs) to localize the adsorption of baby hamster kidney (BHK-21) cells as well as cells from a murine astrocytoma-derived cell line (delayed brain tumor) in linear arrays. We tested the effects of surface chemistries, fibronectin pre-treatments, array dimensions, and cell types on pattern fidelity. Changes in patterns were monitored by phase-contrast microscopy up to 96 h post-plating, followed by digital imaging, and these changes were quantified by measuring an "intrusion distance" or the average distance cells extend beyond the initial adhesive/non-adhesive boundary. Loss of pattern boundaries involved different mechanisms for different cells. Treatment of patterned surfaces with fibronectin prior to plating of cells tended to promote earlier loss of pattern fidelity, and the extent of pattern loss was further augmented for SAMs formed using hydrophobic monolayers. Finally, reduction of gap spacing between adjacent cell arrays promoted pattern loss. PMID:15920741

Endler, Elizabeth E; Nealey, Paul F; Yin, John

2005-07-01

98

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

PubMed

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

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

2014-01-01

99

Efficient definitive endoderm induction from mouse embryonic stem cell adherent cultures: a rapid screening model for differentiation studies.  

PubMed

Definitive endoderm (DE) differentiation from mouse embryonic stem cell (mESC) monolayer cultures has been limited by poor cell survival or low efficiency. Recently, a combination of TGF? and Wnt activation with BMP inhibition improved DE induction in embryoid bodies cultured in suspension. Based on these observations we developed a protocol to efficiently induce DE cells in monolayer cultures of mESCs. We obtained a good cell yield with 54.92% DE induction as shown by Foxa2, Sox17, Cxcr4 and E-Cadherin expression. These DE-cells could be further differentiated into posterior foregut and pancreatic phenotypes using a culture protocol initially developed for human embryonic stem cell (hESC) differentiation. In addition, this mESC-derived DE gave rise to hepatocyte-like cells after exposure to BMP and FGF ligands. Our data therefore indicate a substantial improvement of monolayer DE induction from mESCs and support the concept that differentiation conditions for mESC-derived DE are similar to those for hESCs. As mESCs are easier to maintain and manipulate in culture compared to hESCs, and considering the shorter duration of embryonic development in the mouse, this method of efficient DE induction on monolayer will promote the development of new differentiation protocols to obtain DE-derivatives, like pancreatic beta-cells, for future use in cell replacement therapies. PMID:24239964

Mfopou, Josué Kunjom; Geeraerts, Marloes; Dejene, Roba; Van Langenhoven, Stijn; Aberkane, Asma; Van Grunsven, Leo A; Bouwens, Luc

2014-01-01

100

Adipose cell differentiation in culture  

Microsoft Academic Search

The isolation of preadipocyte cell strains from adipose tissue and from bone marrow, and the establishment of preadipocyte cell lines from embryonic and adult mouse, have been useful tools to study the process of adipose cell differentiation.

G. Ailhaud

1982-01-01

101

Introduction: Rural Cultural Studies  

Microsoft Academic Search

This themed section of Australian Humanities Review seeks to establish the emerging field of 'rural cultural studies' firmly on the agenda of the contemporary humanities and social sciences. This is a timely intervention as rural Australia has featured increasingly over the last decade and especially over the last few years as a topic of national policy attention, public commentary and

David Carter; Kate Darian-Smith; Andrew Gorman-Murray

102

A Comparative Study on Morphochemical Properties and Osteogenic Cell Differentiation within Bone Graft and Coral Graft Culture Systems  

PubMed Central

The objective of this study was to compare the morphological and chemical composition of bone graft (BG) and coral graft (CG) as well as their osteogenic differentiation potential using rabbit mesenchymal stem cells (rMSCs) in vitro. SEM analysis of BG and CG revealed that the pores in these grafts were interconnected, and their micro-CT confirmed pore sizes in the range of 107-315 µm and 103-514 µm with a total porosity of 92% and 94%, respectively. EDS analysis indicated that the level of calcium in CG was relatively higher than that in BG. FTIR of BG and CG confirmed the presence of functional groups corresponding to carbonyl, aromatic, alkyl, and alkane groups. XRD results revealed that the phase content of the inorganic layer comprised highly crystalline form of calcium carbonate and carbon. Atomic force microscopy analysis showed CG had better surface roughness compared to BG. In addition, significantly higher levels of osteogenic differentiation markers, namely, alkaline phosphatase (ALP), Osteocalcin (OC) levels, and Osteonectin and Runx2, Integrin gene expression were detected in the CG cultures, when compared with those in the BG cultures. In conclusion, our results demonstrate that the osteogenic differentiation of rMSCs is relatively superior in coral graft than in bone graft culture system. PMID:24151432

Puvaneswary, Subramaniam; Balaji Raghavendran, Hanumantha Rao; Ibrahim, Nurul Syuhada; Murali, Malliga Raman; Merican, Azhar Mahmood; Kamarul, T.

2013-01-01

103

Interactions of cardiac glycosides with cultured cardiac cells. II. Biochemical and electron microscopic studies on the effects of ouabain on muscle and non-muscle cells.  

PubMed

Electron microscopic and biochemical studies revealed a salient difference in the response to toxic doses of ouabain by cultured cardiac muscle and non-muscle cells from neonatal rats. Progressive cellular injury in myocytes incubated with 1 . 10(-4)--1 . 10(-3) M ouabain ultimately leads to swelling and necrosis. The morphological damage in myocytes was accompanied by a drastic decrease in 14CO2 formation from 14C-labeled stearate or acetate but not glucose. Neither morphological nor biochemical impairments were observed in non-muscle cells. The interaction between ouabain and the cultured cells, using therapeutic doses of ouabain (i.e., less than 1 . 10(-7) M), was characterized. Two binding sites were described in both classes of cells, one site is a saturable K+-sensitive site whereas the other is non-saturable and K+-insensitive. The complexes formed between the sarcolemma receptor(s) and ouabain, at low concentrations of the drug (e.g., 7.52 . 10(-9) M), had Kd values of 8.9 . 10(-8) and 2.3 . 10(-8) M for muscle and non-muscle cells, respectively. The formation and dissociation of the complexes were affected by temperature and potassium ions. PMID:7378406

Friedman, I; Schwab, H; Hallaq, H; Pinson, A; Heller, M

1980-05-23

104

An appropriate selection of a 3D alginate culture model for hepatic Huh-7 cell line encapsulation intended for viral studies.  

PubMed

Three-dimensional (3D) culture systems have been introduced to provide cells with a biomimetic environment that is similar to in vivo conditions. Among the polymeric molecules available, sodium-alginate (Na-alg) salt is a material that is currently employed in different areas of drug delivery and tissue engineering, because it offers biocompatibility and optimal chemical properties, and its gelation with calcium chloride provides calcium-alginate (Ca-alg) scaffolds with mechanical stability and relative permeability. In this work, four different preparations of Ca-alg beads with varying Na-alg viscosity and concentration were used for a human hepatoma cell line (Huh-7) encapsulation. The effects of Ca-alg bead preparation on structural cell organization, liver-specific functions, and the expression of specific receptors implicated in hepatotropic virus permissivity were evaluated. Hepatic cells were cultured in 500??m diameter Ca-alg beads for 7 days under dynamic conditions. For all culture systems, cell viability reached almost 100% at day 7. Cell proliferation was concomitantly followed by hepatocyte organization in aggregates, which adopted two different morphologies (spheroid aggregates or multicellular channel-like structures), depending on Ca-alg bead preparation. These cellular organizations established a real 3D hepatocyte architecture with cell polarity, cell junctions, and abundant bile canaliculi possessing microvillus-lined channels. The functionality of these 3D cultures was confirmed by the production of albumin and the exhibition of CYP1A activity over culture time, which were variable, according to Ca-alg bead condition. The expression of specific receptors of hepatitis C virus by Huh-7 cells suggests encouraging data for the further development of a new viral culture system in Ca-alg beads. In summary, this 3D hepatic cell culture represents a promising physiologically relevant system for further in vitro studies and demonstrates that an adequate encapsulation condition can be selected for each target application in liver tissue engineering, specifically in viral studies. PMID:22889091

Tran, Nhu Mai; Dufresne, Murielle; Duverlie, Gilles; Castelain, Sandrine; Défarge, Christian; Paullier, Patrick; Legallais, Cecile

2013-01-01

105

Optimization and characterization of an in vitro bovine mammary cell culture system to study regulation of milk protein synthesis and mammary differentiation  

SciTech Connect

A long term bovine mammary cell culture system that maintains normal mammary cell function was established and optimized to study milk protein synthesis and secretion and mammary differentiation. This culture system used bovine mammary acini isolated from developing or lactating mammary gland by enzymatic dissociation, and cryopreserved until thawed and plated for growth in vitro for these studies. Cells in M199 with lactogenic hormones {plus minus} fetal calf serum (FCS) were cultured on plastic, 100ul and 500ul type I collagen, and Matrigel, or embedded within type I collagen. Cell morphology, cell number, and total TCA-precipitable {sup 35}S-labelled proteins were monitored. Milk protein ({alpha}{sub s,1}-casein, lactoferrin (LF), {alpha}-lactalbumin, and {beta}-lactoglobulin) secretion and intracellular levels were determined by an ELISA assay.

Talhouk, R.S.

1988-01-01

106

DNA damage and repair measurements from cryopreserved lymphocytes without cell culture--a reproducible assay for intervention studies.  

PubMed

Single-cell gel electrophoresis (the Comet assay) can be used to measure DNA damage and DNA repair capacity (DRC). However, to test DRC of cryopreserved lymphocytes, published methods include steps for cell culturing and phytohemagglutinin stimulation, which may limit use of this assay in intervention studies. We developed a modified Comet assay protocol that allows us to measure DRC from cryopreserved lymphocytes without these in vitro manipulations. Assay reproducibility was evaluated by performing the assay six times on different dates using six aliquots from one blood draw of one individual. The interindividual variation was assessed by performing the assay using one aliquot from six individuals. When gamma-irradiation was used as the mutagen, intra-assay coefficients of variation (CVs.) for baseline DNA damage, damage after gamma-irradiation exposure, and DRC--measured as tail moment--were 8, 31, and 10%, respectively. Interindividual CVs. were higher. When H(2)O(2) was used as the mutagen, intra-assay CVs. for damage measurements were lower for a protocol modification that included damage and repair at 37 degrees C (CVs. ranging from 8 to 35%) than for the more standard 4 degrees C protocol. Analyzing moment arm--the average distance of DNA migration within the tail--yielded similar results. DNA repair was successfully detected in each experiment. Comparing freshly isolated lymphocytes to cryopreserved lymphocytes from the same individuals' blood draw indicated that DRC was highly correlated when determined using moment arm values. This modified protocol extends the use of the Comet assay to measuring DRC in intervention studies (e.g., dietary interventions) in that it assesses cellular response after cryopreservation without cell culture or other extensive manipulation. PMID:16673412

Chang, Jyh-Lurn; Chen, Gang; Lampe, Johanna W; Ulrich, Cornelia M

2006-08-01

107

[Ebola virus reproduction in cell cultures].  

PubMed

Ebola-Zaire virus production in Vero and BGM cells was studied. The CPE developed in both cell cultures. The cell monolayer destruction by 80-90% was seen at a low multiplicity of infection in 7-8 days after virus inoculation. An overlay composition was developed for virus titration using plaque assay. The plaque production was shown to be directly proportional to the virus dose. The curve of Ebola virus production in Vero cell culture fluid was determined. At a multiplicity of infection of 0.01 PFU/cell, the maximum virus titer of 10(6.4) PFU/ml was reached in 7 days postinfection. Specific antisera were generated by inoculation of guinea pigs. Indirect immunofluorescent assay was used for testing of virus-specific antigen and antibody. PMID:1279896

Titenko, A M; Novozhilov, S S; Andaev, E I; Borisova, T I; Kulikova, E V

1992-01-01

108

Isolation and culture of larval cells from C. elegans.  

PubMed

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. PMID:21559335

Zhang, Sihui; Banerjee, Diya; Kuhn, Jeffrey R

2011-01-01

109

Electron microscopic studies of the endocytotic process of cationized ferritin in cultured human retinal pigment epithelial cells  

Microsoft Academic Search

Summary  The endocytotic process in cultured human RPE cells was observed after 1 min, 20 min, and 2 h incubation with cationized ferritin.\\u000a \\u000a Within 1 min the ferritin particles were seen to attach to the cell membrane, especially between microvilli. Uncoated and\\u000a coated pits could be recognized on the cell membranes, and uncoated and coated endocytotic vesicles were found in the

Kiyoshi Akeo; Yasuhiko Tanaka; Tatsuji Fujiwara

1988-01-01

110

Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor  

NASA Technical Reports Server (NTRS)

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

Parks, Kelsey

2009-01-01

111

9 CFR 101.6 - Cell cultures.  

Code of Federal Regulations, 2010 CFR

...2010-01-01 2010-01-01 false Cell cultures. 101.6 Section 101.6...AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to...

2010-01-01

112

9 CFR 101.6 - Cell cultures.  

...2014-01-01 2014-01-01 false Cell cultures. 101.6 Section 101.6...AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to...

2014-01-01

113

9 CFR 101.6 - Cell cultures.  

Code of Federal Regulations, 2013 CFR

...2013-01-01 2013-01-01 false Cell cultures. 101.6 Section 101.6...AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to...

2013-01-01

114

9 CFR 101.6 - Cell cultures.  

Code of Federal Regulations, 2012 CFR

...2012-01-01 2012-01-01 false Cell cultures. 101.6 Section 101.6...AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to...

2012-01-01

115

9 CFR 101.6 - Cell cultures.  

Code of Federal Regulations, 2011 CFR

...2011-01-01 2011-01-01 false Cell cultures. 101.6 Section 101.6...AND VECTORS DEFINITIONS § 101.6 Cell cultures. When used in conjunction with or in reference to cell cultures, which may be referred to...

2011-01-01

116

Differentiated cultures of primary hamster tracheal airway epithelial cells.  

PubMed

Primary airway epithelial cell cultures can provide a faithful representation of the in vivo airway while allowing for a controlled nutrient source and isolation from other tissues or immune cells. The methods used have significant differences based on tissue source, cell isolation, culture conditions, and assessment of culture purity. We modified and optimized a method for generating tracheal epithelial cultures from Syrian golden hamsters and characterized the cultures for cell composition and function. Soon after initial plating, the epithelial cells reached a high transepithelial resistance and formed tight junctions. The cells differentiated into a heterogeneous, multicellular culture containing ciliated, secretory, and basal cells after culture at an air-liquid interface (ALI). The secretory cell populations initially consisted of MUC5AC-positive goblet cells and MUC5AC/CCSP double-positive cells, but the makeup changed to predominantly Clara cell secretory protein (CCSP)-positive Clara cells after 14 d. The ciliated cell populations differentiated rapidly after ALI, as judged by the appearance of beta tubulin IV-positive cells. The cultures produced mucus, CCSP, and trypsin-like proteases and were capable of wound repair as judged by increased expression of matrilysin. Our method provides an efficient, high-yield protocol for producing differentiated hamster tracheal epithelial cells that can be used for a variety of in vitro studies including tracheal cell differentiation, airway disease mechanisms, and pathogen-host interactions. PMID:15780007

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

2004-01-01

117

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

E-print Network

vesicles were contained within these vacuoles; some appeared myelin-like in form and contained electron dense bodies (Figs. Z, 3, 4). The vesicles were sometimes observed continuous with the plasma membrane as though they were in the process of being... inclusions (i) many of which appeared in myelin-like forms (mf). FIG. 3. Dividing cell after completion of logarithmic growth. Section of a 96 h PS cell. Dividing cells were still evi dent after cessation of logarithmic growth. The dividing cells...

Droleskey, Robert Edward

2012-06-07

118

PRECLINICAL STUDY Prolonged mammosphere culture of MCF-7 cells induces an EMT  

E-print Network

expression of junctional proteins (e.g., E-cadherin), loss of apico-basal polarity, and the acquisition o and miR-221. Antisense hairpin RNA inhibitor targeting miR-221 resulted in re-expression of ERa in MCF-7M) and expresses ERa GATA3 and FOXA1, [1, 2]. Luminal A cells also display hallmarks of epithelial organization

Terasaki, Mark

119

Culture and differentiation of embryonic stem cells  

Microsoft Academic Search

Summary Techniques are described for the culture of murine embryonic stem cells in the absence of heterologous feeder cells and for the induction of differentiation programs. The regulatory factor differentiation inhibiting activity\\/ leukaemia inhibitory factor (DIA\\/LIF) is produced at high concentration by transient expression in Cos cells and is used to suppress stem cell differentiation by addition to the culture

Austin G. Smith

1991-01-01

120

Immobilized MWCNT support osteogenic cell culture.  

PubMed

The broad use of versatile, strong, lightweight multi-walled carbon nanotubes (MWCNT) for use in biomaterial applications is tempered by ongoing debate about their safety. Recent reports suggest that factors such as their diameter and surface coating affect their function and cytotoxicity. The cell culture surfaces used in the current study were made of MWCNT immobilized in a high-density polyethylene substrate, differentiating it from most studies of MWCNT cytotoxicity. The purity, chemical functionalization, and immobilization of MWCNT were evaluated to elucidate their effect on MWCNT behavior relative to controls. While purity was found not to be significant in determining the behavior of cells on MWCNT relative to standard controls, the presence of carboxyl functional groups was generally associated with reduced cell metabolic activity, proliferation, and differentiation as measured using the MTS assay, nucleic acid incorporation, and alkaline phosphatase expression, respectively. This study demonstrates that the culture of osteogenic cells on surfaces made of nonfunctionalized and immobilized MWCNT is associated with a level of cell growth and differentiation comparable to those of standard tissue culture controls. PMID:23471503

Emohare, Osa; Rushton, Neil

2013-06-01

121

Primary monolayer cultures of adult rat liver parenchymal cells suitable for study of the regulation of enzyme synthesis  

Microsoft Academic Search

Summary  Parenchymal cells from adult rat liver which had been fully regenerated were isolated and cultured in nonproliferating monolayers\\u000a in vitro. The optimum conditions for attachment of these cells to Falcon plastic dishes were determined. When approximately\\u000a 1.0105 nuclei per cm2 suspended in Ham's F-12 medium with 0.5 ?g of insulin per ml and 25% fetal calf serum were incubated at

Robert J. Bonney; Joyce E. Becker; P. Roy Walker; R. Van Potter

1974-01-01

122

Paclitaxel loaded nanosponges: in-vitro characterization and cytotoxicity study on MCF-7 cell line culture.  

PubMed

Beta cyclodextrin (?-CD) based nanosponges were synthesized and paclitaxel inclusion complex with nanosponges were prepared using techniques of inclusion complex formation. The paclitaxel nanosponge's complexes were evaluated for their release. The nanosponges complexes were also evaluated using DSC, FTIR, and NMR techniques for confirmation of inclusion complex formation between paclitaxel and nanosponges. Particle size and morphology of paclitaxel nanosponge's complex were estimated using SEM, TEM and dynamic light scattering techniques. The particle sizes were found out to be in range of 400 to 600 nm. Cytotoxic efficacy of paclitaxel nanosponge complex was determined against MCF-7 cells and paclitaxel nanosponge's complex was found to be cytotoxic and more effective against this cell line. PMID:21235471

Ansari, Khalid A; Torne, Satyen J; Vavia, Pradeep R; Trotta, Francesco; Cavalli, Roberta

2011-03-01

123

A digitized fluorescence imaging study of intracellular Ca 2+ , pH, and mitochondrial function in primary cultures of rabbit corneal epithelial cells exposed to sodium dodecyl sulfate  

Microsoft Academic Search

Summary  Primary cultures of rabbit corneal epithelial cells have been developed as an in vitro system to predict irritancy potential\\u000a and delayed cytotoxicity of surfactants in our laboratory. The objective of this study was to evaluate the effects of the\\u000a surfactant sodium dodecyl sulfate (SDS), a common ingredient in consumer products, on intracellular Ca2+, pH, and mitochondrial function in this culture

Wei Yang; Daniel Acosta

1995-01-01

124

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

PubMed Central

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

Sato, Mitsuru; Hosokawa, Munetaka; Doi, Motoaki

2011-01-01

125

A cross-cultural study of psychosocial aspects of sickle cell disease in the UK and Nigeria  

Microsoft Academic Search

Pain experience, health service utilization and psychological coping in adult patients with sickle cell disease were compared cross-culturally between the UK and Nigeria. Patients in the UK experienced a significantly greater number of pain episodes and of longer duration, with more frequent visits to accident and emergency departments compared with those in Nigeria. The Nigerian patients, on the other hand,

Kofi A. Anie; Tanya Dasgupta; Pauline Ezenduka; Agnes Anarado; Ifoema Emodi

2007-01-01

126

Chemical potential of Aphelandra sp. cell cultures  

Microsoft Academic Search

Six different callus lines and three different suspension culture lines were established from plants of two Aphelandra species (Acanthaceae). All established lines were analyzed for secondary metabolite accumulation. A discrepancy between secondary\\u000a metabolites accumulated in the plants and in the cell cultures could be observed. All established Aphelandrasp. cell cultures produced verbascoside (acteoside) as the major extractable metabolite. Time course

Lenka Nezbedová; Manfred Hesse; Jaroslav Dušek; Christa Werner

1999-01-01

127

Catechin production in cultured Polygonum hydropiper cells  

Microsoft Academic Search

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

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

1998-01-01

128

Carrot Embryogenesis from Frozen Cultured Cells  

Microsoft Academic Search

MANY plant tissue cultures change in growth rate, chromosome cytology and morphogenic potential during repeated subculture1-3. Controlled freezing and low temperature storage of cultured plant cells might enable the characters of newly initiated cultures to be preserved. Cell preservation at the temperature of liquid N2 (-196° C) has been successful with animal cells4 and preliminary work with plant cells has

K. K. Nag

1973-01-01

129

Enhanced lymphatic transport of bioactive lipids: cell culture study of polymethoxyflavone incorporation into chylomicrons.  

PubMed

Polymethoxyflavones (PMFs) are bioactive flavonoids found in citrus fruits that have been shown to have potential health promoting properties. However, their application as nutraceuticals in functional foods and beverages is currently limited due to their low water solubility and high melting point. The oral bioavailability of lipophilic compounds can be enhanced by promoting their intestinal lymphatic transport through co-administration with digestible lipids. We investigated the effects of chylomicron-mediated intestinal lymphatic transport on the bioavailability of 5-hydroxy-6,7,8,3',4'-pentamethoxylflavone (5-HPMF), one of representative PMFs in Caco-2 cells. Our results demonstrated that oleic acid and bile acid promoted secretion of chylomicrons in Caco-2 cells, with mean diameter ranged from 70 to 150 nm. The intracellular level of 5-HPMF increased 3-fold by co-incubation with the mixed micelle solution. Moreover, the basolateral level of 5-HPMF increased 3-fold due to enhanced chylomicron-mediated transport. Overall, our results demonstrated for the first time that the bioavailability of polymethoxyflavones can be enhanced by promoting their incorporation into chylomicrons. PMID:24084938

Yao, Mingfei; Chen, Jingjing; Zheng, Jinkai; Song, Mingyue; McClements, David Julian; Xiao, Hang

2013-11-01

130

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

PubMed Central

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

Stephens, D S

1989-01-01

131

OA02.01. Fibroblast growth inhibitory and antiinflammatory effect of Curcumin extract: A cell culture study  

PubMed Central

Purpose: Curcumin is an active principle obtained from the plant curcuma longa which is not a new therapeutic tool but proved long time back with lots of medicinal values in traditional medicine. This dried rhizome powder extract had been studied extensively in animal models and proved with antiinflammatory activity. Many human studies had proved its antiinflammatory action and its effectiveness in reducing inflammation in osteoarthritis and its benefits in rheumatoid arthritis. Because this extract widely used for antiinflammatory action in arthritis and help in healing a wound we had planned to compare the effectiveness with known antiinflammatory agent methyl prednesolone. Method: Curcumin extract, fibroblast cells L6 rat skeletal muscle cell line (NCCS, Pune), Medium DMEM (Dulbecco's Modified Eagles Medium) with 10% fetal bovine serum in 5% CO2 at 37°C, Drug Prednisolone and Curcumin successive extract. Curcumin extract: Locally available Curcumin rhizomes was purchased after confirmation with botanical survey department of India, Coimbatore, allowed drying at shade for three to four months after cleaning with water to remove the mud and dust. After three months Rhizomes were powdered. This powdered cumin were used to prepare successive extract as per the standard technique using chloroform and ethanol with the help of Soxhlet apparatus for cell culture study with the varying doses of 100?g, 300?g, 600?g, 900?g and 1200?g using MIT Assay. Result: After 24hrs of the drug treatment, the medium was changed for all the groups and 10 ?l of MTT (5 mg/ml stock solution) was added and the plates were incubated for an additional 4 hrs. The medium was discarded and the formazan blue, which was formed in the cells, was dissolved with 50 ?l of DMSO. The optical density was measured at 570 nm. The percentage toxicity was calculated & IC 50 was calculated by using Grapad PRISM software tool following formula. Conclusion: Curcumin extract has role in chronic inflammation based on the inhibitory role on connective tissue cell fibroblast proliferation when compared with the known established anti-inflammatory agent methyl prednisolone.

Bhuvaneswari, K; Ramanathan, M; Balaji, Prathap

2013-01-01

132

Skeletal muscle satellite cells cultured in simulated microgravity  

NASA Technical Reports Server (NTRS)

Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells. Satellite cells retain the capacity to proliferate and differentiate in vitro and therefore provide a useful model to study postnatal muscle development. Most culture systems used to study postnatal muscle development are limited by the two-dimensional (2-D) confines of the culture dish. Limiting proliferation and differentiation of satellite cells in 2-D could potentially limit cell-cell contacts important for developing the level of organization in skeletal muscle obtained in vivo. Culturing satellite cells on microcarrier beads suspended in the High-Aspect-Ratio-Vessel (HARV) designed by NASA provides a low shear, three-dimensional (3-D) environment to study muscle development. Primary cultures established from anterior tibialis muscles of growing rats (approximately 200 gm) were used for all studies and were composed of greater than 75 % satellite cells. Different inoculation densities did not affect the proliferative potential of satellite cells in the HARV. Plating efficiency, proliferation, and glucose utilization were compared between 2-D flat culture and 3-D HARV culture. Plating efficiency (cells attached - cells plated x 100) was similar between the two culture systems. Proliferation was reduced in HARV cultures and this reduction was apparent for both satellite cells and non-satellite cells. Furthermore, reduction in proliferation within the HARV could not be attributed to reduced substrate availability since glucose levels in media from HARV and 2-D cell culture were similar. Morphologically, microcarrier beads within the HARVS were joined together by cells into three-dimensional aggregates composed of greater than 10 beads/aggregate. Aggregation of beads did not occur in the absence of cells. Myotubes were often seen on individual beads or spanning the surface of two beads. In summary, proliferation and differentiation of satellite cells on microcarrier beads within the HARV bioreactor results in a three dimensional level of organization that could provide a more suitable model to study postnatal muscle development.

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

1993-01-01

133

Hepatotoxicity of salicylates in monolayer cell cultures.  

PubMed

The influence of graded doses of sodium salicylate on rat liver cells cultured in monolayers was assessed by measuring lactic dehydrogenase activity in a culture media after incubation. Morphological alterations were studied by electron microscopy. The influence of different albumin concentrations in the media on toxicity was also evaluated. Lactic dehydrogenase concentrations rose with increasing doses of salicylate up to 40 mg per dl. High concentrations of albumin were associated with reduced salicylate toxicity. These findings suggest that salicylate-induced hepatic injury is dose related and my be influenced by serum albumin levels PMID:620893

Tolman, K G; Peterson, P; Gray, P; Hammar, S P

1978-02-01

134

Induction of heme oxygenase-1 by phenylarsine oxide. Studies in cultured primary liver cells.  

PubMed

Heme oxygenase-1, the major inducible isoform of heme oxygenase (HO), can be induced by heme and numerous other physical and chemical factors, many of which cause cellular 'stress'. This has led to the realization that HO-1 is a major highly conserved stress or heat shock protein. Recent work has implicated activation of mitogen-activated protein kinases and other kinases in the mechanism of induction of HO-1, and suggested that signal transduction pathways through tyrosine kinases are involved in induction of HO-1 gene expression by stress inducers. We hypothesized that phenylarsine oxide (PAO), an inhibitor of protein tyrosine phosphatases (PTPs), might up-regulate the HO-1 gene. Here, we show that a remarkably brief (1-15 min) exposure of normal hepatocytes to low concentrations (0.5-3 microM) of PAO produces a marked increase in mRNA and protein of HO-1. This increase is comparable to the level obtained by addition of heme (20 microM), and occurs without producing changes in cellular glutathione levels or stabilization of HO-1 message. Preincubation of cells with inhibitors of protein synthesis decreased the ability of PAO to increase levels of HO-1 mRNA, suggesting that the inductive effect requires de novo protein synthesis. Addition of thiol donors abrogated the PAO-mediated induction of HO-1 in a dose dependent fashion. Addition of genistein, a tyrosine kinase inhibitor, blunted the induction produced by both PAO and heme. After brief incubations with PAO or heme, cell extracts showed comparable increases in levels of protein tyrosine phosphorylation in general, and specifically in ZAP70 kinase. Our results are consistent with the proposition that induction of HO-1 by PAO involves inhibition of specific PTP(s), and that the mechanisms of induction of HO-1 by PAO and by heme may share some common pathways. PMID:11768235

Gildemeister, O S; Pepe, J A; Lambrecht, R W; Bonkovsky, H L

2001-10-01

135

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

NASA Technical Reports Server (NTRS)

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

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

2000-01-01

136

Study on characteristics of in vitro culture and intracellular transduction of exogenous proteins in fibroblast cell line of Liaoning cashmere goat.  

PubMed

Establishment of fibroblast cell lines of endangered goat breeds and research on the gene or protein functions based on the cells made a significant contribution to the conservation and utilization of genetic resources. In this study, a fibroblast cell line of Liaoning cashmere goat, frozen in 174 cryovials with 5 × 10(6) cells each, was successfully established from 60 goats ear marginal tissues using explant culture and cryopreservation techniques. Biological analysis of in vitro cultured cell line showed that, the cells were morphologically consistent with fibroblasts; the average viability of the cells was 94.9 % before freezing and 90.1 % after thawing; the growth process of cells was consisted of a lag phase, a logarithmic phase and a plateau phase; cell population doubling time was 65.5 h; more than 90 % of cells were diploid prior to the 6th generation; Neither microbial contamination nor cross-contamination was detected. To determine cell permeability, intracellular path and stability of exogenous proteins during the transduction, a TAT protein transduction domain was fused to the C-terminus of enhanced green fluorescent protein, the established fibroblast cell line was treated with the purified exogenous proteins at various concentrations by adding them to the cell culture media for 1-24 h and assayed cell morphology and protein presence, it was found that the purified exogenous proteins readily entered cells at a concentration of 0.1 mg/ml within 1.5 h and some of them could translocate into nucleus, moreover, the exogenous proteins appeared to be stable inside cells for up to 24 h. PMID:23065271

Hu, P F; Guan, W J; Li, X C; Zhang, W X; Li, C L; Ma, Y H

2013-01-01

137

Culture surface influence on T-cell phenotype and function.  

PubMed

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

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

2013-01-01

138

In vitro culture of C. elegans somatic cells.  

PubMed

Because of technical hurdles, large-scale cell culture methods have not been widely exploited until recently for the study of Caenorhabditis elegans. Culturing differentiated cells from larvae and adult worms is probably not technically feasible because of difficulties in removing the animal's cuticle and dissociating cells. In contrast, large numbers of developing embryo cells can be isolated relatively easily. When placed in culture, embryo cells undergo terminal differentiation within 24 h. Cultured embryo cells have been used recently to characterize ion channel function and regulation and to determine cell specific gene expression patterns. This chapter will provide a detailed description of the methods for isolating and culturing C. elegans embryo cells. PMID:16988440

Strange, Kevin; Morrison, Rebecca

2006-01-01

139

Performance of enzymatic fuel cell in cell culture.  

PubMed

Here we present the very first study of an enzymatic fuel cell (EFC) in a cell culture. An EFC with Corynascus thermophilus cellobiose dehydrogenase (CDH) based bioanode and Myrothecium verrucaria bilirubin oxidase (BOx) based biocathode was constructed at the bottom of a medusa cell culture plate. The constructed EFC had a power density of up to 25 ?W cm(-2) at 0.5 V potential in simple buffer solution and in cell culturing medium. L929 murine fibroblast cells were seeded on top of the EFC and possible effects of the EFC on the cells and vice versa were studied. It was shown that on average the power of the EFC drops by about 70% under a nearly confluent layer of cells. The EFC appeared to have a toxic effect on the L929 cell line. It was concluded that the bioanode, consisting of CDH, produced hydrogen peroxide at toxic concentrations. However, the toxic effect was circumvented by co-immobilizing catalase on the bioanode. PMID:24374299

Lamberg, P; Shleev, S; Ludwig, R; Arnebrant, T; Ruzgas, T

2014-05-15

140

Primary cell cultures from Drosophila gastrula embryos.  

PubMed

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells. PMID:21403631

Perrimon, Norbert; Zirin, Jonathan; Bai, Jianwu

2011-01-01

141

Controlled, Scalable Embryonic Stem Cell Differentiation Culture  

Microsoft Academic Search

Embryonic stem (ES) cells are of significant interest as a renewable source of therapeutically useful cells. ES cell aggregation is important for both human and mouse embryoid body (EB) formation and the subse- quent generation of ES cell derivatives. Aggregation between EBs (agglomeration), however, inhibits cell growth and differentiation in stirred or high-cell-den- sity static cultures. We demonstrate that the

STEPHEN M. DANG; SHARON GERECHT-NIR; JINNY CHEN; JOSEPH ITSKOVITZ-ELDOR; PETER W. ZANDSTRAa

2004-01-01

142

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

PubMed

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

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

2014-10-01

143

Three-dimensional perfused cell culture.  

PubMed

Compelling evidence suggests the limitation and shortcomings of the current and well established cell culture method using multi-well plates, flasks and Petri dishes. These are particularly important when cell functions are sensitive to the local microenvironment, cell-cell and cell-extracellular matrix interactions. There is a clear need for advanced cell culture systems which mimic in vivo and more physiological conditions. This review summarises and analyses recent progress in three dimensional (3D) cell culture with perfusion as the next generation cell culture tools, while excluding engineered tissue culture where three dimensional scaffold has to be used for structural support and perfusion for overcoming mass transfer control. Apart from research activities in academic community, product development in industry is also included in this review. PMID:24184152

Li, Zhaohui; Cui, Zhanfeng

2014-01-01

144

Dynamic cell culture system (7-IML-1)  

NASA Technical Reports Server (NTRS)

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.

Cogoli, Augusto

1992-01-01

145

Cell Culture as an Alternative in Education.  

ERIC Educational Resources Information Center

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)

Nardone, Roland M.

1990-01-01

146

Cell culture from sponges: pluripotency and immortality  

Microsoft Academic Search

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 new source of sponge material for cell culture. Stem cells are present in high

Caralt Bosch de S; María J. Uriz; René H. Wijffels

2007-01-01

147

Microcoil-based MRI: feasibility study and cell culture applications using a conventional animal system  

Microsoft Academic Search

Object  The aim of this study was to demonstrate the feasibility of MR microimaging on a conventional 9.4 T horizontal animal MRI\\u000a system using commercial available microcoils in combination with only minor modifications to the system, thereby opening this\\u000a field to a larger community.\\u000a \\u000a \\u000a \\u000a \\u000a Materials and methods  Commercially available RF microcoils designed for high-resolution NMR spectrometers were used in combination with a

Hans Weber; Nicoleta Baxan; Dominik Paul; Julian Maclaren; Daniel Schmidig; Mohammad Mohammadzadeh; Jürgen Hennig; Dominik von Elverfeldt

2011-01-01

148

[Polysaccharides of cell cultures of Silene vulgaris].  

PubMed

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

Giunter, E A; Ovodov, Iu S

2007-01-01

149

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

Microsoft Academic Search

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

Mazen Nayef Salman

2002-01-01

150

Particle Trajectories in Rotating Wall Cell Culture Devices  

NASA Technical Reports Server (NTRS)

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.

Ramachandran N.; Downey, J. P.

1999-01-01

151

Tubulin dynamics in cultured mammalian cells  

PubMed Central

Bovine neurotubulin has been labeled with dichlorotriazinyl- aminofluorescein (DTAF-tubulin) and microinjected into cultured mammalian cells strains PTK1 and BSC. The fibrous, fluorescence patterns that developed in the microinjected cells were almost indistinguishable from the pattern of microtubules seen in the same cells by indirect immunofluorescence. DTAF-tubulin participated in the formation of all visible, microtubule-related structures at all cell cycle stages for at least 48 h after injection. Treatments of injected cells with Nocodazole or Taxol showed that DTAF-tubulin closely mimicked the behavior of endogenous tubulin. The rate at which microtubules incorporated DTAF-tubulin depended on the cell-cycle stage of the injected cell. Mitotic microtubules became fluorescent within seconds while interphase microtubules required minutes. Studies using fluorescence redistribution after photobleaching confirmed this apparent difference in tubulin dynamics between mitotic and interphase cells. The temporal patterns of redistribution included a rapid phase (approximately 3 s) that we attribute to diffusion of free DTAF-tubulin and a second, slower phase that seems to represent the exchange of bleached DTAF-tubulin in microtubules with free, unbleached DTAF- tubulin. Mean half times of redistribution were 18-fold shorter in mitotic cells than they were in interphase cells. PMID:6501419

1984-01-01

152

Ketamine is toxic to chondrocyte cell cultures.  

PubMed

Ketamine has been used in combination with a variety of other agents for intra-articular analgesia, with promising results. However, although it has been shown to be toxic to various types of cell, there is no available information on the effects of ketamine on chondrocytes. We conducted a prospective randomised controlled study to evaluate the effects of ketamine on cultured chondrocytes isolated from rat articular cartilage. The cultured cells were treated with 0.125 mM, 0.250 mM, 0.5 mM, 1 mM and 2 mM of ketamine respectively for 6 h, 24 hours and 48 hours, and compared with controls. Changes of apoptosis were evaluated using fluorescence microscopy with a 490 nm excitation wavelength. Apoptosis and eventual necrosis were seen at each concentration. The percentage viability of the cells was inversely proportional to both the duration and dose of treatment (p = 0.002 and p = 0.009). Doses of 0.5 mM, 1 mM and 2mM were absolutely toxic. We concluded that in the absence of solid data to support the efficacy of intra-articular ketamine for the control of pain, and the toxic effects of ketamine on cultured chondrocytes shown by this study, intra-articular ketamine, either alone or in combination with other agents, should not be used to control pain. Cite this article: Bone Joint J 2014; 96-B:989-94. PMID:24986956

Ozturk, A M; Ergun, M A; Demir, T; Gungor, I; Yilmaz, A; Kaya, K

2014-07-01

153

High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays  

Microsoft Academic Search

Heterogeneity in cell populations poses a major obstacle to understanding complex biological processes. Here we present a microfluidic platform containing thousands of nanoliter-scale chambers suitable for live-cell imaging studies of clonal cultures of nonadherent cells with precise control of the conditions, capabilities for in situ immunostaining and recovery of viable cells. We show that this platform mimics conventional cultures in

Véronique Lecault; Michael VanInsberghe; Sanja Sekulovic; David J H F Knapp; Stefan Wohrer; William Bowden; Francis Viel; Thomas McLaughlin; Asefeh Jarandehei; Michelle Miller; Didier Falconnet; Adam K White; David G Kent; Michael R Copley; Fariborz Taghipour; Connie J Eaves; R Keith Humphries; James M Piret; Carl L Hansen

2011-01-01

154

A morphological study of a human adenocarcinoma cell line (HT29) differentiating in culture. Similarities to intestinal embryonic development.  

PubMed

HT29 cells, a human adenocarcinoma cell line, when grown in Dulbecco's modified Eagle's medium (DMEM), form a multilayer of morphologically undifferentiated and unpolarized cells. However, when DMEM is replaced by RPMI medium, after 1-4 passages, a large amount of intracellular (ICL) and intercellular (ITCL) or secondary lumina (SL) are observed. These are detected in the light microscope and appear in the electron microscope as spherical structures embedded inside a multilayer of cells and bordered with microvilli. After 4-15 passages in RPMI, the cells retain the same pattern of cell growth but in addition exhibit apical brush-border microvilli and reveal a well developed belt of tight junctions. After 15 passages a single layer of polarized cells is clearly observed and a large number of 'domes' appeared. These results show that each of these culture types mimics morphologically specific stages described during intestinal ontogenesis between the 9th and the 16th week in the human embryo. PMID:2271997

Hekmati, M; Polak-Charcon, S; Ben-Shaul, Y

1990-09-01

155

Ascorbic acid transport into cultured pituitary cells  

SciTech Connect

An amidating enzyme designated peptidyl-glycine ..cap alpha..-amidating monooxygenase (PAM) has been studied in a variety of tissues and is dependent on molecular oxygen and stimulated by copper and ascorbic acid. To continue investigating the relationship among cellular ascorbic acid concentrations, amidating ability, and PAM activity, the authors studied ascorbic acid transport in three cell preparations that contain PAM and produce amidated peptides: primary cultures of rat anterior and intermediate pituitary and mouse AtT-20 tumor cells. When incubated in 50 ..mu..M (/sup 14/C)ascorbic acid all three cell preparations concentrated ascorbic acid 20- to 40-fold, producing intracellular ascorbate concentrations of 1 to 2 mM, based on experimentally determined cell volumes. All three cell preparations displayed saturable ascorbic acid uptake with half-maximal initial rates occurring between 9 and 18 ..mu..M ascorbate. Replacing NaCl in the uptake buffer with choline chloride significantly diminished ascorbate uptake in all three preparations. Ascorbic acid efflux from these cells was slow, displaying half-lives of 7 hours. Unlike systems that transport dehydroascorbic acid, the transport system for ascorbic acid in these cells was not inhibited by glucose. Thus, ascorbate is transported into pituitary cells by a sodium-dependent, active transport system.

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

1986-05-01

156

Cell-cell signaling in co-cultures of macrophages and fibroblasts  

PubMed Central

The foreign body response (FBR) comprises a general, ubiquitous host tissue-based reaction to implanted materials. In vitro cell-based models are frequently employed to study FBR mechanisms involving cell signaling responses to materials. However, these models often study only one cell type, identify only limited signals, and cannot accurately represent the complexity of in vivo inflammatory signaling. To address this issue, a cell co-culture system involving two primary effector cells of the FBR, macrophages and fibroblasts, was employed. Cell-cell signaling systems were monitored between these cell types, including long-term 1) culture of one cell type in conditioned media from the other cell type, 2) non-contacting cell co-cultures (paracrine signaling), and 3) contact co-cultures (juxtacrine signaling) of primary- and secondary-derived cells. Cell culture media and cell images were collected on Days 1, 2, 3, 7, 14, and 21 and changes in soluble protein secretion, cellular behavior, and morphology were assessed. Primary- and secondary- derived cells responded uniquely during each signaling scenario and to one another. In general higher in vitro fidelity to FBR-like responses was found in primary cell co-cultures compared to their mono-cultures and all secondary cell cultures. PMID:20932568

Holt, Dolly J.; Chamberlain, Lisa M.; Grainger, David W.

2010-01-01

157

Glucosylation of taxifolin with cultured plant cells.  

PubMed

Cultured plant cells of Eucalyptus perriniana glucosylated taxifolin to its 3'- and 7-O-beta-D-glucosides and 3',7-O-beta-D-diglucoside. On the other hand, taxifolin was converted into 3'- and 7-O-beta-D-glucosides by cultured cells of Nicotiana tabacum and Catharanthus roseus. PMID:23980419

Shimoda, Kei; Kubota, Naoji; Hamada, Manabu; Sugamoto, Masahiro; Ishihara, Kohji; Hamada, Hatsuyuki; Hamada, Hiroki

2013-07-01

158

AMMONIA REMOVAL FROM MAMMALIAN CELL CULTURE MEDIUM  

EPA Science Inventory

Metabolites such as ammonia and lactic formed during mammalian cell culture can frequently be toxic to the cells themselves beyond a threshold concentration of the metabolites. ell culture conducted in the presence of such accumulated metabolites is therefore limited in productiv...

159

Cell viability studies and operation in cellular culture medium of n-type organic field-effect transistors  

NASA Astrophysics Data System (ADS)

The possibility of the fabrication of organic devices suitable to be applied in bio-sensing fields depends largely on the availability of organic compounds displaying robust electrical properties even in aqueous solutions and effective biocompatibility features. In this paper, we report about the good cellular biocompatibility and the electrical response stability in an ionic medium of n-type organic transistors based on the recently developed PDI-8CN2 oligomer. The biocompatibility has been tested by analyzing the adhesion and viability of two different cell lines, human epithelial HeLa cells and murine neuronal F11 cells, on PDI-8CN2 films grown by organic molecular beam deposition (OMBD) on SiO2 substrates. The effect of film thickness on cell attachment was also tested. Uncoated SiO2 substrates were used as control surfaces and sexithiophene (T6) as device testing control. Moreover, the possible toxicity of -CN groups of PDI-8CN2 was tested on HeLa cell cultures, using PDI-8 and T6 molecules as controls. Results showed that, although at high concentration these organic compounds are toxic in solution, if they are presented in form of film, cell lines can attach and grow on them. The electrical response stability of PDI-8CN2 transistors in a cellular culture medium characterized by high concentrations of ionic species has been also investigated. For this purpose, low-voltage operation devices with VGS ranging from -5 V to 5 V, able to strongly reduce the influence of Faradaic currents coming from the electrical operation in an highly ionic environment, have been fabricated on 35 nm thick SiO2 layers and electrically characterized. These results are useful to experimentally define the main critical issues to be further addressed for the fabrication of reliable bio-sensors based on organic transistors.

Barra, M.; Viggiano, D.; Di Capua, R.; Di Girolamo, F.; Santoro, F.; Taglialatela, M.; Cassinese, A.

2012-02-01

160

Biochemical Assays of Cultured Cells  

NASA Technical Reports Server (NTRS)

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

Barlow, G. H.

1985-01-01

161

Structure and function of cultured endometrial epithelial cells.  

PubMed

Uterine endometrial epithelial cells undergo profound changes in structure and function in preparation for blastocyst implantation. The dynamics of this process for human endometrium can be studied only in cell culture. Primary cell cultures started from tissue removed at varying times during the cycle retain some aspects of differentiation as manifest in regulated protein synthesis. Differentiation of endometrial, epithelial cell lines will occur when culture conditions are varied. Domes, gland-like structures, polarized sheets and spheroids can be produced. Studying the process of differentiation in vitro should provide information about differentiation in vivo, particularly about how changing protein synthesis accompanies changing cell structure. Endometrial epithelial cells in culture can also be manipulated to allow study of steroid agonism and antagonism, cancer, menses and regeneration and endometriosis. PMID:10406079

Fleming, H

1999-01-01

162

Mammalian cell cultures for biologics manufacturing.  

PubMed

Biopharmaceuticals represent a growing sector of the pharmaceutical industry, and are used for a wide range of indications, including oncology and rheumatology. Cultured mammalian cells have become the predominant expression system for their production, partly due to their ability to complete the posttranslational modifications required for drug safety and efficacy. Over the past decade, the productivity of mammalian cell culture production processes has growth dramatically through improvements in both volumetric and specific productivities. This article presents an overview of the biologics market, including analysis of sales and approvals; as well as a review of industrial production cell lines and cell culture operations. PMID:24258145

Kantardjieff, Anne; Zhou, Weichang

2014-01-01

163

Antibacterial effect of theaflavin, polyphenon 60 ( Camellia sinensis) and Euphorbia hirta on Shigella spp. — a cell culture study  

Microsoft Academic Search

Antibacterial effect of compounds extracted from Camellia sinensis L. and the methanol extract of Euphorbia hirta L. were studied against dysentery causing Shigella spp. using the Vero cell line. Cytotoxicity studies of the extracts were performed using the cell line and the non-cytotoxic concentration of the extract was tested for antibacterial activity against the cytopathic dose of the pathogen. These

K. Vijaya; S. Ananthan; R. Nalini

1995-01-01

164

Emulsions Containing Perfluorocarbon Support Cell Cultures  

NASA Technical Reports Server (NTRS)

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

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

1990-01-01

165

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing  

PubMed Central

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

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

2014-01-01

166

Generation of a Chemical Gradient Across an Array of 256 Cell Cultures in a Single Chip  

PubMed Central

A microfluidic diffusion diluter to create stable chemical gradients across an array of cell cultures was demonstrated. The device enabled concentration based studies to be conducted at 256 different concentrations across individual, low shear cell cultures. A gradient of staurosporine on cells stained with Mitotracker Deep Red (MTDR) showed a concentration-based effect on cell apoptosis across the cell culture array. PMID:23939026

Somaweera, Himali; Ibragimov, Akif; Pappas, Dimitri

2013-01-01

167

Oxygen Control in Static Cell Cultures  

Microsoft Academic Search

Oxygen control in static cell cultures is one of the most critical parameters of a process, influencing the metabolism of\\u000a the cells and ultimately the final yield of the product. With the aim to improve the cultures of different types of cells,\\u000a both anchorage dependent and suspension, we have carried out several experiments evaluating the behaviour of some types of

Nadia De Bernardi; Edwin Schwander; Antonio Orlandi; Ilaria Tano; Sonia Castiglioni; Elena Muru; Fausto Gaspari; Marta Galgano; Claudia Mattei; Luigi Cavenaghi; Maria Nolli

168

Constructing a High Density Cell Culture System  

NASA Technical Reports Server (NTRS)

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

Spaulding, Glenn F. (Inventor)

1996-01-01

169

Algal culture studies for CELSS  

NASA Technical Reports Server (NTRS)

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

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

1987-01-01

170

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

Code of Federal Regulations, 2010 CFR

...culture media for human ex vivo tissue and cell culture processing applications. 876...culture media for human ex vivo tissue and cell culture processing applications. (a...culture media for human ex vivo tissue and cell culture processing applications...

2010-04-01

171

Relationship between P-glycoprotein expression and cyclosporin A in kidney. An immunohistological and cell culture study.  

PubMed Central

P-glycoprotein (P-gp), encoded in humans by the mdr-1 gene, acts physiologically as an efflux pump to expel hydrophobic substances from cells. This glycoprotein is closely related to multidrug resistance in tumor cells and can be modulated by cyclosporin A (CsA). We investigated the relationship between CsA and P-gp in 52 renal allograft biopsies and in cultures of Madin-Darby canine kidney (MDCK) renal tubule cells to determine whether the intrarenal accumulation of CsA or chronic stimulation with the drug modified the expression of P-gp. Expression of P-gp and CsA was analyzed by immunohistochemistry. Immunostaining was evaluated semiquantitatively. Modulation of P-gp in MDCK cells after chronic stimulation with CsA for 7, 30, and 60 days was analyzed by flow cytometry. P-gp and CsA immunostaining in renal post-transplant biopsies showed considerable overlap in all cases (Spearman's test, r = 0.577, P < 0.001). After 7 days in vitro, the number of cells expressing P-gp increased progressively; a further increase in mean fluorescence was found after 60 days (P < 0.001, Student's t-test). Our findings suggest that in non-neoplastic cells, CsA may stimulate P-gp as a mechanism of detoxification. Individual differences in the adaptive responses to glycoprotein may be responsible for the appearance of nephrotoxicity or a CsA-resistant rejection reaction in cases of overexpression on lymphocytes and macrophages. Images Figure 1 PMID:7856751

Garcia del Moral, R.; O'Valle, F.; Andujar, M.; Aguilar, M.; Lucena, M. A.; Lopez-Hidalgo, J.; Ramirez, C.; Medina-Cano, M. T.; Aguilar, D.; Gomez-Morales, M.

1995-01-01

172

Skeletal muscle satellite cells cultured in simulated microgravity.  

PubMed

Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells. Satellite cells retain the capacity to proliferate and differentiate in vitro and, therefore, provide a useful model to study postnatal muscle development. Most culture systems used to study postnatal muscle development are limited by the two-dimensional (2-D) confines of the culture dish. Limiting proliferation and differentiation of satellite cells in 2-D could potentially limit cell-cell contacts important for developing the level of organization in skeletal muscle obtained in vivo. Culturing satellite cells on microcarrier beads suspended in the High-Aspect-Ratio-Vessel (HARV) designed by NASA provides a low shear, three-dimensional (3-D) environment to study muscle development. Primary cultures established from anterior tibialis muscles of growing rats (approximately 200 gm) were used for all studies and were composed of greater than 75% satellite cells. Different inoculation densities did not affect the proliferative potential of satellite cells in the HARV. Plating efficiency, proliferation, and glucose utilization were compared between 2-D culture and 3-D HARV culture. Plating efficiency (cells attached divided by cells plated x 100) was similar between the two culture systems. Proliferation was reduced in HARV cultures and this reduction was apparent for both satellite cells and nonsatellite cells. Furthermore, reduction in proliferation within the HARV could not be attributed to reduced substrate availability because glucose levels in medium from HARV and 2-D cell culture were similar. Morphologically, microcarrier beads within the HARV were joined together by cells into 3-D aggregates composed of greater than 10 beads/aggregate. Aggregation of beads did not occur in the absence of cells. Myotubes were often seen on individual beads or spanning the surface of two beads. In summary, proliferation and differentiation of satellite cells on microcarrier beads within the HARV bioreactor results in a 3-D level of organization that could provide a more suitable model to study postnatal muscle development than is currently available with standard culture methods. PMID:9196898

Molnar, G; Schroedl, N A; Gonda, S R; Hartzell, C R

1997-05-01

173

Initiation of primary cell culture from amphioxus Branchiostoma belcheri tsingtauense  

NASA Astrophysics Data System (ADS)

Amphioxus, a cephalochordate, is an important model fish for studies in evolution and comparative biology. A successful cell culture from amphioxus tissues in vitro would help understanding some basic issues. To determine the optimal culture conditions for proliferation of amphioxus cells, primary cultures were initiated from buccal cirri, tail, gill, gut and metapleural fold of amphioxus Branchiostoma belcheri tsingtauense. The media tested were L-15, F-12, M 199, MEM, DMEM, PRMI 1640 and LDF, each was supplemented with 20% fetal bovine serum. The optimal conditions include tail tissue cultured in L-15 or F-12 with supplement of 20% FBS and 1.5% NaCl at about 25°C.

Wang, Changliu; Zhang, Shicui; Su, Feng; Wang, Lei; Li, Hongyan

2009-02-01

174

Vectorial Production of Interleukin 1 and Interleukin 6 by Rat Sertoli Cells Cultured in a Dual Culture Compartment System  

Microsoft Academic Search

The bidirectional production of interleukin-1 (IL-1) and IL-6 by Sertoli cells and its regulation by inflammatory and physiological stimuli has been studied using a dual compartment culture system allowing the study of Sertoli cell apical and basal secretory activities. Another Sertoli cell activity, the vectorial transferrin production was also studied in all culture conditions. A low constitutive IL-1 produc- tion

CORINNE CUDICINI; HENRI KERCRET; ANNE-MARIE TOUZALIN; FRANCOIS BALLET; BERNARD JEGOU

1997-01-01

175

21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2010 CFR

...2010-04-01 false Cultured animal and human cells. 864.2280 Section 864.2280... HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification....

2010-04-01

176

Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture  

PubMed Central

Secretagogin is a calcium binding protein (CBP) highly expressed in neuroendocrine cells. It has been shown to be involved in insulin secretion from pancreatic beta cells and is a strong candidate as a biomarker for endocrine tumors, stroke, and eventually psychiatric conditions. Secretagogin has been hypothesized to exert a neuroprotective role in neurodegenerative diseases like Alzheimer’s disease. The expression pattern of Secretagogin is not conserved from rodents to humans. We used brain tissue and primary neuronal cell cultures from rat to further characterize this CBP in rodents and to perform a few functional assays in vitro. Immunohistochemistry on rat brain slices revealed a high density of Secretagogin-positive cells in distinct brain regions. Secretagogin was found in the cytosol or associated with subcellular compartments. We tested primary neuronal cultures for their suitability as model systems to further investigate functional properties of Secretagogin. These cultures can easily be manipulated by treatment with drugs or by transfection with test constructs interfering with signaling cascades that might be linked to the cellular function of Secretagogin. We show that, like in pancreatic beta cells and insulinoma cell lines, also in neurons the expression level of Secretagogin is dependent on extracellular insulin and glucose. Further, we show also for rat brain neuronal tissue that Secretagogin interacts with the microtubule-associated protein Tau and that this interaction is dependent on Ca2+. Future studies should aim to study in further detail the molecular properties and function of Secretagogin in individual neuronal cell types, in particular the subcellular localization and trafficking of this protein and a possible active secretion by neurons. PMID:22888312

Maj, Magdalena; Milenkovic, Ivan; Bauer, Jan; Berggard, Tord; Veit, Martina; Ilhan-Mutlu, Aysegul; Wagner, Ludwig; Tretter, Verena

2012-01-01

177

Bioprocessing technology for plant cell suspension cultures  

Microsoft Academic Search

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

Wei wen Su

1995-01-01

178

Antibacterial effect of theaflavin, polyphenon 60 (Camellia sinensis) and Euphorbia hirta on Shigella spp.--a cell culture study.  

PubMed

Antibacterial effect of compounds extracted from Camellia sinensis L. and the methanol extract of Euphorbia hirta L. were studied against dysentery causing Shigella spp. using the Vero cell line. Cytotoxicity studies of the extracts were performed using the cell line and the non-cytotoxic concentration of the extract was tested for antibacterial activity against the cytopathic dose of the pathogen. These extracts were found to be non-cytotoxic and effective antibacterial agents. PMID:8847884

Vijaya, K; Ananthan, S; Nalini, R

1995-12-01

179

Culture and manipulation of embryonic cells.  

PubMed

The direct manipulation of embryonic cells is an important tool for addressing key questions in cell and developmental biology. C. elegans is relatively unique among genetic model systems in being amenable to manipulation of embryonic cells. Embryonic cell manipulation has allowed the identification of cell interactions by direct means, and it has been an important technique for dissecting mechanisms by which cell fates are specified, cell divisions are oriented, and morphogenesis is accomplished. Here, we present detailed methods for isolating, manipulating and culturing embryonic cells of C. elegans. PMID:22226523

Edgar, Lois G; Goldstein, Bob

2012-01-01

180

Isolation of mitochondria from tissue culture cells.  

PubMed

The number of mitochondria per cell varies substantially from cell line to cell line. For example, human HeLa cells contain at least twice as many mitochondria as smaller mouse L cells. This protocol starts with a washed cell pellet of 1-2 mL derived from ?10(9) cells grown in culture. The cells are swollen in a hypotonic buffer and ruptured with a Dounce or Potter-Elvehjem homogenizer using a tight-fitting pestle, and mitochondria are isolated by differential centrifugation. PMID:25275104

Clayton, David A; Shadel, Gerald S

2014-01-01

181

Hollow fiber culture accelerates differentiation of Caco-2 cells.  

PubMed

Caco-2 cells usually require 21 days of culture for developing sufficient differentiation in traditional two-dimensional Transwell culture, deviating far away from the quick differentiation of enterocytes in vivo. The recently proposed three-dimensional cultures of Caco-2 cells, though imitating the villi/crypt-like microstructure of intestinal epithelium, showed no effect on accelerating the differentiation of Caco-2 cells. In this study, a novel culture of Caco-2 cells on hollow fiber bioreactor was applied to morphologically mimic the human small intestine lumen for accelerating the expression of intestine functions. The porous hollow fibers of polyethersulfone (PES), a suitable membrane material for Caco-2 cell culture, successfully promoted cells to form confluent monolayer on the inner surface. The differentiated functions of Caco-2 cells, represented by alkaline phosphatase, ?-glutamyltransferase, and P-glycoprotein activity, were greatly higher in a 10-day hollow fiber culture than in a 21-day Transwell culture. Moreover, the Caco-2 cells on PES hollow fibers expressed higher F-actin and zonula occludens-1 protein than those on Transwell culture, indicative of an increased mechanical stress in Caco-2 cells on PES hollow fibers. The accelerated differentiation of Caco-2 cells on PES hollow fibers was unassociated with membrane chemical composition and surface roughness, but could be stimulated by hollow fiber configuration, since PES flat membranes with either rough or smooth surface failed to enhance the differentiation of Caco-2. Therefore, the accelerated expression of Caco-2 cell function on hollow fiber culture might show great values in simulation of the tissue microenvironment in vivo and guide the construction of intestinal tissue engineering apparatus. PMID:23689647

Deng, Xudong; Zhang, Guoliang; Shen, Chong; Yin, Jian; Meng, Qin

2013-08-01

182

Kinetic studies on the hydrogen peroxide elimination by cultured PC12 cells: rate limitation by glucose-6-phosphate dehydrogenase.  

PubMed

Oxidative stress is implicated in the pathogenesis of neurodegenerative disorders and brain ischemia, and hydrogen peroxide (H(2)O(2)) plays a central role in the stress. In this study, we have examined the kinetics of H(2)O(2) elimination by PC12 cells as a neuronal model in connection with the enzyme activities supporting the reaction. Similarly to other cell lines previously studied, H(2)O(2) removal kinetics could be divided into two reactions: one apparently following the Michaelis-Menten kinetics (GSH-dependent reaction) and the other following the first-order kinetics (mainly catalyzed by catalase). Based on the enzyme activities in the cell homogenate, it was inferred that glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the GSH- and NADPH-dependent H(2)O(2) elimination by PC12 cells. This is in contrast with fibroblasts and endothelial cells previously examined, in which glutathione reductase (GR) is rate-limiting in the reaction sequence. Treatment of PC12 cells with nerve growth factor increased G6PD activity in the cell homogenate and H(2)O(2) removal activity of the whole cells, with a concomitant increase in the resistance against H(2)O(2) toxicity. These results suggest the importance of G6PD in the antioxidant function of brain and pathogenesis of the oxidative stress-related diseases. PMID:12204336

Hashida, Kanae; Sakakura, Yuki; Makino, Nobuo

2002-08-15

183

Verbascoside production by plant cell cultures  

Microsoft Academic Search

Verbascoside was found to be produced in all calli derived from eleven species that contained the compound in their leaves. Cell suspension cultures were also established in three species, i.e., Leucosceptrum japonicum f. barbinerve, Syringa josikaea, and Sy. vulgaris, all of which were found to produce verbascoside at more than 1 g\\/l. Of the three species, suspension cultures of L.

Nobuyuki Inagaki; Hiroaki Nishimura; Minoru Okada; Hiroshi Mitsuhashi

1991-01-01

184

Haute Culture: Tailoring stem cells  

E-print Network

Biology, Department of Stem Cell and Regenerative Biology, Harvard University Massachusetts General Hospital Fernando Camargo, PhD Assistant Professor of Stem Cell Regenerative Biology, Department of Stem Cell and Regenerative Biology, Harvard University Children's Hospital Boston Stem Cell Program #12

Chou, James

185

Studies with antibodies to cultured rat glomerular epithelial cells. Subepithelial immune deposit formation after in vivo injection.  

PubMed Central

To investigate the role of glomerular epithelial cell (GEC) membrane proteins in the in situ formation of subepithelial immune deposits, the authors raised a rabbit antiserum against GEC that had been grown in culture (anti-GEC). By indirect immunofluorescence (IF) on normal rat kidney, anti-GEC stained proximal tubular brush border (BB). After intravenous injection into animals, granular glomerular capillary wall staining for IgG was present by IE and subepithelial immune deposits were identified by standard transmission and immunoelectron microscopy. Using the latter technique, injected anti-GEC IgG was identified beneath slit diaphragms and in endocytic-coated pits and intracellular vesicles of podocytes. Anti-GEC immunoprecipitated gp330 and two other proteins from radiolabeled BB. These proteins also were identified by sheep anti-rat Fx1A, the antiserum responsible for passive Heymann nephritis. Anti-GEC and anti-Fx1A also immunoprecipitated five identical proteins from surface-labeled GEC. Biosynthetically-labeled but not surface-labeled GEC contained immunoprecipitable gp330. Thus, injection into rats of antibodies raised against cultured GEC can produce subepithelial immune deposits, a disease process classically induced by antibodies to BB or its purified components. In addition to gp330, GEC and BB share other antigenic determinants that may contribute to the formation of these immune deposits. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:2655461

Quigg, R. J.; Abrahamson, D. R.; Cybulsky, A. V.; Badalamenti, J.; Minto, A. W.; Salant, D. J.

1989-01-01

186

Cell Culture on MEMS Platforms: A Review  

E-print Network

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

Ni, Ming

187

Microglial cells in astroglial cultures: a cautionary note  

PubMed Central

Primary rodent astroglial-enriched cultures are the most popular model to study astroglial biology in vitro. From the original methods described in the 1970's a great number of minor modifications have been incorporated into these protocols by different laboratories. These protocols result in cultures in which the astrocyte is the predominant cell type, but astrocytes are never 100% of cells in these preparations. The aim of this review is to bring attention to the presence of microglia in astroglial cultures because, in my opinion, the proportion of and the role that microglial cells play in astroglial cultures are often underestimated. The main problem with ignoring microglia in these cultures is that relatively minor amounts of microglia can be responsible for effects observed on cultures in which the astrocyte is the most abundant cell type. If the relative contributions of astrocytes and microglia are not properly assessed an observed effect can be erroneously attributed to the astrocytes. In order to illustrate this point the case of NO production in activated astroglial-enriched cultures is examined. Lipopolysaccharide (LPS) induces nitric oxide (NO) production in astroglial-enriched cultures and this effect is very often attributed to astrocytes. However, a careful review of the published data suggests that LPS-induced NO production in rodent astroglial-enriched cultures is likely to be mainly microglial in origin. This review considers cell culture protocol factors that can affect the proportion of microglial cells in astroglial cultures, strategies to minimize the proportion of microglia in these cultures, and specific markers that allow the determination of such microglial proportions. PMID:17937799

Saura, Josep

2007-01-01

188

Multiwell cell culture plate format with integrated microfluidic perfusion system  

NASA Astrophysics Data System (ADS)

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

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

2006-01-01

189

Cell culture systems for hepatitis C virus.  

PubMed

Due to the obligatory intracellular lifestyle of viruses, cell culture systems for efficient viral propagation are crucial to obtain a detailed understanding of the virus-host cell interaction. For hepatitis C virus (HCV) the development of permissive and authentic culture models continues to be a challenging task. The first efforts to culture HCV had limited success and range back to before the virus was molecularly cloned in 1989. Since then several major breakthroughs have gradually overcome limitations in culturing the virus and sequentially permitted analysis of viral RNA replication, cell entry, and ultimately the complete replication cycle in cultured cells in 2005. Until today, basic and applied HCV research greatly benefit from these tremendous efforts which spurred multiple complementary cell-based model systems for distinct steps of the HCV replication cycle. When used in combination they now permit deep insights into the fascinating biology of HCV and its interplay with the host cell. In fact, drug development has been much facilitated and our understanding of the molecular determinants of HCV replication has grown in parallel to these advances. Building on this groundwork and further refining our cellular models to better mimic the architecture, polarization and differentiation of natural hepatocytes should reveal novel unique aspects of HCV replication. Ultimately, models to culture primary HCV isolates across all genotypes may teach us important new lessons about viral functional adaptations that have evolved in exchange with its human host and that may explain the variable natural course of hepatitis C. PMID:23463196

Steinmann, Eike; Pietschmann, Thomas

2013-01-01

190

Feeding Frequency Affects Cultured Rat Pituitary Cells in Low Gravity  

NASA Technical Reports Server (NTRS)

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.

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

1996-01-01

191

Synthesis of basement membrane collagen by cultured human endothelial cells  

PubMed Central

Studies were performed to determine if cultured human endothelial cells synthesized basement membrane collagen. In culture, endothelial cells were attached to grossly visible membranous structures which on light microscopy were composed of ribbons of dense, amorphous material. On transmission electron microscopy, these membranous structures consisted of amorphous basement membrane, and material morphologically similar to microfibrils and elastic fibers. By immunofluorescence microscopy, these membranous structures stained brightly with antisera to human glomerular basement membrane. Cultured endothelial cells incorporated [3H]proline into protein; 18% of the incorporated [3H]proline was solubilized by purified collagenase. When endothelial cells were cultured with [14C]proline, 7.1% of the incorporated counts were present as [14C]hydroxyproline. Cultured endothelial cells were labeled with [3H]glycine and [3H]proline and digested with pepsin. The resulting fractions on analysis by SDS-polyacrylamide gel electrophoresis contained two radioactive protein peaks of mol wt 94,200 and 120,500. Both these peaks disappeared after digestion with purified collagenase. The peak of mol wt 120,500 corresponds to that of alpha1 (IV) collagen; the peak of the mol wt 94,200 probably corresponds to that of alpha1 (III) collagen. Thus, cultured human endothelial cells synthesize material which is morphologically and immunologically like amorphous basement membrane and biochemically like basement membrane collagen. Cultured endothelial cells probably also synthesize material which is morphologically similar to microfibrils and elastic fibers. PMID:58957

1976-01-01

192

Hypergravity signal transduction and gene expression in cultured mammalian cells  

NASA Technical Reports Server (NTRS)

A number of studies have been conducted during space flight and with clinostats and centrifuges, suggesting that gravity effects the proliferation and differentiation of mammalian cells in vitro. However, little is known about the mechanisms by which mammalian cells respond to changes in gravitational stress. This paper summarizes studies designed to clarify the effects of hypergravity on the cultured human HeLa cells and to investigate the mechanism of hypergravity signal transduction in these cells.

Kumei, Y.; Whitson, P. A.

1994-01-01

193

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

Microsoft Academic Search

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder\\u000a cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study,\\u000a five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem\\u000a cell lines

Veronika Akopian; Peter W. Andrews; Stephen Beil; Nissim Benvenisty; Jennifer Brehm; Megan Christie; Angela Ford; Victoria Fox; Paul J. Gokhale; Lyn Healy; Frida Holm; Outi Hovatta; Barbara B. Knowles; Tenneille E. Ludwig; Ronald D. G. McKay; Takamichi Miyazaki; Norio Nakatsuji; Steve K. W. Oh; Martin F. Pera; Janet Rossant; Glyn N. Stacey; Hirofumi Suemori

2010-01-01

194

Growth of melanocytes in human epidermal cell cultures  

SciTech Connect

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.

Staiano-Coico, L.; Hefton, J.M.; Amadeo, C.; Pagan-Charry, I.; Madden, M.R.; Cardon-Cardo, C. (Cornell Medical College, New York, NY (USA))

1990-08-01

195

Cultural Studies, Multiculturalism, and Media Culture  

Microsoft Academic Search

Radio, television, film, and the other products of media culture provide materials out of which we forge our very identities; our sense of selfhood; our notion of what it means to be male or female; our sense of class, of ethnicity and race, of nationality, of sexuality; and of \\

Douglas Kellner

196

Automated adherent human cell culture (mesenchymal stem cells).  

PubMed

Human cell culture processes developed at research laboratory scale need to be translated to large-scale production processes to achieve commercial application to a large market. To allow this transition of scale with consistent process performance and control of costs, it will be necessary to reduce manual processing and increase automation. There are a number of commercially available platforms that will reduce manual process intervention and improve process control for different culture formats. However, in many human cell-based applications, there is currently a need to remain close to the development format, usually adherent culture on cell culture plastic or matrix-coated wells or flasks due to deterioration of cell quality in other environments, such as suspension. This chapter presents an example method for adherent automated human stem cell culture using a specific automated flask handling platform, the CompacT SelecT. PMID:22057466

Thomas, Robert; Ratcliffe, Elizabeth

2012-01-01

197

Microfabricated polymeric vessel mimetics for 3-D cancer cell culture  

PubMed Central

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

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

2013-01-01

198

Osteogenic potential of human umbilical cord-derived mesenchymal stromal cells cultured with umbilical cord blood-derived fibrin: a preliminary study.  

PubMed

This study examined the potential for osteogenesis via regenerative medicine using autologous tissues (umbilical cord (UC) and umbilical cord blood (UCB)) in nude mice. The study was designed to provide the three elements required for regenerative medicine (cell, scaffold, and growth factor) and autoserum for culture by means of autologous tissues. Mesenchymal stromal cells were obtained from UC (UC-MSCs). Fibrin, platelet-rich-plasma, and autoserum were obtained from UCB as scaffold, growth factor and serum for culture respectively. UC-MSCs were obtained from Wharton jelly and cultured with UCB-derived fibrin (UCB-fibrin) for 3-4 weeks to induce their differentiation into osteoblasts. They were implanted subcutaneously into the dorsum of male nude mice for 6 weeks prior to undergoing assessment. The assessments performed were haematoxylin and eosin, and alizarin red staining, immunohistochemical staining of human mitochondria, scanning electron microscopy, scanning electron microscopy with energy dispersive X-ray spectrometry and real-time reverse transcriptase-polymerase chain reaction to assess the expressions of osteoblast markers. Consequently, the differentiation of UC-MSCs into osteoblasts and the production of hydroxyapatite were verified. This study suggested the possible formation of bone tissue using biomedical materials obtained from UC and UCB. PMID:23465638

Baba, Kyoko; Yamazaki, Yasuharu; Ishiguro, Masashi; Kumazawa, Kenichi; Aoyagi, Kazuya; Ikemoto, Shigehiro; Takeda, Akira; Uchinuma, Eiju

2013-12-01

199

Disposable Bioreactors for Plant Micropropagation and Mass Plant Cell Culture  

Microsoft Academic Search

\\u000a Different types of bioreactors are used at Nestlé R&D Centre – Tours for mass propagation of selected plant varieties by somatic\\u000a embryogenesis and for large scale culture of plants cells to produce metabolites or recombinant proteins. Recent studies have\\u000a been directed to cut down the production costs of these two processes by developing disposable cell culture systems. Vegetative\\u000a propagation of

Jean-Paul Ducos; Bénédicte Terrier; Didier Courtois

2010-01-01

200

Large-scale insect and plant cell culture.  

PubMed

Currently, insect and plant cell cultures are not widely used to make products of commercial interest, largely because the development of large-scale cultivation methods is still in its infancy. With the advances made over the past year, some of the limitations associated with scale-up of these two types of expression system have been addressed. Increasing the oxygen supply and the concentration of various nutrients supplied to insect cells after infection has enabled high specific protein production to be maintained to higher cell densities than ever before, improving overall volumetric yields. Detailed work has focused on the capacity of insect cells to carry out complex post-translational modifications; however, as yet, evidence is conflicting as to the extent of protein processing and complex glycosylation possible in infected cells. In plant cell culture, the accepted axioms concerning large-scale culture have been re-examined. Recent studies have assessed culture at high cell densities and the constraints in reactor design resulting from the 'shear sensitivity' of plant cells. Results show that, as cell densities increase, alterations occur in the pathways of secondary metabolism, leading to decreases in specific productivity. The use of nutrient supplements and a medium cycling strategy shows promise for increasing and sustaining product formation. Furthermore, the importance of dissolved gas composition has been clearly demonstrated by use of a gas recirculation reactor. Reports of taxol and vindoline production in vitro demonstrate the potential and the necessity for further research in scale-up of plant cell culture. PMID:7764795

Taticek, R A; Lee, C W; Shuler, M L

1994-02-01

201

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

PubMed Central

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

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

2014-01-01

202

Glycosylation of hesperetin by plant cell cultures  

Microsoft Academic Search

The biotransformation of hesperetin by cultured cells of Ipomoea batatas and Eucalyptus perriniana was investigated. Three glycosides, hesperetin 3?-O-?-d-glucopyranoside (33?g\\/g fr. wt of cells), hesperetin 3?,7-O-?-d-diglucopyranoside (217?g\\/g fr. wt of cells), and hesperetin 7-O-[6-O-(?-d-glucopyranosyl)]-?-d-glucopyranoside (?-gentiobioside, 22?g\\/g fr. wt of cells), together with three hitherto known glycosides, hesperetin 5-O-?-d-glucopyranoside (23?g\\/g fr. wt of cells), hesperetin 7-O-?-d-glucopyranoside (57?g\\/g fr. wt of cells),

Kei Shimoda; Hatsuyuki Hamada; Hiroki Hamada

2008-01-01

203

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

PubMed

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

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

2013-11-01

204

Cultural Education--Iroquois Cultural Study for Elementary Grades.  

ERIC Educational Resources Information Center

Presenting a sequenced cultural education program, this curriculum guide for an Iroquois cultural study for elementary grades concentrates on providing a supplemental classroom program to an existing social studies curriculum, though it is also aimed at teaching culture in Native American classes. Program objectives are to provide students with…

Steele, Catherine

205

Henrietta Lacks, HeLa cells, and cell culture contamination.  

PubMed

Henrietta Lacks died in 1951 of an aggressive adenocarcinoma of the cervix. A tissue biopsy obtained for diagnostic evaluation yielded additional tissue for Dr George O. Gey's tissue culture laboratory at Johns Hopkins (Baltimore, Maryland). The cancer cells, now called HeLa cells, grew rapidly in cell culture and became the first human cell line. HeLa cells were used by researchers around the world. However, 20 years after Henrietta Lacks' death, mounting evidence suggested that HeLa cells contaminated and overgrew other cell lines. Cultures, supposedly of tissues such as breast cancer or mouse, proved to be HeLa cells. We describe the history behind the development of HeLa cells, including the first published description of Ms Lacks' autopsy, and the cell culture contamination that resulted. The debate over cell culture contamination began in the 1970s and was not harmonious. Ultimately, the problem was not resolved and it continues today. Finally, we discuss the philosophical implications of the immortal HeLa cell line. PMID:19722756

Lucey, Brendan P; Nelson-Rees, Walter A; Hutchins, Grover M

2009-09-01

206

Detection of contaminants in cell cultures, sera and trypsin.  

PubMed

The aim of this study was standardization and application of polymerase chain reaction (PCR) for the detection of contaminants in cell cultures, sera and trypsin. Five PCR protocols were standardized to assess the presence of genetic material from mycoplasma, porcine circovirus 1 (PCV1), bovine leukemia virus (BLV) or bovine viral diarrhea virus (BVDV) in cell culture samples. PCR reactions for the genes GAPDH and beta-actin were used to evaluate the efficiency of nucleic acid extraction. The PCR protocols were applied to 88 cell culture samples from eight laboratories. The tests were also used to assess potential contamination in 10 trypsin samples and 13 fetal calf serum samples from different lots from five of the laboratories. The results showed the occurrence of the following as DNA cell culture contaminants: 34.1% for mycoplasma, 35.2% for PCV1, 23.9% for BVDV RNA and 2.3% for BLV. In fetal calf sera and trypsin samples BVDV RNA and PCV1 DNA was detected. The results demonstrated that cell culture, sera and trypsin used by different laboratories show a high rate of contaminants. The results highlight the need for monitoring cell cultures and controlling for biological contaminants in laboratories and cell banks working with these materials. PMID:24071554

Pinheiro de Oliveira, Tatiana Flávia; Fonseca, Antônio Augusto; Camargos, Marcelo Fernandes; de Oliveira, Anapolino Macedo; Pinto Cottorello, Ana Cláudia; Souza, Antonizete Dos Reis; de Almeida, Iassudara Garcia; Heinemann, Marcos Bryan

2013-11-01

207

The impact of cell culture equipment on energy loss.  

PubMed

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. PMID:23568625

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

2014-01-01

208

Rosmarinic acid production in Coleus cell cultures  

Microsoft Academic Search

Cell suspension cultures of Coleus blumei Benth. have been found to accumulate 8–11% of their dry weight as rosmarinic acid (a-O-caffeoyl-3,4-dihydroxyphenyl-lactic acid). Actively-growing tissue converts >20% of exogenously supplied phenylalanine and tyrosine to the caffeoyl ester and this high rate of synthesis coincides with an increase in phenylalanine ammonia-lyase specific activity. Administration to the cultures of known phenylpropanoid precursors of

A. Razzaque; B. E. Ellis

1977-01-01

209

Chromosome preparation from cultured cells.  

PubMed

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births(1,2), 60-80% of all miscarriages(3,4), 10% of stillbirths(2,5), 13% of individuals with congenital heart disease(6), 3-6% of infertility cases(2), and in many patients with developmental delay and birth defects(7). Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance(8,9). Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents(10-13). Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy's fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)(14,15). PMID:24513647

Howe, Bradley; Umrigar, Ayesha; Tsien, Fern

2014-01-01

210

Studies on the Production of Digitalis Cardenolides by Plant Tissue Culture: II. EFFECT OF LIGHT AND PLANT GROWTH SUBSTANCES ON DIGITOXIN FORMATION BY UNDIFFERENTIATED CELLS AND SHOOT-FORMING CULTURES OF DIGITALIS PURPUREA L. GROWN IN LIQUID MEDIA.  

PubMed

Undifferentiated, highly chlorophyllous cell cultures; undifferentiated white cell cultures; green, shoot-forming cultures; and white, shoot-forming cultures of Digitalis purpurea L. were established and subcultured every 3 weeks in liquid media in the light or in the dark. The digitoxin content, the chlorophyll content, and the ribulose bisphosphate carboxylase activity of these cultures were assayed. The light-grown, green, shoot-forming cultures accumulated considerable amounts of digitoxin (about 20 to 40 micrograms per gram dry weight), and the white, shoot-forming cultures without chloroplasts accumulated about one-third that amount of digitoxin. The chlorophyll content and the ribulose bisphosphate carboxylase activity of the undifferentiated green cells were about the same as they were in the green, shoot-forming cultures, but the digitoxin content of the former was extremely low (about 0.05 to 0.2 microgram per gram dry weight), which is about the same as that in undifferentiated white cells without chloroplasts. Thus, it was concluded that the chloroplasts are not essential for the synthesis of digitoxin in Digitalis cells. The optimum concentrations of the tested compounds for accumulation of digitoxin were: benzyladenine, 0.01 to 1 milligram per liter; indoleacetic acid, 0.1 to 1 milligram per liter; alpha-naphthaleneacetic acid; 0.1 milligram per liter; and 2,4-dichlorophenoxyacetic acid, 0.01 milligram per liter. PMID:16662267

Hagimori, M; Matsumoto, T; Obi, Y

1982-03-01

211

Pure cultures and characterization of yak Sertoli cells.  

PubMed

The culture of primary Sertoli cells has become an important resource in the study of their function. However, their use is limited because of contamination of isolated cells with other testicular cells, mainly germ cells. The aim was to establish technique to obtain pure yak Sertoli cells as well as to study the growth kinetics and biological characteristics of Sertoli cells in vitro. Two-step enzyme digestion was used to separate and culture yak Sertoli cells. Cultured using starvation method and the hypotonic treatment were also invented to get pure yak Sertoli cells. Furthermore, the purification of Yak Sertoli cells were identified according to their characteristics, such as bipolar corpuscular around the nucleus and expression of Fasl, in addition to their morphology. The average viability of the Sertoli cells was 97% before freezing and 94.5% after thawing, indicating that cryopreservation in liquid nitrogen had little influence on the viability of Sertoli cells. The growth tendency of yak Sertoli cells was similar to an S-shaped growth curve. Purified yak Sertoli cells frequently exhibited bipolar corpuscula in nucleus after Feulgen staining, and did have a positive reaction of Fasl by the immunocytochemical identification. After recovery chromosomal analysis of Sertoli cells had a normal chromosomal number of 60, comprising 29 pairs of autosomes and one pair of sex chromosomes. Assays for bacteria, fungi and mycoplasmas were negative. In conclusion, yak Sertoli cells have been successfully purified and cultured in vitro, and maintain stable biological characteristics after thawing. Therefore, it will not only preserve the genetic resources of yaks at the cellular level, but also provide valuable materials for transgenic research and feeder layer and nuclear donor cells in yak somatic cell cloning technology. PMID:23938058

Zhang, Hua; Liu, Ben; Qiu, Yuan; Fan, Jiang feng; Yu, Si jiu

2013-12-01

212

Cell Culture on MEMS Platforms: A Review  

PubMed Central

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

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

2009-01-01

213

Eotaxin-2 in sputum cell culture to evaluate asthma inflammation  

Microsoft Academic Search

The aim of the present study was to elucidate whether the culture of cells recovered from induced sputum may represent a suitable model to evaluate cytokine and chemokine production by airway inflammatory cells. Sputum induction was performed in 21 normal subjects and 30 asthmatic patients. A total of 21 out of the 30 asthmatic patients were taking inhaled corticosteroids, while

M. E. Scheicher; M. M. Teixeira; F. Q. Cunha; A. L. Teixeira Jr; J. T. Filho; E. O. Vianna

2007-01-01

214

Fundamentals of microfluidic cell culture in controlled microenvironments  

E-print Network

of microfluidic device design and operation. We also discuss the most recent advances in microfluidic cell culture with the potential to make significant impact on cell biology research. The ability to manipulate small volumes with microfluidics has recently become a major focus within the scientific community. The initial motivation to study

Beebe, David J.

215

Robotic cell culture system for stem cell assays  

Microsoft Academic Search

Purpose – The purpose of this paper is to focus on the advantages of a robotic time-lapsed microscopic imaging system for tracking stem cells in in vitro biological assays which measure stem cell activities. Design\\/methodology\\/approach – The unique aspects of the system include robotic movement of stem cell culture flasks which enables selection of a large number of regions of

Benjamin T. Schmidt; Joseph M. Feduska; Ashley M. Witt; Bridget M. Deasy

2008-01-01

216

Cell sheet transplantation of cultured mesenchymal stem cells enhances bone formation in a rat nonunion model  

Microsoft Academic Search

Orthopedic surgeons have long been troubled by cases involving nonunion of fractured bones. This study aimed to enhance bone union by cell sheet transplantation of mesenchymal stem cells. A nonunion model was made in rat femur, and rat bone marrow cells were cultured in medium containing dexamethasone and ascorbic acid phosphate to create a cell sheet that could be scraped

Akifumi Nakamura; Manabu Akahane; Hideki Shigematsu; Mika Tadokoro; Yusuke Morita; Hajime Ohgushi; Yoshiko Dohi; Tomoaki Imamura; Yasuhito Tanaka

2010-01-01

217

Electromagnetic field and other physical methods influencing cell growth in mammal cell culture systems  

Microsoft Academic Search

The growth rate of cultured mammalian cells can be influenced by chemical and physical methods such as electromagnetic fields (EMF), light, temperature and plasma. These physical methods have a number of well documented effects on mammalian cells including modification of gene expression, cell cycle, invasion, motility, cell viability, proliferation, apoptosis and mammosphere numbers. A study of the existing literature confirms

Abbas Z. Kouzani; Akif Kaynak; Christophe Lefevre

2011-01-01

218

Cell wall proteomics of sugarcane cell suspension cultures.  

PubMed

The use of cell walls to produce cellulosic ethanol from sugarcane bagasse is a new challenge. A better knowledge of proteins involved in cell wall remodelling is essential to improve the saccharification processes. Cell suspension cultures were used for this first cell wall proteomics study of sugarcane. Proteins extracted from cell walls were identified using an adapted protocol. They were extracted using 0.2 M CaCl2 and 2 M LiCl after purification of cell walls. The proteins were then identified by the innovative nanoACQUITY UPLC MS/MS technology and bioinformatics using the translated SUCEST EST cluster database of sugarcane. The experiments were reproduced three times. Since Sorghum bicolor is the closest plant with a fully sequenced genome, homologous proteins were searched for to complete the annotation of proteins, that is, prediction of subcellular localization and functional domains. Altogether, 69 different proteins predicted to be secreted were identified among 377 proteins. The reproducibility of the experiments is discussed. These proteins were distributed into eight functional classes. Oxidoreductases such as peroxidases were well represented, whereas glycoside hydrolases were scarce. This work provides information about the proteins that could be manipulated through genetic transformation, to increase second-generation ethanol production. PMID:24436144

Calderan-Rodrigues, Maria Juliana; Jamet, Elisabeth; Bonassi, Maria Beatriz Calderan Rodrigues; Guidetti-Gonzalez, Simone; Begossi, Amanda Carmanhanis; Setem, Laís Vaz; Franceschini, Livia Maria; Fonseca, Juliana Guimarães; Labate, Carlos Alberto

2014-03-01

219

A novel closed cell culture device for fabrication of corneal epithelial cell sheets.  

PubMed

Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25?µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300?µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets. Copyright © 2012 John Wiley & Sons, Ltd. PMID:23239605

Nakajima, Ryota; Kobayashi, Toyoshige; Moriya, Noboru; Mizutani, Manabu; Kan, Kazutoshi; Nozaki, Takayuki; Saitoh, Kazuo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

2012-12-14

220

Detection of Mycoplasma contamination in cell cultures.  

PubMed

Mycoplasma contamination of cell lines is a major problem in cell culture technology. This unit presents protocols involving either the polymerase chain reaction (PCR) or fluorescent in situ hybridization (FISH) to provide independent, fast, and sensitive techniques to monitor mycoplasma contamination in laboratory cultures. Special emphasis is placed on the integration of control reactions to prevent false-negative as well as false-positive results due to reaction inhibition or contamination and background staining, respectively. Curr. Protoc. Mol. Biol. 106:28.4.1-28.4.14. © 2014 by John Wiley & Sons, Inc. PMID:24733240

Uphoff, Cord C; Drexler, Hans G

2014-01-01

221

ISOLATION, CULTURE AND CHARACTERIZATION OF ENDOMETRIAL EPITHELIAL CELLS IN BUFFALO (BUBALUS BUBALIS)  

Microsoft Academic Search

In the present study, the authors isolated and developed the culture of endometrial epithelial cells from buffalo uterus as well as evaluated functional properties of epithelial cells. In primary culture, epithelial cells appeared cuboidal or columnar and showed contact inhibition at the stage of confluence. Protein and DNA concentrations were found to increase with the time in culture. PGF2? concentrations

S. Mondal; S. Nandi; I. J. Reddy; K. P. Suresh

2009-01-01

222

The uptake of two androgen substrates by cultured human gingival fibroblasts in response to minocycline and metabolic studies using a cell-free system  

Microsoft Academic Search

The uptake of two androgen substrates by cultured fibroblasts and the anabolic response of homogenates of cultured gingival fibroblasts to minocycline were investigated. Monolayer cultures of confluent fibroblasts were incubated with [14C]testosterone\\/[14C]4-androstenedione for timed intervals in the presence or absence of optimal concentrations of minocycline. The intracellular uptake of androgens was quantified by radio-isotope counts on cell digests. Confluent gingival

M Soory; H Virdi

1999-01-01

223

Media, media practice and cultural studies  

E-print Network

Media, media practice and cultural studies MPhil, PhD Normally a Masters degree with a Merit and Media, MA in Media and Cultural Studies, PhD in Media and Cultural Studies IELTS 7.0, with not less than MA in Digital Media MA in Filmmaking MA in Gender and Media MA in Media and Cultural Studies MA

Sussex, University of

224

Electron microscopic appearance of cells from carcinoma of the prostate in monolayer tissue culture  

Microsoft Academic Search

An electron microscopic study of human prostate cells grown in monolayer tissue cultures is presented. The cultures were established from tissue of 4 patients with adenocarcinomas of the prostate. The predominant cell type was identified as epithelial. These cells were characterized by close cell growth with many desmosomal junctions, formation of “lumina” between cells and atypical mitochondria. The ultrastructural appearance

B. Brehmer; J. F. Riemann; J. M. B. Bloodworth; P. O. Madsen

1973-01-01

225

Induction of growth in resting fibroblastic cell cultures by Ca++.  

PubMed Central

Of all the components of the culture medium, only CaCl2 induces DNA replication when added to resting cultures of Balb/c 3T3 cells. The effect is present even in a serum-free medium. Increasing the Ca++ concentration above the standard 1.8 mM in the medium of a new culture increases the total number of cells ultimately produced, without affecting the initial cell growth rate. This effect is synergistic with that of serum. The elevated Ca++ concentration also induces striking morphological changes. The Ca++ effect could not be reproduced by a Ca++ ionophore. These observations afford a new tool for studying how the various intracellular events following the addition of growth factors to resting cultures are involved in the control of cellular growth. Images PMID:165515

Dulbecco, R; Elkington, J

1975-01-01

226

Scaffold-free culture of mesenchymal stem cell spheroids in suspension preserves multilineage potential.  

PubMed

While traditional cell culture methods have relied on growing cells as monolayers, three-dimensional (3D) culture systems can provide a convenient in vitro model for the study of complex cell-cell and cell-matrix interactions in the absence of exogenous substrates and may benefit the development of regenerative medicine strategies. In this study, mesenchymal stem cell (MSC) spheroids, or "mesenspheres", of different sizes, were formed using a forced aggregation technique and maintained in suspension culture for extended periods of time thereafter. Cell proliferation and differentiation potential within mesenspheres and dissociated cells retrieved from spheroids were compared to conventional adherent monolayer cultures. Mesenspheres maintained in growth medium exhibited no evidence of cell necrosis or differentiation, while mesenspheres in differentiation media exhibited differentiation similar to conventional 2D culture methods based on histological markers of osteogenic and adipogenic commitment. Furthermore, when plated onto tissue culture plates, cells that had been cultured within mesenspheres in growth medium recovered morphology typical of cells cultured continuously in adherent monolayers and retained their capacity for multi-lineage differentiation potential. In fact, more robust matrix mineralization and lipid vacuole content were evident in recovered MSCs when compared to monolayers, suggesting enhanced differentiation by cells cultured as 3D spheroids. Thus, this study demonstrates the development of a 3D culture system for mesenchymal stem cells that may circumvent limitations associated with conventional monolayer cultures and enhance the differentiation potential of multipotent cells. PMID:21833761

Baraniak, Priya R; McDevitt, Todd C

2012-03-01

227

Aquatic toxicity evaluated using human and monkey cell culture assays  

SciTech Connect

A new bioassay using cultured mammalian cells was reported. In this report, the author describes the use of a cell culture assay using growing cells as a screen for aquatic toxicity. Cadmium, copper and zinc toxicity were measured.

Mochida, K.

1986-04-01

228

The culture of human embryonic stem cells in microchannel perfusion bioreactors  

NASA Astrophysics Data System (ADS)

The culture of human Embryonic Stem (ES) cells in microchannel bioreactors can be highly beneficial for ES cell biology studies and ES tissue engineering applications. In the present study we examine the use of Human Foreskin Fibroblasts (HFF) cells as feeder cells for human ES culture in a microchannel perfusion bioreactor. PDMS microchannels (depth:130 micron) were fabricated using conventional soft-lithography techniques. The channels were sterilized, coated with a human fibronectin solution and seeded with cells. Following a period of static incubation, culture medium was perfused through the channels at various flow rates and cell growth was monitored throughout the culture process. Mass transport and fluid mechanics models were used to evaluate the culture conditions (shear stress, oxygen levels within the micro-bioreactor as a function of the medium flow rate. The conditions for successful long-term culture (>7 days) of HFF under flow were established. Experiments with human embryonic stem cells cultured in microchannels show that the conditions essential to co-culture human ES cell on HFF cells under perfusion differ from the conditions necessary for HFF cell culture. Human ES cells were found to be highly sensitive to flow and culture conditions and did not grow under flow rates which were suitable for HFF long-term culture. Successful culture of undifferentiated human ES cell colonies in a perfusion micro-bioreactor is a basic step towards utilizing microfluidic techniques to explore stem cell biology.

Korin, Natanel; Bransky, Avishay; Dinnar, Uri; Levenberg, Shulamit

2007-12-01

229

Cell culture conditions affect RPE phagocytic function  

Microsoft Academic Search

Background  Changes in the phenotype of retinal pigment epithelium (RPE) cells in vitro are associated with medium conditions and changes\\u000a in function. Main goals in RPE tissue engineering are cell propagation in serum-free defined culture conditions, resulting\\u000a in cells exhibiting differentiated morphology and functioning in vitro.\\u000a \\u000a \\u000a \\u000a Methods  To compare the effects of various media and supplements on cell function, an optimized high-throughput

Mike O. Karl; Monika Valtink; Jürgen Bednarz; Katrin Engelmann

2006-01-01

230

Implantation of cultured human leptomeningeal cells into rat brain.  

PubMed

Since previous studies have shown that cells cultured from human leptomeninges can express neuronal and glial antigens under appropriate culture conditions [DeGiorgio L. A. et al. (1994) J. Neurol. Sci. 124, 141 148; Bernstein J. J. et al. (1996) Int. J. Derl Neurosci. 14(5), 681 687], we have studied the developmental characteristics of these cells further by grafting them into young adult rat brains. Cells were labeled in culture with Fast Blue and were identified unequivocally by hybridization with nick-translated human DNA. Intensely Fast Blue positive human leptomeningeal cells were concentrated in the implant pocket and adjacent rat leptomeninges al one and two weeks postimplant. Human and rat leptomeningeal cells were similar morphologically and were equally immunopositive for vimentin and fibronectin. Implanted human cells did not express the neuronal and glial proteins they had in vitro. Cells which hybridized with human DNA corresponded to the intensely Fast Blue positive cells. Small groups of human DNA hybridizing cells were also observed in the choroid plexus. Less intensely Fast Blue positive neurons and glia were found in the brain but these hybridized with rat DNA. A minority of human leptomeningeal cells implanted into rat brain are subsequently found in host leptomeninges where they demonstrate properties characteristic of leptomeningeal fibroblasts. Small numbers of implanted cells can survive for two weeks. PMID:9178041

DeGiorgio, L A; Bernstein, J J; Blass, J P

1997-04-01

231

Biodegradable thermogelling poly[(R)-3-hydroxybutyrate]-based block copolymers: micellization, gelation, and cytotoxicity and cell culture studies.  

PubMed

A series of biodegradable multiblock poly(ester urethane)s having poly[(R)-3-hydroxybutyrate] (PHB), poly(ethylene glycol) (PEG), and poly(propylene glycol) (PPG) segments was prepared. The critical micellization concentration (CMCs) of these water-soluble poly(ester urethane)s were determined at different temperatures in order to calculate the thermodynamic parameters for the process of micelle formation. The process for micelle formation was found to be entropy-driven. The thermogelling behavior of the aqueous polymer solution was studied by (1)H and (13)C NMR spectroscopy at different temperatures. We obtained valuable molecular level information regarding the state of the copolymer in solution based on the variation of the peak widths. Cytotoxicity studies performed on the extracts of the copolymer gel indicate good cell compatibility. Cells attach on the surface of the gel much better than on the commercially available PEG-PPG-PEG triblock copolymer. These studies indicate a potential for the copolymer gel to be used for tissue engineering applications. PMID:19663517

Loh, Xian Jun; Goh, Suat Hong; Li, Jun

2009-09-01

232

Hormonal growth control of cells in culture  

Microsoft Academic Search

Summary  Serum is the last undefined component in cell culture media. Our results indicate that the primary role of serum is to provide\\u000a hormones and that serum can be replaced by a group of hormones. A rat pituitary cell line, GH3, can grow in serum-free medium if the medium is supplemented with 3,3?,5-triiodothyronine, TSH-releasing hormone, transferrin,\\u000a parathyroid hormone, insulin and three

I. Hayashi; J. Larner; G. Sato

1978-01-01

233

Rice suspension cultured cells are evaluated as a model system to study salt responsive networks in plants using a combined proteomic and metabolomic profiling approach.  

PubMed

Salinity is one of the major abiotic stresses affecting plant productivity but surprisingly, a thorough understanding of the salt-responsive networks responsible for sustaining growth and maintaining crop yield remains a significant challenge. Rice suspension culture cells (SCCs), a single cell type, were evaluated as a model system as they provide a ready source of a homogenous cell type and avoid the complications of multicellular tissue types in planta. A combination of growth performance, and transcriptional analyses using known salt-induced genes was performed on control and 100 mM NaCl cultured cells to validate the biological system. Protein profiling was conducted using both DIGE- and iTRAQ-based proteomics approaches. In total, 106 proteins were identified in DIGE experiments and 521 proteins in iTRAQ experiments with 58 proteins common to both approaches. Metabolomic analysis provided insights into both developmental changes and salt-induced changes of rice SCCs at the metabolite level; 134 known metabolites were identified, including 30 amines and amides, 40 organic acids, 40 sugars, sugar acids and sugar alcohols, 21 fatty acids and sterols, and 3 miscellaneous compounds. Our results from proteomic and metabolomic studies indicate that the salt-responsive networks of rice SCCs are extremely complex and share some similarities with thee cellular responses observed in planta. For instance, carbohydrate and energy metabolism pathways, redox signaling pathways, auxin/indole-3-acetic acid pathways and biosynthesis pathways for osmoprotectants are all salt responsive in SCCs enabling cells to maintain cellular function under stress condition. These data are discussed in the context of our understanding of in planta salt-responses. PMID:23661342

Liu, Dawei; Ford, Kristina L; Roessner, Ute; Natera, Siria; Cassin, Andrew M; Patterson, John H; Bacic, Antony

2013-06-01

234

How are pluripotent cells captured in culture?  

E-print Network

tetracyclin (Tet) inducible Oct3/4 transgene. Tet addition into the culture 1 media results in rapid loss of Oct3/4 protein and differentiation into 2 trophectoderm cells through de-repressing the Cdx2 and Eomesodermin 3 genes [78]. 4 5 5.2 Sox2 6 Sox2 is a...

Kinoshita, Masaki

2014-01-01

235

Phenotypic diversity in cultured cerebral microvascular endothelial cells  

Microsoft Academic Search

Summary  Diversity exists in both the structure and function of the endothelial cells (EC) that comprise the microvasculature of different\\u000a organs. Studies of EC have been aided by our ability to first isolate and subsequently establish cultures from microvascularized\\u000a tissue. After the isolation of microvessel endothelial cells (MEC) derived from rat cerebrum, we observed morphologic differences\\u000a in colonies of cells that

Maria A. Rupnick; Andrew Carey; Stuart K. Williams

1988-01-01

236

Cell culturing of Caenorhabditis elegans glial cells for the assessment of cytosolic Ca²? dynamics.  

PubMed

Cell culture has emerged as an important research method for studying various types of primary cells, including neurons and glial cells. This method has been especially instrumental in assessing intracellular Ca(2+) dynamics of neural cells. The invertebrate model organism Caenorhabditis elegans has been extensively used in neurobiology to study wide-spread issues ranging from gene regulation to behavior. We present some of the basic morphological characteristics of the four C. elegans glial cells residing in the cephalic sensilla of the worm, followed by a description of cell culturing methods for these glial cells. We describe the combined genetic and fluorescence microscopy approaches for identification of C. elegans glial cells in culture and assessment of their cytosolic Ca(2+) dynamics. PMID:22144307

Stout, Randy F; Parpura, Vladimir

2012-01-01

237

Chinese Cultural Studies: Bibliographical Guide  

NSDL National Science Digital Library

This site offers a number of references of use to Asian Studies scholars. Created by Paul Halsall (formerly of Brooklyn College), it is organized by subject, including Classic Chinese Sources in Translation, Gender, Chinese Women, Philosophy and Religion, and Movies Addressing Chinese History and Culture. Historical texts are divided into periods, from the Zhou Dynasty to Post-Mao China. A Guide to Chinese Philosophical Texts for Inclusion in Introductory Courses is also featured.

Halsall, Paul.

1997-01-01

238

Establishing a stem cell culture laboratory for clinical trials  

PubMed Central

Adult stem/progenitor cells are found in different human tissues. An in vitro cell culture is needed for their isolation or for their expansion when they are not available in a sufficient quantity to regenerate damaged organs and tissues. The level of complexity of these new technologies requires adequate facilities, qualified personnel with experience in cell culture techniques, assessment of quality and clear protocols for cell production. The rules for the implementation of cell therapy centers involve national and international standards of good manufacturing practices. However, such standards are not uniform, reflecting the diversity of technical and scientific development. Here standards from the United States, the European Union and Brazil are analyzed. Moreover, practical solutions encountered for the implementation of a cell therapy center appropriate for the preparation and supply of cultured cells for clinical studies are described. Development stages involved the planning and preparation of the project, the construction of the facility, standardization of laboratory procedures and development of systems to prevent cross contamination. Combining the theoretical knowledge of research centers involved in the study of cells with the practical experience of blood therapy services that manage structures for cell transplantation is presented as the best potential for synergy to meet the demands to implement cell therapy centers. PMID:23049427

Sekiya, Eliseo Joji; Forte, Andresa; Kuhn, Telma Ingrid Borges de Bellis; Janz, Felipe; Bydlowski, Sergio Paulo; Alves, Adelson

2012-01-01

239

Cell Culture Assay for Human Noroviruses [response  

SciTech Connect

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

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

240

Boron Deficiency in Cultured Pine Cells 1  

PubMed Central

A pronounced interaction between calcium, magnesium, and boron was found in growth studies with Pinus radiata cell cultures. Quantitative isoactivity data for the interaction was analyzed in terms of selected simple and plausible theoretical models. The data was found to be consistent with a model in which a critical acceptor molecule is activated only by binding both Ca and B at separate sites; Mg competitively displaces Ca to inactivate the acceptor. It was found that B is, surprisingly, not bound strongly (Kdiss = 450 ± 80 micromolar) and that the affinity for Ca is two orders of magnitude stronger than for Mg. Therefore only a small proportion of the acceptor will be boronated under natural conditions. Moderate levels of mannitol were found to aggravate B deficiency due to its effective removal by direct chemical complexation. At higher concentrations of mannitol (or other sugars), where osmotic contribution is significant, little B was needed to overcome growth inhibition—a result consistent with B having a primary role in cell wall biosynthesis. PMID:16667559

Teasdale, Robert Dixon; Richards, Dianne Katherine

1990-01-01

241

Metabolic measurements in cell culture and tissue constructs  

NASA Astrophysics Data System (ADS)

This paper concerns the study and use of biological cells in which there is a need for sensors and assemblies for the measurement of a diverse range of physical and chemical variables. In this field cell culture is used for basic research and for applications such as protein and drug synthesis, and in cell, tissue and organ engineering. Metabolic processes are fundamental to cell behaviour and must therefore be monitored reliably. Basic metabolic studies measure the transport of oxygen, glucose, carbon dioxide, lactic acid to, from, or within cells, whilst more advanced research requires examination of energy storage and utilisation. Assemblies are designed to incorporate bioreactor functions for cell culture together with appropriate sensing devices. Oxygen consumption by populations of cells is achieved in a flowthrough assembly that incorporates O2 micro-sensors based on either amperometry or fluorescence. Measurements in single cell are possible with intra-cellular fluorophores acting as biosensors together with optical stimulation and detection. Near infra-red spectroscopy (NIRS) is used for analysis within culture fluid, for example for estimation of glucose levels, as well as within cell populations, for example to study the respiratory enzymes.Â#

Rolfe, P.

2008-10-01

242

Scalable GMP compliant suspension culture system for human ES cells.  

PubMed

Suspension bioreactors are an attractive alternative to static culture of human embryonic stem cells (hESCs) for the generation of clinically relevant cell numbers in a controlled system. In this study, we have developed a scalable suspension culture system using serum-free defined media with spinner flasks for hESC expansion as cell aggregates. With optimized cell seeding density and splitting interval, we demonstrate prolonged passaging and expansion of several hESC lines with overall expansion, yield, viability and maintenance of pluripotency equivalent to adherent culture. Human ESCs maintained in suspension as aggregates can be passaged at least 20 times to achieve over 1×10(13) fold calculated expansion with high undifferentiation rate and normal karyotype. Furthermore, the aggregates are able to differentiate to cardiomyocytes in a directed fashion. Finally, we show that the cells can be cryopreserved in serum-free medium and thawed into adherent or suspension cultures to continue passaging and expansion. We have successfully used this method under cGMP or cGMP-equivalent conditions to generate cell banks of several hESC lines. Taken together, our suspension culture system provides a powerful approach for scale-up expansion of hESCs under defined and serum-free conditions for clinical and research applications. PMID:22459095

Chen, Vincent C; Couture, Sylvana M; Ye, Jingjing; Lin, Ziguang; Hua, Giau; Huang, Hsiao-I P; Wu, Jun; Hsu, David; Carpenter, Melissa K; Couture, Larry A

2012-05-01

243

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

NASA Technical Reports Server (NTRS)

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-based investigations simulating the conditions expected in the flight experiment. Several parameters including cell concentration, time between cell loading and activation, and storage temperature on cell survival were examined to characterise cell response and optimise the experiments to be flown aboard the Space Shuttle. Results indicate that the objectives of the experiments could be met with delays up to 5 days between cell loading into the hardware and initial in flight experiment activation, without the need for medium exchange. Experiment hardware of this kind, which is adaptable to a wide range of cell types and can be easily interfaced to different spacecraft facilities, offers the possibility for a wide range of experimenters successfully and easily to utilise future flight opportunities.

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

1998-01-01

244

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

PubMed

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-based investigations simulating the conditions expected in the flight experiment. Several parameters including cell concentration, time between cell loading and activation, and storage temperature on cell survival were examined to characterise cell response and optimise the experiments to be flown aboard the Space Shuttle. Results indicate that the objectives of the experiments could be met with delays up to 5 days between cell loading into the hardware and initial in flight experiment activation, without the need for medium exchange. Experiment hardware of this kind, which is adaptable to a wide range of cell types and can be easily interfaced to different spacecraft facilities, offers the possibility for a wide range of experimenters successfully and easily to utilise future flight opportunities. PMID:9671231

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

1998-08-01

245

Novel Micropatterned Cardiac Cell Cultures with Realistic Ventricular Microstructure  

PubMed Central

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

Badie, Nima; Bursac, Nenad

2009-01-01

246

Microinjection of Single Cells in Culture Louise Cramer December 1995  

E-print Network

, then 1x with culture media. 3. Plate cells. #12;Observation Media To observe cells on a microscope we use a media optimal for preserving cell health. Maintaining pH outside of a tissue culture incubator is mostMicroinjection of Single Cells in Culture Louise Cramer December 1995 Introduction We typically

Mitchison, Tim

247

Ten commandments for preventing contamination of primary cell cultures  

Microsoft Academic Search

Procedures for preventing contamination in primary cell cultures must be carefully defined and strictly followed in order to obtain healthy cells. Protocols have been developed and refined in our laboratory for establishing primary cultures of muscle and fat stem cells without contamination from a variety of animals. Contamination of cell cultures is not only frustrating, but is also very expensive

Janet L. Vierck; Katherine Byrne; Priya S. Mir; Michael V. Dodson

2000-01-01

248

Plant cell cultures: bioreactors for industrial production.  

PubMed

The recent biotechnology boom has triggered increased interest in plant cell cultures, since a number of firms and academic institutions investigated intensively to rise the production of very promising bioactive compounds. In alternative to wild collection or plant cultivation, the production of useful and valuable secondary metabolites in large bioreactors is an attractive proposal; it should contribute significantly to future attempts to preserve global biodiversity and alleviate associated ecological problems. The advantages of such processes include the controlled production according to demand and a reduced man work requirement. Plant cells have been grown in different shape bioreactors, however, there are a variety of problems to be solved before this technology can be adopted on a wide scale for the production of useful plant secondary metabolites. There are different factors affecting the culture growth and secondary metabolite production in bioreactors: the gaseous atmosphere, oxygen supply and CO2 exchange, pH, minerals, carbohydrates, growth regulators, the liquid medium rheology and cell density. Moreover agitation systems and sterilization conditions may negatively influence the whole process. Many types ofbioreactors have been successfully used for cultivating transformed root cultures, depending on both different aeration system and nutrient supply. Several examples of medicinal and aromatic plant cultures were here summarized for the scale up cultivation in bioreactors. PMID:21520713

Ruffoni, Barbara; Pistelli, Laura; Bertoli, Alessandra; Pistelli, Luisa

2010-01-01

249

Region Specific Response of Intervertebral Disc Cells to Complex Dynamic Loading: An Organ Culture Study Using a Dynamic Torsion-Compression Bioreactor  

PubMed Central

The spine is routinely subjected to repetitive complex loading consisting of axial compression, torsion, flexion and extension. Mechanical loading is one of the important causes of spinal diseases, including disc herniation and disc degeneration. It is known that static and dynamic compression can lead to progressive disc degeneration, but little is known about the mechanobiology of the disc subjected to combined dynamic compression and torsion. Therefore, the purpose of this study was to compare the mechanobiology of the intervertebral disc when subjected to combined dynamic compression and axial torsion or pure dynamic compression or axial torsion using organ culture. We applied four different loading modalities [1. control: no loading (NL), 2. cyclic compression (CC), 3. cyclic torsion (CT), and 4. combined cyclic compression and torsion (CCT)] on bovine caudal disc explants using our custom made dynamic loading bioreactor for disc organ culture. Loads were applied for 8 h/day and continued for 14 days, all at a physiological magnitude and frequency. Our results provided strong evidence that complex loading induced a stronger degree of disc degeneration compared to one degree of freedom loading. In the CCT group, less than 10% nucleus pulposus (NP) cells survived the 14 days of loading, while cell viabilities were maintained above 70% in the NP of all the other three groups and in the annulus fibrosus (AF) of all the groups. Gene expression analysis revealed a strong up-regulation in matrix genes and matrix remodeling genes in the AF of the CCT group. Cell apoptotic activity and glycosaminoglycan content were also quantified but there were no statistically significant differences found. Cell morphology in the NP of the CCT was changed, as shown by histological evaluation. Our results stress the importance of complex loading on the initiation and progression of disc degeneration. PMID:24013824

Chan, Samantha C. W.; Walser, Jochen; Kappeli, Patrick; Shamsollahi, Mohammad Javad; Ferguson, Stephen J.; Gantenbein-Ritter, Benjamin

2013-01-01

250

Teaching Culture. The Long Revolution in Cultural Studies.  

ERIC Educational Resources Information Center

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

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

251

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

PubMed

An imbalance in medium osmolarity is a determinant that affects cell culture longevity. Even in humidified incubators, evaporation of water leads to a gradual increase in osmolarity over time. We present a simple replica-moulding strategy for producing self-sealing lids adaptable to standard, small-size cell-culture vessels. They are made of polydimethylsiloxane (PDMS), a flexible, transparent and biocompatible material, which is gas-permeable but largely impermeable to water. Keeping cell cultures in a humidified 5% CO2 incubator at 37 degrees C, medium osmolarity increased by +6.86 mosmol/kg/day in standard 35 mm Petri dishes, while PDMS lids attenuated its rise by a factor of four to changes of +1.72 mosmol/kg/ day. Depending on the lid membrane thickness,pH drifts at ambient CO2 levels were attenuated by a factor of 4 to 9. Comparative evaporation studies at temperatures below 60 degrees C yielded a 10-fold reduced water vapour flux of 1.75 g/day/ dm 2 through PDMS lids as compared with 18.69 g/day/dm 2 with conventional Petri dishes. Using such PDMS lids,about 2/3 of the cell cultures grew longer than 30 days in vitro. Among these,the average survival time was 69 days with the longest survival being 284 days under otherwise conventional cell culture conditions. PMID:19430119

Blau, Axel; Neumann, Tanja; Ziegler, Christiane; Benfenati, Fabio

2009-03-01

252

Specimen Sample Preservation for Cell and Tissue Cultures  

NASA Technical Reports Server (NTRS)

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

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

1996-01-01

253

Preparation of neuronal co-cultures with single cell precision.  

PubMed

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (<10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (>85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision. PMID:24894871

Dinh, Ngoc-Duy; Chiang, Ya-Yu; Hardelauf, Heike; Waide, Sarah; Janasek, Dirk; West, Jonathan

2014-01-01

254

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

PubMed Central

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

2014-01-01

255

Derivation of Pluripotent Stem Cells from Cultured Human Primordial Germ Cells  

Microsoft Academic Search

Human pluripotent stem cells would be invaluable for in vitro studies of aspects of human embryogenesis. With the goal of establishing pluripotent stem cell lines, gonadal ridges and mesenteries containing primordial germ cells (PGCs, 5-9 weeks postfertilization) were cultured on mouse STO fibroblast feeder layers in the presence of human recombinant leukemia inhibitory factor, human recombinant basic fibroblast growth factor,

Michael J. Shamblott; Joyce Axelman; Shunping Wang; Elizabeth M. Bugg; John W. Littlefield; Peter J. Donovan; Paul D. Blumenthal; George R. Huggins; John D. Gearhart

1998-01-01

256

Characterization of cultured multipotent zebrafish neural crest cells.  

PubMed

The neural crest is a unique cell population associated with vertebrate evolution. Neural crest cells (NCCs) are characterized by their multipotent and migratory potentials. While zebrafish is a powerful genetic model organism, the isolation and culture of zebrafish NCCs would provide a useful adjunct to fully interrogate the genetic networks that regulate NCC development. Here we report for the first time the isolation, in vitro culture, and characterization of NCCs from zebrafish embryos. NCCs were isolated from transgenic sox10:egfp embryos using fluorescence activated cell sorting and cultured in complex culture medium without feeder layers. NCC multilineage differentiation was determined by immunocytochemistry and real-time qPCR, cell migration was assessed by wound healing assay, and the proliferation index was calculated by immunostaining against the mitosis marker phospho-histone H3. Cultured NCCs expressed major neural crest lineage markers such as sox10, sox9a, hnk1, p75, dlx2a, and pax3, and the pluripotency markers c-myc and klf4. We showed that the cultured NCCs can be differentiated into multiple neural crest lineages, contributing to neurons, glial cells, smooth muscle cells, melanocytes, and chondrocytes. We applied the NCC in vitro model to study the effect of retinoic acid on NCC development. We showed that retinoic acid had a profound effect on NCC morphology and differentiation, significantly inhibited proliferation and enhanced cell migration. The availability of high numbers of NCCs and reproducible functional assays offers new opportunities for mechanistic studies of neural crest development, in genetic and chemical biology applications. PMID:24326414

Kinikoglu, Beste; Kong, Yawei; Liao, Eric C

2014-02-01

257

Studies of transepithelial Cl- transport in cultured cauda epididymal cells of rats by the short-circuit current method.  

PubMed Central

1. Monolayer cultures of cauda epididymides from male Sprague-Dawley rats (210-230 g) were studied by the short-circuit current (ISC) technique to characterize the properties of the transepithelial chloride transport. In HCO(3-)-free, HEPES-buffered solution, adrenaline (0.23 microM) added to the basolateral side led to an increase in ISC and transepithelial conductance (gt). 2. Decreasing apical chloride concentration ([Cl-]a) progressively from 126.7 to 0 mM by substituting chloride with gluconate increased the ISC response to adrenaline (delta ISC) in a linear fashion with a slope of -1.6 x 10(-3) mu equiv h-1 cm-2 per millimolar change in [Cl-]a. Pretreating the tissue with a chloride channel blocker diphenylamine-2-carboxylate (DPC) on the apical side significantly reduced the slope to -4.9 x 10(-4) mu equiv h-1 cm-2 per millimolar change in [Cl-]a. 3. By substituting apical chloride with various anions and measuring the change in ISC upon adrenaline stimulation, the selectivity sequence of the apical anion conductance was found to be NO3- approximately Br- > Cl- > I- > gluconate > isethionate. 4. When the monolayers were bathed with Krebs-Henseleit solution containing 25 mM HCO3- and 5% CO2, the delta ISC at each [Cl-]a as well as the dependence of delta ISC on [Cl-]a (slope = -3.3 x 10(-3) mu equiv h-1 cm-2 per millimolar change in [Cl-]a) were significantly greater than the HCO(3-)-free counterpart. Addition of 0.1 mM acetazolamide or 0.5 mM SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) to the basolateral side significantly reduced the effects of HCO3- and CO2. 5. When the tissues were bathed on both sides with HCO(3-)-free, HEPES-buffered solution and were clamped at various transepithelial potential differences (PDt) from +30 mV (lumen positive) to -30 mV (lumen negative), the relationship between the clamping current response to adrenaline (delta ICL) and the PDt applied was linear. Zero clamping current response was found at -6 mV. Decreasing [Cl-]a to 0 mM reduced the dependence of delta ICL on PDt and delta ICL was positive at all PDt tested. The response of the transepithelial conductance to adrenaline (delta gt) did not depend on the PDt applied but was reduced with decreasing apical chloride concentration.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1297839

Leung, A Y; Wong, P Y

1992-01-01

258

The birth of therapy with cultured cells.  

PubMed

Long ago, I set out to solve a problem, but something happened along the way: I was diverted by an unexpected observation. Thereafter, the direction of my research was guided at each stage by increasing familiarity with the experimental material and what could be done with it. The result was the birth of therapy with cultured keratinocytes. Subsequent developments soon led to the formation of the company Biosurface Technology (later taken over by the Genzyme Corporation), which provided autologous cultures for burn victims in many parts of the world. Further progress by others led to new therapeutic applications of cultured keratinocytes, such as treatment of an ocular disease and gene therapy. Unfortunately, there have developed serious regulatory problems that are a danger to future progress. As described in this brief history, the initial stages of development of cell therapy for the treatment of human disease were possible only because there was no restraint by committees or governmental regulations. PMID:18693268

Green, Howard

2008-09-01

259

Gene Delivery Through Cell Culture Substrate Adsorbed DNA Complexes  

PubMed Central

Efficient gene delivery is a fundamental goal of biotechnology and has numerous applications in both basic and applied science. Substrate-mediated delivery and reverse transfection enhance gene transfer by increasing the concentration of DNA in the cellular microenvironment through immobilizing a plasmid to a cell culture substrate prior to cell seeding. In this report, we examine gene delivery of plasmids that were complexed with cationic polymers (polyplexes) or lipids (lipoplexes) and subsequently immobilized to cell culture or biomaterial substrates by adsorption. Polyplexes and lipoplexes were adsorbed to either tissue culture polystyrene or serum-adsorbed tissue culture polystyrene. The quantity of DNA immobilized increased with time of exposure, and the deposition rate and final amount deposited depended upon the properties of the substrate and complex. For polyplexes, serum modification enhanced reporter gene expression up to 1500-fold relative to unmodified substrates and yielded equivalent or greater expression compared to bolus delivery. For lipoplexes, serum modification significantly increased the number of transfected cells relative to unmodified substrates yet provided similar levels of expression. Immobilized complexes transfect primary cells with improved cellular viability relative to bolus delivery. Finally, this substrate-mediated delivery approach was extended to a widely used biomaterial, poly(lactide-co-glycolide). Immobilization of DNA complexes to tissue culture polystyrene substrates can be a useful tool for enhancing gene delivery for in vitro studies. Additionally, adapting this system to biomaterials may facilitate application to fields such as tissue engineering. PMID:15800863

Bengali, Zain; Pannier, Angela K.; Segura, Tatiana; Anderson, Brian C.; Jang, Jae-Hyung; Mustoe, Thomas A.; Shea, Lonnie D.

2008-01-01

260

Eradication of Mycoplasma contaminations from cell cultures.  

PubMed

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

Uphoff, Cord C; Drexler, Hans G

2014-01-01

261

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

PubMed Central

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

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

2014-01-01

262

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

PubMed

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

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

2014-04-01

263

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

Microsoft Academic Search

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

I. Hakman; L. C. Fowke

1987-01-01

264

Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices.  

PubMed

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. PMID:25105943

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

2015-01-15

265

Cell population evolution in tissue cultures from embryo barley ( Hordeum vulgare L.) after caffeine treatment  

Microsoft Academic Search

Summary Over a period of time a comparative study of the evolution of the different number of nuclei as well as the different chromosome number in the cells of severalin vitro tissue cultures of barley (Hordeum vulgare L.) was carried out. The cultures were obtained by culturing embryos in a control culture medium or in a media supplemented with 0.5

M. L. Ruiz; A. M. VikzQurz

1981-01-01

266

Oxygen consumption of human heart cells in monolayer culture.  

PubMed

Tissue engineering in cardiovascular regenerative therapy requires the development of an efficient oxygen supply system for cell cultures. However, there are few studies which have examined human cardiomyocytes in terms of oxygen consumption and metabolism in culture. We developed an oxygen measurement system equipped with an oxygen microelectrode sensor and estimated the oxygen consumption rates (OCRs) by using the oxygen concentration profiles in culture medium. The heart is largely made up of cardiomyocytes, cardiac fibroblasts, and cardiac endothelial cells. Therefore, we measured the oxygen consumption of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), cardiac fibroblasts, human cardiac microvascular endothelial cell and aortic smooth muscle cells. Then we made correlations with their metabolisms. In hiPSC-CMs, the value of the OCR was 0.71±0.38pmol/h/cell, whereas the glucose consumption rate and lactate production rate were 0.77±0.32pmol/h/cell and 1.61±0.70pmol/h/cell, respectively. These values differed significantly from those of the other cells in human heart. The metabolism of the cells that constitute human heart showed the molar ratio of lactate production to glucose consumption (L/G ratio) that ranged between 1.97 and 2.2. Although the energy metabolism in adult heart in vivo is reported to be aerobic, our data demonstrated a dominance of anaerobic glycolysis in an in vitro environment. With our measuring system, we clearly showed the differences in the metabolism of cells between in vivo and in vitro monolayer culture. Our results regarding cell OCRs and metabolism may be useful for future tissue engineering of human heart. PMID:25218502

Sekine, Kaori; Kagawa, Yuki; Maeyama, Erina; Ota, Hiroki; Haraguchi, Yuji; Matsuura, Katsuhisa; Shimizu, Tatsuya

2014-09-26

267

Behaviour of cell cultures from human amniotic fluid  

Microsoft Academic Search

The growth pattern of cell cultures originating from 11 amniotic fluid specimens have been observed. From each specimen 2 to 12 primary cultures were set up. In most cases growth started simultaneously in the primary cultures originating from one sample. The primary cultures lasted from 7 to 30 days. A variation was found both between cultures from different pregnancies as

L Hasholt

1976-01-01

268

Regulation of expression of glucose transporters by glucose: a review of studies in vivo and in cell cultures  

Microsoft Academic Search

Glucose transporters are membrane- embedded proteins that mediate the uptake of glucose from the surrounding medium into the cell. Glucose is the main fuel for most cells, and its uptake is rate-limiting for glucose utilization. For this reason, it is expected that glu- cose transport is tightly regulated. Whereas rapid regula- tion of glucose transporters by hormones has been known

AMIRA KLIP; THEODOROS TSAKIRIDIS; S ANDRE MARETTE; PHILLIP A. ORTIZ

269

21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2012 CFR

... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a)...

2012-04-01

270

21 CFR 864.2280 - Cultured animal and human cells.  

... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a)...

2014-04-01

271

21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2013 CFR

... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a)...

2013-04-01

272

Primary porcine brain microvessel endothelial cell isolation and culture.  

PubMed

Cell culture models of the blood-brain barrier (BBB) are useful tools to study the functionality of the BBB in health and disease. Several good in vitro BBB models are available from different species. However, most brain endothelial cells lose some of their in vivo BBB phenotype in culture. Porcine brain endothelial cells (PBECs) tend to retain most of their in vivo BBB characteristics and usually give higher transendothelial electrical resistance (TEER, representing functional well-developed tight junctions) compared to brain endothelial cells from other species. The protocol described in this unit gives detailed instructions for isolation and culture of PBECs from fresh porcine brains. This porcine BBB model generates high TEER without the need for co-culture with astrocytes. However, astrocyte-derived factors can be introduced to the system through the use of astrocyte-conditioned medium or co-culture with astrocytes, which may be necessary for further enhancing the BBB phenotype for certain complex studies. Curr. Protoc. Neurosci. 69:3.27.1-3.27.17. © 2014 by John Wiley & Sons, Inc. PMID:25297692

Patabendige, Adjanie; Abbott, N Joan

2014-01-01

273

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

NASA Technical Reports Server (NTRS)

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.

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

2000-01-01

274

Biotransformation of salicylaldehyde to salicin using Varthemia persica cell suspension cultures  

Microsoft Academic Search

Cell cultures of Varthemia persica DC. have been studied to evaluate their abilities in biotransformation of aromatic and aliphatic precursors. V. Persica (Asteraceae) is an aromatic plant growing in Iran. V. persica contain different terpens but its cell culture does not posses these compounds. Callus cultures of V. persica was established from seedlings and healthy suspensions were grown using Murashige

Gholamreza Asghari; Maryam Mosaeybi

275

Convenient three-dimensional cell culture in supermolecular hydrogels.  

PubMed

A convenient three-dimensional cell culture was developed by employing high swelling property of hybrid hydrogels coassembled from C2-phenyl-based supermolecular gelators and sodium hyaluronate. Imaging and spectroscopic analysis by scanning electron microscopy (SEM), atomic force microscopy (AFM), transform infrared (FT-IR) spectra confirm that the hybrid gelators can self-assemble into nanofibrous hydrogel. The high swelling property of dried gel ensures cell migration and proliferation inside bulk of the hydrogels, which provides a facial method to study disease models, the effect of drug dosages, and tissue culture in vitro. PMID:24802591

Li, Ping; Yin, Zongqi; Dou, Xiao-Qiu; Zhou, Guangdong; Feng, Chuan-Liang

2014-05-28

276

Natural products from plant cell cultures  

Microsoft Academic Search

Plants produce complex small molecules — natural products — that exhibit anticancer, antimalarial and antimicrobial activity.\\u000a These molecules play a key role in human medicine. However, plants typically produce these compounds in low quantities, and\\u000a harvesting plant natural products is frequently expensive, time-consuming and environmentally damaging. Plant cell culture\\u000a provides a renewable, easily scalable source of plant material. In this

Elizabeth McCoy; Sarah E. O’Connor

277

Striatal interneurons in dissociated cell culture  

Microsoft Academic Search

In addition to the well-characterized direct and indirect projection neurons there are four major interneuron types in the\\u000a striatum. Three contain GABA and either parvalbumin, calretinin or NOS\\/NPY\\/somatostatin. The fourth is cholinergic. It might\\u000a be assumed that dissociated cell cultures of striatum (typically from embryonic day E18.5 in rat and E14.5 for mouse) contain\\u000a each of these neuronal types. However,

S. C. Schock; K. S. Jolin-Dahel; P. C. Schock; W. A. Staines; M. Garcia-Munoz; Gordon W. Arbuthnott

2010-01-01

278

Finite Element Analyses of Fluid Flow Conditions in Cell Culture  

PubMed Central

Numerous studies in tissue engineering and biomechanics use fluid flow stimulation, both unidirectional and oscillatory, to analyze the effects of shear stresses on cell behavior. However, it has typically been assumed that these shear stresses are uniform and that cell and substrate properties do not adversely affect these assumptions. With the increasing utilization of fluid flow in cell biology, it would be beneficial to determine the validity of various experimental protocols. Because it is difficult to determine the velocity profiles and shear stresses empirically, we used the finite element method (FEM). Using FEM, we determined the effects of cell confluence on fluid flow, the effects of cell height on the uniformity of shear stresses, apparent shear stresses exhibited by cells cultured on various substrates, and the effects of oscillatory fluid flow relative to the unidirectional flow. FEM analyses could successfully analyze flow patterns over cells for various cell confluence and shape and substrate characteristics. Our data suggest the benefits of the utilization of oscillatory fluid flow and the use of substrates that stimulate cell spreading in the distribution of more uniform shear stresses across the surface of cells. Also we demonstrated that the cells cultured on nanotopographies were exposed to greater apparent shear stresses than cells on flat controls when using the same fluid flow conditions. FEM thus provides an excellent tool for the development of experimental protocols and the design of bioreactor systems. PMID:19778171

Salvi, Joshua D.; Lim, Jung Yul

2010-01-01

279

Long-Term Culture of Capillary Endothelial Cells  

Microsoft Academic Search

Capillary endothelial cells from rats, calves, and humans, have been carried in long-term culture. Bovine capillary endothelial cells have been cloned and maintained by serial passage for longer than 8 months. This prolonged culture was accomplished by using tumor-conditioned medium, gelatin-coated plates, and a method of enriching cells in primary culture. Cultured bovine capillary endothelial cells produce Factor VIII antigen

Judah Folkman; Christian C. Haudenschild; Bruce R. Zetter

1979-01-01

280

Recombinant protein production and insect cell culture and process  

NASA Technical Reports Server (NTRS)

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 the cultured insect cells as host for a virus encoding the described polypeptide such as baculovirus. The insect cells can also be a host for viral production.

Spaulding, Glenn (inventor); Prewett, Tacey (inventor); Goodwin, Thomas (inventor); Francis, Karen (inventor); Andrews, Angela (inventor); Oconnor, Kim (inventor)

1993-01-01

281

Perfusion enhanced polydimethylsiloxane based scaffold cell culturing system for multi-well drug screening platform.  

PubMed

Conventional two-dimensional cultures in monolayer and sandwich configuration have been used as a model for in vitro drug testing. However, these culture configurations do not present the actual in vivo liver cytoarchitecture for the hepatocytes cultures and thus they may compromise the cells liver-specific functions and their cuboidal morphology over longer term culture. In this study, we present a three-dimensional polydimethylsiloxane (PDMS) scaffold with interconnected spherical macropores for the culturing of rat liver cells (hepatocytes). The scaffolds were integrated into our perfusion enhanced bioreactor to improve the nutrients and gas supply for cell cultures. The liver-specific functions of the cell culture were assessed by their albumin and urea production, and the changes in the cell morphology were tracked by immunofluorescence staining over 9 days of culture period. N-Acetyl-Para-Amino-Phenol (acetaminophen) was used as drug model to investigate the response of cells to drug in our scaffold-bioreactor system. Our experimental results revealed that the perfusion enhanced PDMS-based scaffold system provides a more conducive microenvironment with better cell-to-cell contacts among the hepatocytes that maintains the culture specific enzymatic functions and their cuboidal morphology during the culturing period. The numerical simulation results further showed improved oxygen distribution within the culturing chamber with the scaffold providing an additional function of shielding the cell cultures from the potentially detrimental fluid induced shear stresses. In conclusion, this study could serve a crucial role as a platform for future preclinical hepatotoxicity testing. PMID:24399780

Tania, Marshella; Hsu, Myat Noe; Png, Si Ning; Leo, Hwa Liang; Toh, Guoyang William; Birgersson, Erik

2014-01-01

282

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

NASA Astrophysics Data System (ADS)

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

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

2014-08-01

283

Microfluidic-based 3D cell culture for studies of biophysical and biochemical regulation of endothelial function  

E-print Network

New and more biologically relevant in vitro models are needed for use in drug development, regenerative medicine, and fundamental scientific investigations. The ultimate challenge lies in replicating the native cell/tissue ...

Vickerman, Vernella V. V. (Vernella Velonie Verlin)

2012-01-01

284

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

DOEpatents

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.

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

2000-01-01

285

An Introductory Undergraduate Course Covering Animal Cell Culture Techniques  

ERIC Educational Resources Information Center

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

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

2004-01-01

286

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

PubMed Central

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

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

2014-01-01

287

Poverty (lab) oratory: Rastafari and cultural studies  

Microsoft Academic Search

This paper presents Rastafari Experience in Jamaica as one of the first cultural studies projects. This cultural studies project is located as originating in the 1930s in Kingston and in 1960 within the University College of the West Indies. It is argued that the Rastafari approach was demonstrative of a faculty of cultural studies at work – its members being

Jalani Niaah

2003-01-01

288

Culture of cells from two life stages of Schistosoma mansoni  

Microsoft Academic Search

The ability to culture continuously proliferating cell lines of various organisms in vitro has provided numerous advantages in experimental approaches toward the understanding of basic biology and disease. Although in vitro approaches are common in many disciplines, this methodology has proven difficult to exploit in the study of helminthic parasites. A major cause of parasitic disease, particularly in tropical countries,

Christopher J. Bayne; David W. Barnes

1997-01-01

289

Culturing with trehalose produces viable endothelial cells after cryopreservation.  

PubMed

Dimethylsulfoxide, the most commonly employed cryoprotectant for cells, has well documented cytotoxic effects in patients. Among the compounds available that may provide protection to cells and tissues during preservation with less cytotoxicity is trehalose. Some animals, such as brine shrimp and tardigrades, accumulate trehalose during periods of extreme environmental stress. In this study, experiments were performed to evaluate the effects of culturing a bovine endothelial cell line (ATCC #CCL-209) in the presence of trehalose prior to preservation by freezing. A number of factors were shown to contribute to cell retention of metabolic activity and proliferative potential including cell culture time with trehalose and the solution conditions during cryopreservation. Using an optimized protocol consisting of 24 h of cell culture with 0.2 M trehalose followed by cryopreservation with 0.2-0.4 M trehalose in sodium bicarbonate buffered Eagles minimum essential medium at pH 7.4 resulted in 87±4% post-preservation cell metabolic activity expressed as relative fluorescence based upon reduction of resazurin to resorufin. This new method provides an alternative preservation strategy to the more classical preservation methods employing dimethylsulfoxide available for cells and tissues. PMID:22366172

Campbell, Lia H; Brockbank, Kelvin G M

2012-06-01

290

Cytotoxicity of Voriconazole on Cultured Human Corneal Endothelial Cells?  

PubMed Central

The purpose of the present study was to evaluate the toxicity of voriconazole on cultured human corneal endothelial cells (HCECs). HCECs were cultured and exposed to various concentrations of voriconazole (5.0 to 1,000 ?g/ml). Cell viability was measured using a Cell Counting Kit-8 (CCK-8) and live/dead viability/cytotoxicity assays. Cell damage was assessed using phase-contrast microscopy after 24 h of exposure to voriconazole. To analyze the effect of voriconazole on the intercellular barrier, immunolocalization of zonula occludens 1 (ZO1) was performed. A flow cytometric assay was performed to evaluate the apoptotic and necrotic effects of voriconazole on HCECs. Cytotoxicity tests demonstrated the dose-dependent toxic effect of voriconazole on HCECs. Voriconazole concentrations of ?100 ?g/ml led to a significant reduction in cell viability. The morphological characteristics of HCECs also changed in a dose-dependent manner. Increasing concentrations of voriconazole resulted in fading staining for ZO1. Higher concentrations of voriconazole resulted in an increased number of propidium iodide (PI)-positive cells, indicating activation of the proapoptotic pathway. In conclusion, voriconazole may have a dose-dependent toxic effect on cultured HCECs. The results of this study suggest that although voriconazole concentrations of up to 50 ?g/ml do not decrease cell viability, intracameral voriconazole concentrations of ?100 ?g/ml may increase the risk of corneal endothelial damage. PMID:21768517

Han, Sang Beom; Shin, Young Joo; Hyon, Joon Young; Wee, Won Ryang

2011-01-01

291

Immunocytochemical localization of endothelin in cultured bovine endothelial cells  

Microsoft Academic Search

To investigate the intracellular localization of endothelin in cultured endothelial cells, an immunocytochemical study was carried out by the post-embedding protein A-gold technique with endothelin-specific antiserum. Gold particles were seen on the rough endoplasmic reticulum, the Golgi cisternae, the Golgi vesicles, small vesicles beneath the cell membrane, and the lysosomes. By contrast, no secretory granules were observed. These results suggest

S. Nakamura; M. Naruse; K. Naruse; H. Demura; H. Uemura

1990-01-01

292

Vibrational spectroscopy characterization of low level laser therapy on mammary culture cells: a micro-FTIR study  

NASA Astrophysics Data System (ADS)

Low level laser therapy (LLLT) is an emerging therapeutic approach for several clinical conditions. The clinical effects induced by LLLT presumably go from the photobiostimulation/photobioinibition at cellular level to the molecular level. The detailed mechanism underlying this effect is still obscure. This work is dedicated to quantify some relevant aspects of LLLT related to molecular and cellular variations. This goal was attached by exposing malignant breast cells (MCF7) to spatially filtered light of a He-Ne laser (633 nm) with 28.8 mJ/cm2 of fluency. The cell viability was evaluated by microscopic observation using Trypan Blue viability test. The vibrational spectra of each experimental group (micro- FTIR technique) were used to identify the relevant biochemical alterations occurred due the process. The red light had influence over RNA, phosphate and serine/threonine/tyrosine bands. Light effects on cell number or viability were not detected. However, the irradiation had direct influence on metabolic activity of cells.

Magrini, Taciana D.; Villa dos Santos, Nathalia; Pecora Milazzotto, Marcella; Cerchiaro, Giselle; da Silva Martinho, Herculano

2011-03-01

293

CELL GROWTH IN PLANT CULTURES: AN INTERPRETATION OF THE INFLUENCE OF INITIAL WEIGHT IN CADMIUM AND COPPER TOXICITY TESTS  

EPA Science Inventory

The authors present an approach for conducting and interpreting results of newly established plant cell culture in toxicity studies. xtended culturing produces uniform suspension and facilities sampling. rimary (new) cultures are more representative of all responses of their plan...

294

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

PubMed Central

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

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

2010-01-01

295

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

NASA Astrophysics Data System (ADS)

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

Chang, Jiyoung; Yoon, Sang-Hee; Mofrad, Mohammad R. K.; Lin, Liwei

2011-05-01

296

Three-Dimensional Cultures of Mouse Mammary Epithelial Cells  

PubMed Central

The mammary gland is an ideal “model organism” for studying tissue specificity and gene expression in mammals: it is one of the few organs that develop after birth and it undergoes multiple cycles of growth, differentiation and regression during the animal’s lifetime in preparation for the important function of lactation. The basic “functional differentiation” unit in the gland is the mammary acinus made up of a layer of polarized epithelial cells specialized for milk production surrounded by myoepithelial contractile cells, and the two-layered structure is surrounded by basement membrane. Much knowledge about the regulation of mammary gland development has been acquired from studying the physiology of the gland and of lactation in rodents. Culture studies, however, were hampered by the inability to maintain functional differentiation on conventional tissue culture plastic. We now know that the microenvironment, including the extracellular matrix and tissue architecture, plays a crucial role in directing functional differentiation of organs. Thus, in order for culture systems to be effective experimental models, they need to recapitulate the basic unit of differentiated function in the tissue or organ and to maintain its three-dimensional (3D) structure. Mouse mammary culture models evolved from basic monolayers of cells to an array of complex 3D systems that observe the importance of the microenvironment in dictating proper tissue function and structure. In this chapter, we focus on how 3D mouse mammary epithelial cultures have enabled investigators to gain a better understanding of the organization, development and function of the acinus, and to identify key molecular, structural, and mechanical cues important for maintaining mammary function and architecture. The accompanying chapter of Vidi et al. describes 3D models developed for human cells. Here, we describe how mouse primary epithelial cells and cell lines—essentially those we use in our laboratory—are cultured in relevant 3D microenvironments. We focus on the design of functional assays that enable us to understand the intricate signaling events underlying mammary gland biology, and address the advantages and limitations of the different culture settings. Finally we also discuss how advances in bioengineering tools may help towards the ultimate goal of building tissues and organs in culture for basic research and clinical studies. PMID:23097110

Mroue, Rana; Bissell, Mina J.

2013-01-01

297

Cultural Studies and Classroom Assignments I.  

ERIC Educational Resources Information Center

This paper presents ways in which critical thinking can be taught in a communication studies class using cultural studies. The understanding of culture provided by E. Hall (who stressed the importance of self-reflexivity) and R. Williams (whose theory of culture involves relations between elements in a whole way of life) is useful to communication…

Peterson, Valerie

298

Cultural Studies in the English Classroom.  

ERIC Educational Resources Information Center

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

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

299

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

Microsoft Academic Search

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

Kenji Sato; Mamoru Nakayama; Jun-ichi Shigeta

1996-01-01

300

Ocular surface changes in limbal stem cell deficiency caused by chemical injury: a histologic study of excised pannus from recipients of cultured corneal epithelium  

Microsoft Academic Search

PurposeTo report histopathologic changes of the ocular surface pannus in patients with severe limbal stem cell deficiency (LSCD).MethodsCorneal and conjunctival pannus tissues from 29 patients undergoing ocular reconstruction with cultured limbal cell transplantation were included. The medical records of these patients were reviewed for demographics, aetiologic diagnosis, type of injury, interval between the initial insult and excision of pannus, and

A Fatima; G Iftekhar; V S Sangwan; G K Vemuganti

2008-01-01

301

Microfabricated polyester conical microwells for cell culture applications.  

PubMed

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

Selimovi?, Seila; Piraino, Francesco; Bae, Hojae; Rasponi, Marco; Redaelli, Alberto; Khademhosseini, Ali

2011-07-21

302

Cultural studies of science education  

NASA Astrophysics Data System (ADS)

In response to Stetsenko's [2008, Cultural Studies of Science Education, 3] call for a more unified approach in sociocultural perspectives, this paper traces the origins of the use of sociocultural ideas in New Zealand from the 1970s to the present. Of those New Zealanders working from a sociocultural perspective who responded to our query most had encountered these ideas while overseas. More recently activity theory has been of interest and used in reports of work in early childhood, workplace change in the apple industry, and in-service teacher education. In all these projects the use of activity theory has been useful for understanding how the elements of a system can transform the activity. We end by agreeing with Stetsenko that there needs to be a more concerted approach by those working from a sociocultural perspective to recognise the contribution of others in the field.

Higgins, Joanna; McDonald, Geraldine

2008-07-01

303

Rotating bio-reactor cell culture apparatus  

NASA Technical Reports Server (NTRS)

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

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

1991-01-01

304

Differentiated cultures of primary hamster tracheal airway epithelial cells  

Microsoft Academic Search

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

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

2004-01-01

305

Cultured buffalo umbilical cord matrix cells exhibit characteristics of multipotent mesenchymal stem cells.  

PubMed

Recent findings have demonstrated umbilical cord, previously considered as a biomedical waste, as a source of stem cells with promising therapeutic applications in human as well as livestock species. The present study was carried out to isolate the umbilical cord matrix cells and culture for a prolonged period, cryopreserve these cells and test their post-thaw viability, characterize these cells for expression of stem cell markers and differentiation potential in vitro. The intact umbilical cord was taken out of the amniotic sac of a fetus and then incised longitudinally to remove umbilical vessels. Wharton's jelly containing tissue was diced into small pieces and placed in tiny drops of re-calcified buffalo plasma for establishing their primary culture. Confluent primary culture was trypsinized and passaged with a split ratio of 1:2 for multiplication of cells. Cryopreservation of cells was performed at three different passages in cryopreservation medium containing 15%, 20% and 25% fetal bovine serum (FBS). A significant increase in post-thaw viability was observed in cells cryopreserved in freezing medium with higher concentration of FBS. After re-culturing, frozen-thawed cells started adhering, and spike formation occurred within 4-6 h with similar morphology to their parent representative cultures. The normal karyotype and positive expression of alkaline phosphatase and pluripotency genes OCT4, NANOG and SOX2 were observed at different passages of culture. When induced, these cells differentiated into adipogenic and osteogenic cells as confirmed by oil red O and alizarin red stains, respectively. This study indicates that buffalo umbilical cord matrix cells have stemness properties with mesenchymal lineage restricted differentiation and limited proliferation potential in vitro. PMID:23708916

Singh, Jarnail; Mann, Anita; Kumar, D; Duhan, J S; Yadav, P S

2013-06-01

306

Using Living Radical Polymerization to Enable Facile Incorporation of Materials in Microfluidic Cell Culture Devices  

PubMed Central

High throughput screening tools are expediting cell culture studies with applications in drug discovery and tissue engineering. This contribution demonstrates a method to incorporate 3D cell culture sites into microfluidic devices and enables the fabrication of high throughput screening tools with uniquely addressable culture environments. Contact Lithographic Photopolymerization (CLiPP) was used to fabricate microfluidic devices with two types of 3D culture sites: macroporous rigid polymer cell scaffolds and poly(ethylene glycol) (PEG) encapsulated cell matrices. Cells were cultured on-device with both types of culture sites, demonstrating material cytocompatibility. Multilayer microfluidic devices were fabricated with channels passing the top and bottom sides of a series of rigid porous polymer scaffolds. Cells were seeded and cultured on-device, demonstrating the ability to deliver cells and culture cells on multiple scaffolds along the length of a single channel. Flow control through these rigid porous polymer scaffolds was demonstrated. Finally, devices were modified by grafting of PEG methacrylate from surfaces to prevent non-specific protein adsorption and ultimately cell adhesion to channel surfaces. The living radical component of this CLiPP device fabrication platform enables facile incorporation of 3D culture sites into microfluidic cell culture devices, which can be utilized for high throughput screening of cell material interactions. PMID:18294686

Simms, Helen M.; Bowman, Christopher M.; Anseth, Kristi S.

2008-01-01

307

In vitro culture of embryonic cells from the freshwater prawn Macrobrachium rosenbergii  

Microsoft Academic Search

Studies were carried out to develop cell line cultures from embryonic tissue of Macrobrachium rosenbergii. Good yields of dissociated, uncontaminated, viable cell suspensions were obtained by physical disruption of harvested eggs in the presence of buffered iodophore disinfectant and malachite green. Primary cultures in the form of proliferating foci of cells were readily initiated using a wide range of mammalian

G. N. Frerichs

1996-01-01

308

Culture, Development, and Social Theory: On Cultural Studies and the Place of Culture in Development  

Microsoft Academic Search

Debates in development theory have recently swung back to taking seriously the relationship of culture to development, especially in the face of manifest failures of conventional approaches to economic growth and social transformation. This has happened at a moment when, especially within anthropology, the concept of culture itself is undergoing critical examination, and when cultural studies has emerged as a

John Clammer

2005-01-01

309

Embryonic stem cell derived motoneurons provide a highly sensitive cell culture model for botulinum neurotoxin studies, with implications for high-throughput drug discovery  

PubMed Central

Botulinum neurotoxins (BoNTs) inhibit cholinergic synaptic transmission by specifically cleaving proteins that are crucial for neurotransmitter exocytosis. Due to the lethality of these toxins, there are elevated concerns regarding their possible use as bioterrorism agents. Moreover, their widespread use for cosmetic purposes, and as medical treatments, has increased the potential risk of accidental overdosing and environmental exposure. Hence, there is an urgent need to develop novel modalities to counter BoNT intoxication. Mammalian motoneurons are the main target of BoNTs, however, due to the difficulty and poor efficiency of the procedures required to isolate the cells, they are not suitable for high-throughput drug screening assays. Here, we explored the suitability of embryonic stem (ES) cell-derived motoneurons as a renewable, reproducible, and physiologically relevant system for BoNT studies. We found that the sensitivity of ES-derived motoneurons to BoNT/A intoxication is comparable to that of primary mouse spinal motoneurons. Additionally, we demonstrated that several BoNT/A inhibitors protected SNAP-25, the BoNT/A substrate, in the ES-derived motoneuron system. Furthermore, this system is compatible with immunofluorescence-based high-throughput studies. These data suggest that ES-derived motoneurons provide a highly sensitive system that is amenable to large-scale screenings to rapidly identify and evaluate the biological efficacies of novel therapeutics. PMID:21353660

Kiris, Erkan; Nuss, Jonathan E.; Burnett, James C.; Kota, Krishna P.; Koh, Dawn C.; Wanner, Laura M.; Torres-Melendez, Edna; Gussio, Rick; Tessarollo, Lino; Bavari, Sina

2011-01-01

310

Cardiac Cells Beating in Culture: A Laboratory Exercise  

ERIC Educational Resources Information Center

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…

Weaver, Debora

2007-01-01

311

Adult human aortic cells in primary culture: heterogeneity in shape  

Microsoft Academic Search

Summary Adult human aortic cells have different shapes in situ. To determine whether populations of cultured aortic cells are also polymorphic, a technique for separation of cells from the intimal and medial layers of the human aorta by enzymatic dispersion of the vascular tissue was employed. It was established that aortic cells are polymorphic in primary culture, at least within

Alexander N. Orekhov; Anatoly V. Krushinsky; Elena R. Andreeva; Vadim S. Repin; Vladimir N. Smirnov

1986-01-01

312

A microfluidic localized, multiple cell culture array using vacuum actuated cell seeding: integrated anticancer drug testing.  

PubMed

In this study, we introduced a novel and convenient approach to culture multiple cells in localized arrays of microfluidic chambers using one-step vacuum actuation. In one device, we integrated 8 individually addressable regions of culture chambers, each only requiring one simple vacuum operation to seed cell lines. Four cell lines were seeded in designated regions in one device via sequential injection with high purity (99.9 %-100 %) and cultured for long-term. The on-chip simultaneous culture of HuT 78, Ramos, PC-3 and C166-GFP cells for 48 h was demonstrated with viabilities of 92 %+/-2 %, 94 %+/-4 %, 96 %+/-2 % and 97 %+/-2 %, respectively. The longest culture period for C166-GFP cells in this study was 168 h with a viability of 96 %+/-10 %. Cell proliferation in each individual side channel can be tracked. Mass transport between the main channel and side channels was achieved through diffusion and studied using fluorescein solution. The main advantage of this device is the capability to perform multiple cell-based assays on the same device for better comparative studies. After treating cells with staurosporine or anti-human CD95 for 16 h, the apoptotic cell percentage of HuT 78, CCRF-CEM, PC-3 and Ramos cells were 36 %+/-3 %, 24 %+/-4 %, 12 %+/-2 %, 18 %+/-4 % for staurosporine, and 63 %+/-2 %, 45 %+/-1 %, 3 %+/-3 %, 27 %+/-12 % for anti-human CD95, respectively. With the advantages of enhanced integration, ease of use and fabrication, and flexibility, this device will be suitable for long-term multiple cell monitoring and cell based assays. PMID:23813077

Gao, Yan; Li, Peng; Pappas, Dimitri

2013-12-01

313

High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays.  

PubMed

Heterogeneity in cell populations poses a major obstacle to understanding complex biological processes. Here we present a microfluidic platform containing thousands of nanoliter-scale chambers suitable for live-cell imaging studies of clonal cultures of nonadherent cells with precise control of the conditions, capabilities for in situ immunostaining and recovery of viable cells. We show that this platform mimics conventional cultures in reproducing the responses of various types of primitive mouse hematopoietic cells with retention of their functional properties, as demonstrated by subsequent in vitro and in vivo (transplantation) assays of recovered cells. The automated medium exchange of this system made it possible to define when Steel factor stimulation is first required by adult hematopoietic stem cells in vitro as the point of exit from quiescence. This technology will offer many new avenues to interrogate otherwise inaccessible mechanisms governing mammalian cell growth and fate decisions. PMID:21602799

Lecault, Véronique; Vaninsberghe, Michael; Sekulovic, Sanja; Knapp, David J H F; Wohrer, Stefan; Bowden, William; Viel, Francis; McLaughlin, Thomas; Jarandehei, Asefeh; Miller, Michelle; Falconnet, Didier; White, Adam K; Kent, David G; Copley, Michael R; Taghipour, Fariborz; Eaves, Connie J; Humphries, R Keith; Piret, James M; Hansen, Carl L

2011-07-01

314

RINm5F cell culture on Sephadex derivatives.  

PubMed

Interactions between polystyrene sodium sulfonate and insulin-secreting RINm5F cells have been previously described. When cultured on these microcarriers, cells exhibited normal growth, altered morphology, and inhibition of the insulin secretion was observed. For the sake of comparison, interactions of RINm5F cells with Sephadex derivatives, namely, carboxymethyl Sephadex (CM Seph), benzylaminated CM Seph (CMB Seph), and sulfonated CMB Seph (CMBS Seph), as well Sephadex, were studied. Cells attached poorly and did not spread onto Sephadex and CM Seph microcarriers, but all other characteristics were normal. In contrast, with cells cultured on CMB Seph and CMBS Seph microcarriers cell attachment, morphology, and growth rate were comparable to those of cells grown on classic plastic wells. But, in the latter case, surprisingly, insulin secretion was enhanced. This effect is composition of the microcarriers dependent. The insulin secretion per cell-microcarriers composition relationship suggests a specific interaction between an unknown membrane receptor of RINm5F cells and a composite ligand site made of a combination of different chemical functional groups present at the microcarriers surface. PMID:7691825

Oturan, N; Serne, H; Reach, G; Jozefowicz, M

1993-06-01

315

Cross-cultural study of expressive avatars  

Microsoft Academic Search

Avatars play an important role in international online communities. While certain simple expressions, such as facial emotional expressions are cultural independent, more complex expressions might not be. Therefore we conducted a cross-cultural study to investigate the influence of the users' cultural background (Japanese or Dutch) on their perception of avatar's expressions in terms of perceived arousal and valence. A significant

Christoph Bartneck; Toru Takahashi; Yasuhiro Katagiri

2004-01-01

316

Neonatal rat heart cells cultured in simulated microgravity  

NASA Technical Reports Server (NTRS)

In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two-dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by nonmyocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA-designed High-Aspect Ratio Vessel (HARV) bioreactors provide a low shear environment that allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells cultured in HARVs adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARVs using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar; however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissuelike organization of cardiac cells in vitro.

Akins, R. E.; Schroedl, N. A.; Gonda, S. R.; Hartzell, C. R.

1997-01-01

317

Disposable Bioreactors for Plant Micropropagation and Mass Plant Cell Culture  

NASA Astrophysics Data System (ADS)

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

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

318

Tumor necrosis factor (cachetin) decreases adipose cell differentiation in primary cell culture  

SciTech Connect

Cachetin has been shown to effect gene product expression in the established adipose cell line 3T3-L1. Expression of messenger RNA for lipoprotein lipase is suppressed in cultured adipocytes. The purpose of this study was to determine the effect of Cachetin on adipose cell differentiation in primary cell culture. Stromalvascular cells obtained from the inguinal fat pad of 4-5 week old Sprague-Dawley rats were grown in culture for two weeks. During the proliferative growth phase all cells were grown on the same medium and labelled with /sup 3/H-thymidine. Cachetin treatment (10/sup -6/ to 10/sup -10/ M) was initiated on day 5, the initial phase of preadipocyte differentiation. Adipocytes and stromal cells were separated using density gradient, and /sup 3/H-thymidine was determined for both cell types. Thymidine incorporation into adipose cells was decreased maximally (approx. 50%) at 10/sup -10/ M. Stromalvascular cells were not influenced at any of the doses tested. Adipose cell lipid content as indicated by oil red-O staining was decreased by Cachetin. Esterase staining by adipose cells treated with Cachetin was increased indicating an increase in intracellular lipase. These studies show that Cachetin has specific effects on primary adipose cell differentiation.

Martin, R.J.; Jones, D.D.; Jewell, D.E.; Hausman, G.J.

1986-03-05

319

Salt tolerance in cultured cells of Spartina pectinata  

Microsoft Academic Search

Suspension cultures with cell doubling times of ca. 2 days were developed from the halophytic grass Spartina pectinata. Maximum rates of exponential growth measured by direct cell counts and by total culture packed-cell-volume were not significantly reduced by NaCl up to 200 mM but dropped beyond this point. In contrast, total cell production over a one week culture cycle, by

R. Scott Warren; Lisa M. Baird; Angela K. Thompson

1985-01-01

320

Recombinant Protein Production and Insect Cell Culture and Process  

NASA Technical Reports Server (NTRS)

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.

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

321

Cultured stem cells as tools for toxicological assays.  

PubMed

In the last 2 decades, cell culture techniques for both mammalian embryonic stem cells and adult stem cells have developed and improved, and are now widely available. These stem cells are either pluri- or multi-potent, which makes them favorable for use in vitro developmental toxicity assays. Recent studies have reported several applications for embryonic and adult stem cells in cytotoxicity and developmental toxicity testing. These applications have the potential to provide alternative assessment techniques for evaluating toxic substances, and possibly reveal novel toxic and developmental effects that are difficult to investigate in humans because of ethical considerations. In this review, we describe some of the new approaches that use mammalian embryonic and adult stem cells in toxicological safety testing. PMID:23827858

Mori, Hideki; Hara, Masayuki

2013-12-01

322

Unique cell culture systems for ground based research  

NASA Technical Reports Server (NTRS)

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

Lewis, Marian L.

1990-01-01

323

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

PubMed Central

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

Sun, Xiuzhi S.; Nguyen, Thu A.

2013-01-01

324

Peptide hydrogelation and cell encapsulation for 3D culture of MCF-7 breast cancer cells.  

PubMed

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

Huang, Hongzhou; Ding, Ying; Sun, Xiuzhi S; Nguyen, Thu A

2013-01-01

325

Differential marker expression by cultures rich in mesenchymal stem cells  

PubMed Central

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

2013-01-01

326

Inositol-Containing Lipids in Suspension-Cultured Plant Cells  

PubMed Central

Polar lipids were extracted from suspension-cultured tomato (Lycopersicon esculentum Mill.) cells and analyzed by thin layer chromatography. Four major inositol-containing compounds were found, and incorporation of [32P]orthosphosphate, [2-3H]glycerol, and myo-[2-3H]inositol was studied. Results showed that phosphatidylinositol-monophosphate is the phospholipid in these cells displaying the most rapid incorporation of [32P]orthophosphate. We suggest that the tracer is incorporated primarily into the phosphomonoester group. Two inositol-containing lipids showed chromatographic behavior similar to phosphatidylinositol-4,5-bisphosphate when using standard thin layer chromatography techniques. The labeling pattern of these compounds, however, reveals that it is unlikely that either of these is identical to phosphatidylinositol-4,5-bisphosphate. Should phosphatidylinositol-bisphosphate be present in suspension cultured plant cells, our data indicate chemical abundancies substantially lower than previously reported. Images Fig. 1 PMID:16666106

Dr?bak, Bj?rn K.; Ferguson, Ian B.; Dawson, Alan P.; Irvine, Robin F.

1988-01-01

327

Effect of hypotonic treatment on sertoli cell purity and function in culture  

Microsoft Academic Search

Summary  Commonly used enzymic methods for the isolation of rat Seroli cells yield populations containing ?15% germ cells. Although\\u000a the germ cells become eliminated after several media changes, they could interferen with the use of Sertoli cells for critical\\u000a studies during the first several days of culture. A brief treatment of Sertoli cell monolayer cultures with 20 mM Tris-HCl (pH 7.4)

J. R. Wagle; J. J. Heindel; A. Steinberger; B. M. Sanborn

1986-01-01

328

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

PubMed Central

Mild hypothermia condition in mammalian cell culture technology has been one of the main focuses of research for the development of breeding strategies to maximize productivity of these production systems. Despite the large number of studies that show positive effects of mild hypothermia on specific productivity of r-proteins, no experimental approach has addressed the indirect effect of lower temperatures on specific cell growth rate, nor how this condition possibly affects less specific productivity of r-proteins. To separately analyze the effects of mild hypothermia and specific growth rate on CHO cell metabolism and recombinant human tissue plasminogen activator productivity as a model system, high dilution rate (0.017 h?1) and low dilution rate (0.012 h?1) at two cultivation temperatures (37 and 33°C) were evaluated using chemostat culture. The results showed a positive effect on the specific productivity of r-protein with decreasing specific growth rate at 33°C. Differential effect was achieved by mild hypothermia on the specific productivity of r-protein, contrary to the evidence reported in batch culture. Interestingly, reduction of metabolism could not be associated with a decrease in culture temperature, but rather with a decrease in specific growth rate. PMID:24699760

Vergara, Mauricio; Becerra, Silvana; Berrios, Julio; Osses, Nelson; Reyes, Juan; Rodriguez-Moya, Maria; Gonzalez, Ramon; Altamirano, Claudia

2014-01-01

329

New method for selection of hydrogen peroxide adapted bifidobacteria cells using continuous culture and immobilized cell technology  

Microsoft Academic Search

BACKGROUND: Oxidative stress can severely compromise viability of bifidobacteria. Exposure of Bifidobacterium cells to oxygen causes accumulation of reactive oxygen species, mainly hydrogen peroxide, leading to cell death. In this study, we tested the suitability of continuous culture under increasing selective pressure combined with immobilized cell technology for the selection of hydrogen peroxide adapted Bifidobacterium cells. Cells of B. longum

Valeria Mozzetti; Franck Grattepanche; Déborah Moine; Bernard Berger; Enea Rezzonico; Leo Meile; Fabrizio Arigoni; Christophe Lacroix

2010-01-01

330

From Bedside to Bench Drug-induced Tubulointerstitial Disease Cyclosporine Nephropathy Study from Models of Cultured Renal Epithelial Cells  

Microsoft Academic Search

Cyclosporine (CsA) is a potent immunosuppressant used in the prevention of transplant- ed organ rejection. CsA is associated with sodium retention, hypertension, hyperkalemia, interstitial fibrosis, and progressive renal failure in transplant recipients. The cellular mecha- nisms, responding to these complications, were revealed in recent studies. CsA decreased the expression iNOS and production of the nitric oxide (NO) in mouse medullary

Mai-Szu Wu

331

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

E-print Network

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

Li, Leon Daliang

2009-01-01

332

The Effect of Spaceflight on Bone Cell Cultures  

NASA Technical Reports Server (NTRS)

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

Landis, William J.

1999-01-01

333

Small SSEA-4-positive cells from human ovarian cell cultures: related to embryonic stem cells and germinal lineage?  

PubMed Central

Background It has already been found that very small embyronic-like stem cells (VSELs) are present in adult human tissues and organs. The aim of this study was to find if there exists any similar population of cells in cell cultures of reproductive tissues and embryonic stem cells, and if these cells have any relation to pluripotency and germinal lineage. Methods and results Here we report that a population of small SSEA-4-positive cells with diameters of up to 4 ?m was isolated by fluorescence-activated cell sorting (FACS) from the human ovarian cell cultures after enzymatic degradation of adult cortex tissues. These small cells – putative ovarian stem cells – were also observed during cell culturing of up to 6 months and more. In general, small putative ovarian stem cells, isolated by FACS, showed a relatively low gene expression profile when compared to human embryonic stem cells (hESCs) and human adult fibroblasts; this may reflect the quiescent state of these cells. In spite of that, small putative ovarian stem cells expressed several genes related to primordial germ cells (PGCs), pluripotency and germinal lineage, including VASA. The PGC-related gene PRDM1 was strongly expressed in small putative ovarian stem cells; in both hESCs and fibroblasts it was significantly down-regulated. In addition, putative ovarian stem cells expressed other PGC-related genes, such as PRDM14 and DPPA3. Most of the pluripotency and germinal lineage-related genes were up-regulated in hESCs (except VASA). When compared to fibroblasts, there were several pluripotency-related genes, which were up-regulated in small putative ovarian stem cells. Similar populations of small cells were also isolated by FACS from human testicular and hESC cultures. Conclusions Our results confirm the potential embryonic-like character of small putative stem cells isolated from human adult ovaries and their possible relation to germinal lineage. PMID:23570331

2013-01-01

334

Characterization of retinal pigment epithelial cells cultured on microporous filters.  

PubMed

Retinal pigment epithelium (RPE) cultured on microporous filter supports was compared to RPE cultured on plastic and evaluated for features characteristic of RPE in vivo. RPE cells grown on filters were cuboidal, formed junctional complex structures between cells, and had elaborate microvilli and basal infoldings similar to RPE in vivo, while RPE grown on plastic also formed intercellular junctions but appeared squamous and had few microvilli and basal infoldings. RPE grown on filters or plastic secreted an extracellular matrix at the basal surface and ingested isolated rat rod outer segments at the apical surface. RPE grown on filters coated with laminin or fibronectin became confluent more rapidly than RPE grown on uncoated filters, while RPE grown at the same density on filters coated with collagen type I did not become confluent. The laminin and fibronectin coatings did not alter the RPE cell morphology; however, cells seeded on collagen-coated filters grew in large disorganized clusters. RPE grown on laminin-coated filters formed functional tight junctions as evidenced by the capacity of RPE monolayers to prevent the bulk flow of medium and the passage of trypan blue across the filter. Radiolabeled sucrose and inulin were used to measure the paracellular flux through the tight junctions between cells. The passage of these tracers was linear over time, with the lower molecular weight tracer, sucrose, passing through the monolayer more readily than inulin. Values for the flux of radiolabeled bovine serum albumin across RPE monolayers fell between values for sucrose and inulin. The results from these studies show that RPE monolayers cultured on laminin-coated filters maintain a morphology similar to that of RPE in vivo, are capable of ingesting rod outer segments, and form a selectively permeable barrier to various tracers. This culture system should be useful for studies of transepithelial transport, secretion, endocytosis and exocytosis that require independent control of the extracellular environment at the apical and basolateral cell surfaces. PMID:3311644

Heth, C A; Yankauckas, M A; Adamian, M; Edwards, R B

1987-08-01

335

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

NASA Technical Reports Server (NTRS)

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

Krikorian, Abraham D.

1993-01-01

336

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

PubMed

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

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

1985-10-01

337

Attachment of human bone cells to tissue culture polystyrene and to unmodified polystyrene: the effect of surface chemistry upon initial cell attachment  

Microsoft Academic Search

Cell culture studies have often been used in the determination of the suitability of biomaterials as surfaces for the attachment and growth of cells. For such studies of surfaces for potential use in bone implants, cells derived from bone may be maintained in culture on tissue culture polystyrene (TCPS). We have determined the contribution that serum fibronectin (FN) or vitronectin

John G. Steele; Clive McFarland; B. Ann Dalton; Graham Johnson; Margaret D. M. Evans; C. Rolfe Howlett; P. Anne Underwood

1994-01-01

338

Development of mouse embryos co-cultured with polarized or non-polarized uterine epithelial cells using sequential culture media.  

PubMed

This study investigated the effects of the in vitro co-culture of mouse embryos with non-polarized or polarized uterine epithelial cells, using sequential culture media, on their development to blastocysts, blastocyst quality (blastocyst diameter and cell number), apoptosis, Bcl-2 and Bax gene expression. There were three treatments, all of which used sequential culture media. The treatments were no co-culture (control), non-polarized or polarized epithelial cell monolayer co-culture in 24-well tissue culture plates. Mouse uterine epithelial cells were isolated enzymatically and were seeded either on the surface of the culture plate (non-polarized monolayer) or on a Millipore filter insert coated with extra-cellular matrix extract (polarized monolayer) that was then placed in the culture plate. Two-cell mouse embryos were cultured in G-1 ver3 medium to the eight-cell stage when they were randomly assigned to the treatments. The culture medium was G-2 ver3 during the treatment phase of the study. Significances of differences were evaluated by the one-way analysis of variance for continuous data. The epithelial cells cultured on Millipore filters became polarized and their morphology compared favorably with those cultured on the surface of the culture plate and in vivo uterine epithelial cells. After 96 h on the treatments, the polarized monolayer had supported the development of significantly more hatched blastocysts (80.0%; P<0.05) than the non-polarized monolayer (63.4%) or the control (61.4%) culture treatments. Co-culture resulted in the production of blastocysts with significantly more cells (non-polarized monolayer 56.7+/-2.1, polarized monolayer 61.9+/-2.1) than the control culture (42.8+/-2.6; P<0.05) but the diameter and shape of the blastocysts were not significantly different. The proportion of blastocysts with apoptotic blastomere was higher for the control culture (94.4%) than for the non-polarized (68.2%) or polarized (66.7%) co-culture systems (P<0.05). Moreover, the apoptotic index was significantly higher in control blastocysts (5.6+/-0.9; P<0.05) than in non-polarized (1.7+/-0.3) or polarized (1.5+/-0.3) co-culture. In the control, Bax mRNA was strongly expressed when compared to co-culture treatments (P<0.05), whereas, the relative abundance of Bcl-2 mRNA to the beta-tubulin was lower than co-culture treatments (P<0.05). It is concluded that a co-culture system involving polarized uterine epithelial cells and sequential culture media is a promising method of producing mouse embryos. PMID:16876344

Azadbakht, Mehri; Valojerdi, Mojtaba Rezazadeh; Mowla, Sayed Javad

2007-07-01

339

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

Microsoft Academic Search

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

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

2000-01-01

340

Production of calves by transfer of nuclei from cultured inner cell mass cells.  

PubMed Central

We report here the isolation and in vitro culture of bovine inner cell mass (ICM) cells and the use of ICM cells in nuclear transfer to produce totipotent blastocysts that resulted in calves born. Of 15 cell lines represented in this study, 13 were derived from immunosurgically isolated ICM of 3 in vitro produced day 9-10 bovine blastocysts, while 2 lines were derived from single blastocysts. Approximately 70% of attempted cell lines became established cell lines when started from 3 ICMs. The ability to establish cell lines was dependent on the number of ICMs starting the line. Sire differences were noted in the ability of ICMs to establish cell lines and to form blastocysts. The cell lines were cultured as a low cell density suspension in the medium CR1aa plus selenium, insulin, and transferrin (SIT) and 5% fetal calf serum (FCS) for 6-101 days before use in nuclear transfer, at which time some had multiplied to more than 2000 cells. If allowed to aggregate, cells of established cell lines formed embryoid bodies. A total of 659 nuclear transfer clones were made by fusing the ES cells into enucleated oocytes with polyethylene glycol; 460 of these fused, based on cleavage (70%). After culture of the clones for 7 days in vitro in CR1aa/SIT/5% FCS, 109 (24%) of those fused became blastocysts. Thirty-four blastocysts were transferred into uteri of 27 cows, and 13 cows (49%) became pregnant. Four of the 13 cows gave birth to 4 normal calves. DNA typing showed the calves to be derived from the respective sires of the cell lines. The calves were derived from cultures of less than 28 days. Images PMID:8016127

Sims, M; First, N L

1994-01-01

341

Reversible morphological and functional abnormalities of RINm5F cells cultured on polystyrene sulfonate beads.  

PubMed

RINm5F cells (an insulin-secreting cell line) were cultured on PSSO3Na microbeads under static conditions. The cell growth rate was either identical to that of cells grown on plastic wells or slower, depending on the initial cell concentration. With both supports, it was similarly influenced by the fetal calf serum concentration in the culture medium, and protein content per cell was identical. However, no spreading was observed when cells were cultured on microbeads. RINm5F cells cultured on plastic wells responded to arginine + theophylline and to leucine + theophylline by a significant increase in insulin secretion. By contrast, in cells cultured on PSSO3Na microbeads, the increase in this secretion was only slight or nil. All these abnormalities were reversible. Thus, when cells cultured on microbeads were detached and seeded on plastic wells, normal spreading and insulin secretion were observed. Lastly, PSSO3Na beads had an acute suppressive effect on insulin secretion by cells cultured on plastic wells. This study provides an example of cell-biomaterial interaction in which cell growth is possible, but with altered cell function. PMID:3034913

Aubert, N; Reach, G; Serne, H; Jozefowicz, M

1987-05-01

342

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

PubMed

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

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

2013-08-28

343

Co-culture with periodontal ligament stem cells enhances osteogenic gene expression in de-differentiated fat cells.  

PubMed

In recent decades, de-differentiated fat cells (DFAT cells) have emerged in regenerative medicine because of their trans-differentiation capability and the fact that their characteristics are similar to bone marrow mesenchymal stem cells. Even so, there is no evidence to support the osteogenic induction using DFAT cells in periodontal regeneration and also the co-culture system. Consequently, this study sought to evaluate the DFAT cells co-culture with periodontal ligament stem cells (PDLSCs) in vitro in terms of gene expression by comparing runt-related transcription factor 2 (RUNX2) and Peroxisome proliferator-activated receptor gamma 2 (PPAR?2) genes. We isolated DFAT cells from mature adipocytes and compared proliferation with PDLSCs. After co-culture with PDLSCs, we analyzed transcriptional activity implying by DNA methylation in all adipogenic gene promoters using combined bisulfite restriction analysis. We compared gene expression in RUNX2 gene with the PPAR?2 gene using quantitative RT-PCR. After being sub-cultured, DFAT cells demonstrated morphology similar to fibroblast-like cells. At the same time, PDLSCs established all stem cell characteristics. Interestingly, the co-culture system attenuated proliferation while enhancing osteogenic gene expression in RUNX2 gene. Using the co-culture system, DFAT cells could trans-differentiate into osteogenic lineage enhancing, but conversely, their adipogenic characteristic diminished. Therefore, DFAT cells and the co-culture system might be a novel cell-based therapy for promoting osteogenic differentiation in periodontal regeneration. PMID:24573839

Tansriratanawong, Kallapat; Tamaki, Yuichi; Ishikawa, Hiroshi; Sato, Soh

2014-10-01

344

Imaging the division process in living tissue culture cells  

PubMed Central

We detail some of the pitfalls encountered when following live cultured somatic cells by light microscopy during mitosis. Principle difficulties in this methodology arise from the necessity to compromise between maintaining the health of the cell while achieving the appropriate temporal and spatial resolutions required for the study. Although the quality of the data collected from fixed cells is restricted only by the quality of the imaging system and the optical properties of the specimen, the major limiting factor when viewing live cells is radiation damage induced during illumination. We discuss practical considerations for minimizing this damage, and for maintaining the general health of the cell, while it is being followed by multi-mode or multi-dimensional light microscopy. PMID:16343936

Khodjakov, Alexey; Rieder, Conly L.

2008-01-01

345

Roles of adherent myogenic cells and dynamic culture in engineered muscle function and maintenance of satellite cells.  

PubMed

Highly functional engineered skeletal muscle constructs could serve as physiological models of muscle function and regeneration and have utility in therapeutic replacement of damaged or diseased muscle tissue. In this study, we examined the roles of different myogenic cell fractions and culturing conditions in the generation of highly functional engineered muscle. Fibrin-based muscle bundles were fabricated using either freshly-isolated myogenic cells or their adherent fraction pre-cultured for 36 h. Muscle bundles made of these cells were cultured in both static and dynamic conditions and systematically characterized with respect to early myogenic events and contractile function. Following 2 weeks of culture, we observed both individual and synergistic benefits of using the adherent cell fraction and dynamic culture on muscle formation and function. In particular, optimal culture conditions resulted in significant increase in the total cross-sectional muscle area (- 3-fold), myofiber size (- 1.6-fold), myonuclei density (- 1.2-fold), and force generation (- 9-fold) compared to traditional use of freshly-isolated cells and static culture. Curiously, we observed that only a simultaneous use of the adherent cell fraction and dynamic culture resulted in accelerated formation of differentiated myofibers which were critical for providing a niche-like environment for maintenance of a satellite cell pool early during culture. Our study identifies key parameters for engineering large-size, highly functional skeletal muscle tissues with improved ability for retention of functional satellite cells. PMID:25154662

Juhas, Mark; Bursac, Nenad

2014-11-01

346

Glycosylation-mediated phenylpropanoid partitioning in Populus tremuloides cell cultures  

Microsoft Academic Search

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

Raja S Payyavula; Benjamin A Babst; Matthew P Nelsen; Scott A Harding; Chung-Jui Tsai

2009-01-01

347

Giant eosinophil colonies from cultures of bone marrow cells  

Microsoft Academic Search

Summary  To date, the small size and slow growth of eosinophil colonies in vitro has hampered study of cloned eosinophils. We found\\u000a enhanced eosinophil colony size and numbers in methylcellulose cultures of bone marrow cells utilizing defined supplemented\\u000a bovine calf serum (DSBCS) in combination with EL4 conditioned medium (EL4-CM). At days 9, 16 and 23 significantly more eosinophil\\u000a colonies and more

J. H. Butterfield; D. Weiler

1986-01-01

348

Influence of Compensatory Renal Growth on Susceptibility of Primary Cultures of Renal Cells to Chemically Induced Injury  

Microsoft Academic Search

Primary cultures of rat renal proximal tubular (PT) and distal tubular (DT) cells from control and uninephrectomized (NPX) Sprague-Dawley rats were established to study whether the altered toxicological responses identified in freshly isolated cells are maintained in culture. Previous work showed that primary cultures of PT cells from hypertrophied rat kidneys maintained their differentiated properties, as evidenced by their high

Lawrence H. Lash; David A. Putt; Rudolfs K. Zalups

2006-01-01

349

Cultural Studies as Performative Politics  

Microsoft Academic Search

This article addresses the role public intellectuals and cultural workers might play in challenging the pervasive institutional and ideological influ ence of neoliberalism as it continues to attack all public spaces and social services not governed by the logic of the market. The author takes up this challenge by articulating a relationship between the political and peda gogical that is

Henry A. Giroux

2001-01-01

350

Cultural Studies Meets Religious Education  

ERIC Educational Resources Information Center

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

Parker, Evelyn

2006-01-01

351

Visually guided whole cell patch clamp of mouse supraoptic nucleus neurons in cultured and acute conditions.  

PubMed

Recent advances in neuronal culturing techniques have supplied a new set of tools for studying neural tissue, providing effective means to study molecular aspects of regulatory elements in the supraoptic nucleus of the hypothalamus (SON). To combine molecular biology techniques with electrophysiological recording, we modified an organotypic culture protocol to permit transfection and whole cell patch-clamp recordings from SON cells. Neonatal mouse brain coronal sections containing the SON were dissected out, placed on a filter insert in culture medium, and incubated for at least 4 days to allow attachment to the insert. The SON was identifiable using gross anatomical landmarks, which remained intact throughout the culturing period. Immunohistochemical staining identified both vasopressinergic and oxytocinergic cells present in the cultures, typically appearing in well-defined clusters. Whole cell recordings from these cultures demonstrated that certain properties of the neonatal mouse SON were comparable to adult mouse magnocellular neurons. SON neurons in both neonatal cultures and acute adult slices showed similar sustained outward rectification above -60 mV and action potential broadening during evoked activity. Membrane potential, input resistance, and rapidly inactivating potassium current density (IA) were reduced in the cultures, whereas whole cell capacitance and spontaneous synaptic excitation were increased, perhaps reflecting developmental changes in cell physiology that warrant further study. The use of the outlined organotypic culturing procedures will allow the study of such electrophysiological properties of mouse SON using whole cell patch-clamp, in addition to various molecular, techniques that require longer incubation times. PMID:16469834

Stachniak, Tevye J E; Bourque, Charles W

2006-07-01

352

Development of 3-D Hydrogel Culture Systems With On-Demand Cell Separation  

PubMed Central

Recently there has been an increased interest in the effects of paracrine signaling between groups of cells, particularly in the context of better understanding how stem cells contribute to tissue repair. Most current 3-D co-culture methods lack the ability to effectively separate 2 cell populations after the culture period, which is important for simultaneously analyzing the reciprocal effects of each cell type on the other. Here, we detail the development of a 3-D hydrogel co-culture system that allows us to culture different cell types for up to 7 days and subsequently separate and isolate the different cell populations using enzyme-sensitive glues. Separable 3-D co-culture laminates were prepared by laminating PEG-based hydrogels with enzyme-degradable hydrogel adhesives. Encapsulated cell populations exhibited good segregation with well-defined interfaces. Furthermore, constructs can be separated on-demand upon addition of the appropriate enzyme and cell viability remains high throughout the culture period, even after laminate separation. This platform offers great potential for a variety of basic cell signaling studies as the incorporation of an enzyme-sensitive adhesive interface allows the on-demand separation of individual cell populations for immediate analysis or further culture to examine persistence of co-culture effects and paracrine signaling on cell populations. PMID:23447378

Hamilton, Sharon K.; Bloodworth, Nathaniel C.; Massad, Christopher S.; Hammoudi, Taymour M.; Suri, Shalu; Yang, Peter J.; Lu, Hang; Temenoff, Johnna S.

2013-01-01

353

Development of an intestinal cell culture model to obtain smooth muscle cells and myenteric neurones  

PubMed Central

This paper reports on the development of an entirely new intestinal smooth muscle cell (ISMC) culture model using rat neonates for use in pharmacological research applications. Segments of the duodenum, jejunum and ileum were obtained from Sprague-Dawley rat neonates. The cell extraction technique consisted of ligating both ends of the intestine and incubating (37 ºC) in 0.25% trypsin for periods of 30–90 min. Isolated cells were suspended in DMEM-HEPES, plated and allowed to proliferate for 7 days. Cell culture quality was assessed via a series of viability tests using the dye exclusion assay. In separate experiments, tissues were exposed to trypsin for varying durations and subsequently histological procedures were applied. Cell purification techniques included differential adhesion technique for minimizing fibroblasts. Selective treatments with neurotoxin scorpion venom (30 µg mL?1) and anti-mitotic cytosine arabinoside (6 µm) were also applied to purify respectively ISMC and myenteric neurones selectively. The different cell populations were identified in regard to morphology and growth characteristics via immunocytochemistry using antibodies to smooth muscle ?-actin, ?-actinin and serotonin-5HT3 receptors. Based on both viability and cell confluence experiments, results demonstrated that intestinal cells were best obtained from segments of the ileum dissociated in trypsin for 30 min. This provided the optimum parameters to yield highly viable cells and confluent cultures. The finding was further supported by histological studies demonstrating that an optimum incubation time of 30 min is required to isolate viable cells from the muscularis externae layer. When cell cultures were treated with cytosine arabinoside, the non-neuronal cells were abolished, resulting in the proliferation of cell bodies and extended neurites. Conversely, cultures treated with scorpion venom resulted in complete abolition of neurones and proliferation of increasing numbers of ISMC, which were spindle-shaped and uniform throughout the culture. When characterized by immunocytochemistry, neurones were stained with antibody to 5HT3 receptors but not with antibodies to ?-smooth muscle actin and ?-actinin. Conversely, ISMC were stained with antibodies to ?-smooth muscle actin and ?-actinin but not with antibody to 5HT3 receptors. The present study provides evidence that our method of dissociation and selectively purifying different cell populations will allow for pharmacological investigation of each cell type on different or defined mixtures of different cell types. PMID:17979953

Batista Lobo, S; Denyer, M; Britland, S; Javid, F A

2007-01-01

354

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

NASA Technical Reports Server (NTRS)

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).

Richmond, Robert C.

2001-01-01

355

Primary culture of astrocytic glial cells from rainbow trout, Salmo gairdneri L., brain.  

PubMed

A method is described for the preparation and maintenance of rainbow trout brain astrocytes in primary culture. A dissociated cell suspension was prepared from brains from young fish by mechanical sieving through nylon guazes, and the cells cultured in polylysine-treated plastic tissue culture flasks at 22 degrees C. The resultant cultures were characterised by specific immunofluorescent staining using glial fibrillary acidic protein (GFAP) for astrocytes and Thy 1 for fibroblastic cells. The cultures were greater than 95% astrocytes with fibroblasts the only other cell type present. These highly enriched astrocyte cultures provide a unique system for various neurochemical, neurophysiological and biochemical studies of fish neural cells. Polyunsaturated fatty acid metabolism will be an area of particular interest. PMID:1700235

Tocher, D R; Wilson, R

1990-08-01

356

Greek and Roman Studies Discovering the cultural  

E-print Network

Greek and Roman Studies Discovering the cultural richness and variety of the world is the best. You will study periods that were foundational for these cultures, when Greeks and Romans were asking themselves, What is a citizen? a family? a god? What is art? What is law? What is freedom? These questions

357

Action of staphylococcal exfoliative toxins on epidermal cell cultures and organotypic skin.  

PubMed

In the staphylococcal scalded skin syndrome, spontaneous intraepithelial cleavages are due to the exfoliative toxins A or B (ETA or ETB). Until now, these toxins have been studied either on epidermis or on organotypic skin cultures. In the present study, we compare the effects of these toxins on human keratinocyte cell cultures to those on human and mouse organotypic skin cultures. With concentrations of ETA or ETB of 1 mg/ml for 3 hours, spontaneous intraepithelial cleavages were noted in both cell and organotypic cultures. Keratinocyte cell cultures were as sensitive as organotypic skin cultures to these toxins. Since keratohyaline granules may represent a possible binding site for ETA or ETB, we tried to correlate the expression of keratohyaline granules with the appearance of intraepithelial clefts due to the toxins. However, when cultured in liquid medium, epithelia were not differentiated enough to allow the detection of the binding site of ETA-ETB. PMID:1703553

Gentilhomme, E; Faure, M; Piemont, Y; Binder, P; Thivolet, J

1990-09-01

358

Preparation of Feeder plates for ES cell culture Gelatinize Tissue Culture Plates  

E-print Network

Preparation of Feeder plates for ES cell culture Gelatinize Tissue Culture Plates Gelatinize plates Cells with Mitomycin C 1. Aspirate off media from the STO cell plate. 2. Mix 120 µl of mitomycin C (0.5 mg/ml in PBS) with 6 ml of STO media. 3. Add to the STO cells (6 ml/10 cm) and incubate at 37°C

Oliver, Douglas L.

359

Effects of pulsatile flow on cultured vascular endothelial cell morphology.  

PubMed

Endothelial cells (EC) appear to adapt their morphology and function to the in vivo hemodynamic environment in which they reside. In vitro experiments indicate that similar alterations occur for cultured EC exposed to a laminar steady-state flow-induced shear stress. However, in vivo EC are exposed to a pulsatile flow environment; thus, in this investigation, the influence of pulsatile flow on cell shape and orientation and on actin microfilament localization in confluent bovine aortic endothelial cell (BAEC) monolayers was studied using a 1-Hz nonreversing sinusoidal shear stress of 40 +/- 20 dynes/cm2 (type I), 1-Hz reversing sinusoidal shear stresses of 20 +/- 40 and 10 +/- 15 dynes/cm2 (type II), and 1-Hz oscillatory shear stresses of 0 +/- 20 and 0 +/- 40 dynes/cm2 (type III). The results show that in a type I nonreversing flow, cell shape changed less rapidly, but cells took on a more elongated shape than their steady flow controls long-term. For low-amplitude type II reversing flow, BAECs changed less rapidly in shape and were always less elongated than their steady controls; however, for high amplitude reversal, BAECs did not stay attached for more than 24 hours. For type III oscillatory flows, BAEC cell shape remained polygonal as in static culture and did not exhibit actin stress fibers, such as occurred in all other flows. These results demonstrate that EC can discriminate between different types of pulsatile flow environments.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1875686

Helmlinger, G; Geiger, R V; Schreck, S; Nerem, R M

1991-05-01

360

ARSENIC EXPOSURE INDUCES THE WARBURG EFFECT IN CULTURED HUMAN CELLS  

PubMed Central

Understanding how arsenic exacts its diverse, global disease burden is hampered by a limited understanding of the particular biological pathways that are disrupted by arsenic and underlie pathogenesis. A reductionist view would predict that a small number of basic pathways are generally perturbed by arsenic, and manifest as diverse diseases. Following an initial observation that arsenite-exposed cells in culture acidify their media more rapidly than control cells, the report here shows that low level exposure to arsenite (75 ppb) is sufficient to induce aerobic glycolysis (the Warburg effect) as a generalized phenomenon in cultured human primary cells and cell lines. Expanded studies in one such cell line, the non-malignant pulmonary epithelial line, BEAS-2B, established that the arsenite-induced Warburg effect was associated with increased accumulation of intracellular and extracellular lactate, an increased rate of extracellular acidification, and inhibition by the non-metabolized glucose analog, 2-deoxyglucose. Associated with the induction of aerobic glycolysis was a pathway-wide induction of glycolysis gene expression, as well as protein accumulation of an established glycolysis master-regulator, hypoxia-inducible factor 1?. Arsenite-induced alteration of energy production in human cells represents the type of fundamental perturbation that could extend to many tissue targets and diseases. PMID:23648393

Zhao, Fei; Severson, Paul; Pacheco, Samantha; Futscher, Bernard W.; Klimecki, Walter T.

2013-01-01

361

Optimization of Storage Temperature for Cultured ARPE-19 Cells.  

PubMed

Purpose. The establishment of future retinal pigment epithelium (RPE) replacement therapy is partly dependent on the availability of tissue-engineered RPE cells, which may be enhanced by the development of suitable storage methods for RPE. This study investigates the effect of different storage temperatures on the viability, morphology, and phenotype of cultured RPE. Methods. ARPE-19 cells were cultured under standard conditions and stored in HEPES-buffered MEM at nine temperatures (4°C, 8°C, 12°C, 16°C, 20°C, 24°C, 28°C, 32°C, and 37°C) for seven days. Viability and phenotype were assessed by a microplate fluorometer and epifluorescence microscopy, while morphology was analyzed by scanning electron microscopy. Results. The percentage of viable cells preserved after storage was highest in the 16°C group (48.7% ± 9.8%; P < 0.01 compared to 4°C, 8°C, and 24°C-37°C; P < 0.05 compared to 12°C). Ultrastructure was best preserved at 12°C, 16°C, and 20°C. Expression of actin, ZO-1, PCNA, caspase-3, and RPE65 was maintained after storage at 16°C compared to control cells that were not stored. Conclusion. Out of nine temperatures tested between 4°C and 37°C, storage at 12°C, 16°C, and 20°C was optimal for maintenance of RPE cell viability, morphology, and phenotype. The preservation of RPE cells is critically dependent on storage temperature. PMID:24251032

Pasovic, Lara; Utheim, Tor Paaske; Maria, Rima; Lyberg, Torstein; Messelt, Edward B; Aabel, Peder; Chen, Dong Feng; Chen, Xiangjun; Eidet, Jon Roger

2013-01-01

362

Microfluidic cell culture systems with integrated sensors for drug screening  

NASA Astrophysics Data System (ADS)

Cell-based testing is a key step in drug screening for cancer treatments. A microfluidic platform can permit more precise control of the cell culture microenvironment, such as gradients in soluble factors. These small-scale devices also permit tracking of low cell numbers. As a new screening paradigm, a microscale system for integrated cell culture and drug screening promises to provide a simple, scalable tool to apply standardized protocols used in cellular response assays. With the ability to dynamically control the microenvironment, we can create temporally varying drug profiles to mimic physiologically measured profiles. In addition, low levels of oxygen in cancerous tumors have been linked with drug resistance and decreased likelihood of successful treatment and patient survival. Our work also integrates a thin-film oxygen sensor with a microfluidic oxygen gradient generator which will in future allow us to create spatial oxygen gradients and study effects of hypoxia on cell response to drug treatment. In future, this technology promises to improve cell-based validation in the drug discovery process, decreasing the cost and increasing the speed in screening large numbers of compounds.

Grist, Samantha; Yu, Linfen; Chrostowski, Lukas; Cheung, Karen C.

2012-03-01

363

Anti-PrP antibodies block PrPSc replication in prion infected cell cultures  

E-print Network

Anti-PrP antibodies block PrPSc replication in prion infected cell cultures by accelerating Pr promising strategy for the treatment of prion diseases. In the present study, we screened various anti-PrP antibodies, with the aim to identify those that will block PrPSc replication in prion infected cell culture

Paris-Sud XI, Université de

364

An Optically Controlled 3D Cell Culturing System  

PubMed Central

A novel 3D cell culture system was developed and tested. The cell culture device consists of a microfluidic chamber on an optically absorbing substrate. Cells are suspended in a thermoresponsive hydrogel solution, and optical patterns are utilized to heat the solution, producing localized hydrogel formation around cells of interest. The hydrogel traps only the desired cells in place while also serving as a biocompatible scaffold for supporting the cultivation of cells in 3D. This is demonstrated with the trapping of MDCK II and HeLa cells. The light intensity from the optically induced hydrogel formation does not significantly affect cell viability. PMID:22701475

Ishii, Kelly S.; Hu, Wenqi; Namekar, Swapnil A.; Ohta, Aaron T.

2012-01-01

365

Fluorescent speckle microscopy in cultured cells.  

PubMed

After slightly more than a decade since it was first established, fluorescent speckle microscopy (FSM) has been intensively used to investigate macromolecular dynamics, such as microtubule flux in mitosis and meiosis, microtubule translocation in neurons, microtubule-binding proteins, and focal adhesion proteins, as well as the assembly of actin filaments. This state-of-the-art technique is based on nonuniform distribution of fluorescently labeled subunits diluted in the endogenous, unlabeled ones, resulting in microscopy-detectable speckled patterns. In order to enable sufficient contrast between neighboring diffraction-limited image regions, a low ratio between labeled and endogenous molecules is required, which can be achieved either by microinjection or by expression of limited amounts of fluorescently labeled subunits in cells. Over the years, the initial settings for FSM have been significantly improved by introduction of more sensitive cameras and spinning-disk confocal units, as well as by the development of specialized algorithms for image analysis. In this chapter, we describe our current FSM setup and detail on the necessary experimental approaches for its use in cultured cells, while discussing the present and future challenges of this powerful technique. PMID:22264533

Barisic, Marin; Pereira, António J; Maiato, Helder

2012-01-01

366

Potassium currents in cultured rabbit retinal pigment epithelial cells.  

PubMed

Membrane potential and ionic currents were studied in cultured rabbit retinal pigment epithelial (RPE) cells using whole-cell patch clamp and perforated-patch recording techniques. RPE cells exhibited both outward and inward voltage-dependent currents and had a mean membrane capacitance of 26 +/- 12 pF (SD, n = 92). The resting membrane potential averaged -31 +/- 15 mV (n = 37), but it was as high as -60 mV in some cells. When K+ was the principal cation in the recording electrode, depolarization-activated outward currents were apparent in 91% of cells studied. Tail current analysis revealed that the outward currents were primarily K+ selective. The most frequently observed outward K+ current was a voltage- and time-dependent outward current (IK) which resembled the delayed rectifier K+ current described in other cells. IK was blocked by tetraethylammonium ions (TEA) and barium (Ba2+) and reduced by 4-aminopyridine (4-AP). In a few cells (3-4%), depolarization to -50 mV or more negative potentials evoked an outwardly rectifying K+ current (IKt) which showed more rapid inactivation at depolarized potentials. Inwardly rectifying K+ current (IKI) was also present in 41% of cells. IKI was blocked by extracellular Ba2+ or Cs+ and exhibited time-dependent decay, due to Na+ blockade, at negative potentials. We conclude that cultured rabbit RPE cells exhibit at least three voltage-dependent K+ currents. The K+ conductances reported here may provide conductive pathways important in maintaining ion and fluid homeostasis in the subretinal space. PMID:7807515

Tao, Q; Rafuse, P E; Kelly, M E

1994-08-01

367

Apoptosis in batch cultures of Chinese Hamster Ovary cells  

Microsoft Academic Search

One of the main problems in the culture of Chinese Hamster Ovary (CHO) cells continues to be the inability to maintain the viability of the cultures over an extended period of time. The rapid decline in viability at the end of the culture is exacerbated by the absence of serum. In trying to reduce the extent of death in these

J. Goswami; A. J. Sinskey; H. Steller; G. N. Stephanopoulos; D. I. C. Wang

1999-01-01

368

A method for culturing human hair follicle cells.  

PubMed

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. PMID:7006669

Weterings, P J; Vermorken, A J; Bloemendal, H

1981-01-01

369

Conversion of viable but nonculturable enteric bacteria to culturable by co-culture with eukaryotic cells.  

PubMed

Viable but nonculturable (VBNC) Vibrio cholerae non-O1/non-O139, V. parahaemolyticus, enterohemorrhagic Escherichia coli, enterotoxigenic E. coli, enteropathogenic E. coli, Shigella flexneri, and Salmonella enterica were converted to the culturable state by co-culture with selected eukaryotic cells, e.g., HT-29, Caco-2, T84, HeLa, Intestine 407, and CHO cells. PMID:22537150

Senoh, Mitsutoshi; Ghosh-Banerjee, Jayeeta; Ramamurthy, Thandavarayan; Colwell, Rita R; Miyoshi, Shin-Ichi; Nair, G Balakrish; Takeda, Yoshifumi

2012-05-01

370

Long-term culture and analysis of cashmere goat Sertoli cells.  

PubMed

Sertoli cells have important functions in the testis for spermatogenesis. Thus, Sertoli cell culture systems have been established in many animals, such as rat, mouse, human, dog, cow, and pig, but a goat culture has not been reported. This study describes the isolation and culture of Sertoli cells from 3- to 4-month-old cashmere goat (Capra hircus) testes. These proliferative cells were expanded for 20 passages and repeatedly cryopreserved in vitro, in contrast to previous study in human, of which maintain steady growth for up to seven passages and only passages 1 to 5 could be refrozen. The microstructure and ultrastructure of the culture were typical of Sertoli cells, bearing irregular nuclei and a cytoplasm that was rich in smooth and rough endoplasmic reticulum, mitochondria, Golgi, lysosomes, lipid drops, and glycogenosomes. By immunofluorescence analysis, the all cells expressed SRY-related HMG box gene 9 (Sox9). Growth curves and 5-bromo-2'-deoxyuridine (BrdU) incorporation were used to analyze the proliferation of the cultured cells. With increasing passage times, the proliferation of the Sertoli cells declined, but the transcription of glial cell-derived neurotrophic factor (GDNF), stem cell factor (SCF), and ?1-integrin was constant. By flow cytometry, the cells retained the ability to proliferate after 5 yr of cryopreservation. Thus, cashmere goat Sertoli cells have significant proliferative potential in vitro, expressing germ cell regulatory factors and have important applications in studying Sertoli cell-germ cell interactions, spermatogenesis, reproductive toxicology, and male infertility. PMID:25164184

Su, Huimin; Luo, Fenhua; Bao, Jiajing; Wu, Sachula; Zhang, Xueming; Zhang, Yan; Duo, Shuguang; Wu, Yingji

2014-12-01

371

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

PubMed

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

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

2014-08-01

372

Development of Scalable Culture Systems for Human Embryonic Stem Cells  

PubMed Central

The use of human pluripotent stem cells, including embryonic and induced pluripotent stem cells, in therapeutic applications will require the development of robust, scalable culture technologies for undifferentiated cells. Advances made in large-scale cultures of other mammalian cells will facilitate expansion of undifferentiated human embryonic stem cells (hESCs), but challenges specific to hESCs will also have to be addressed, including development of defined, humanized culture media and substrates, monitoring spontaneous differentiation and heterogeneity in the cultures, and maintaining karyotypic integrity in the cells. This review will describe our current understanding of environmental factors that regulate hESC self-renewal and efforts to provide these cues in various scalable bioreactor culture systems. PMID:20161686

Azarin, Samira M.; Palecek, Sean P.

2009-01-01

373

Automated and Online Characterization of Adherent Cell Culture Growth in a Microfabricated Bioreactor  

PubMed Central

Adherent cell lines are widely used across all fields of biology, including drug discovery, toxicity studies, and regenerative medicine. However, adherent cell processes are often limited by a lack of advances in cell culture systems. While suspension culture processes benefit from decades of development of instrumented bioreactors, adherent cultures are typically performed in static, noninstrumented flasks and well-plates. We previously described a microfabricated bioreactor that enables a high degree of control on the microenvironment of the cells while remaining compatible with standard cell culture protocols. In this report, we describe its integration with automated image-processing capabilities, allowing the continuous monitoring of key cell culture characteristics. A machine learning–based algorithm enabled the specific detection of one cell type within a co-culture setting, such as human embryonic stem cells against the background of fibroblast cells. In addition, the algorithm did not confuse image artifacts resulting from microfabrication, such as scratches on surfaces, or dust particles, with cellular features. We demonstrate how the automation of flow control, environmental control, and image acquisition can be employed to image the whole culture area and obtain time-course data of mouse embryonic stem cell cultures, for example, for confluency. PMID:24692228

Jaccard, Nicolas; Macown, Rhys J.; Super, Alexandre; Griffin, Lewis D.; Veraitch, Farlan S.

2014-01-01

374

Rapid single-cell electroporation for labeling organotypic cultures  

E-print Network

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

Steinmeyer, Joseph D. (Joseph Daly)

2010-01-01

375

Rhamnolipids elicit the same cytotoxic sensitivity between cancer cell and normal cell by reducing surface tension of culture medium.  

PubMed

Biosurfactant rhamnolipids have been claimed to show biological activities of inhibiting the proliferation of cancer cells. In this study, the cytotoxicity of rhamnolipids was examined on four cancer cells (HepG2, Caco-2, Hela, MCF-7 cells) and two normal cells (HK-2 cell, primary hepatocyte). Interestingly, both cancer cells and normal cells exhibited similar sensitivities to the addition of rhamnolipids in culture medium, and the cytotoxicity was largely attenuated by the presence of fetal bovine serum (FBS) in culture medium. In correlation of the mono-/di-rhamnolipid cytotoxicity with the surface tension of culture medium, it was found that rhamnolipids triggered cytotoxicity whenever the surface tension of culture medium decreased below 41 mN/m irrespective of the FBS content in culture medium, cell line, or rhamnolipid congener. Similarly, each chemical surfactant (Tween-80, sodium dodecyl sulfate, and sodium dodecyl benzene sulfonate) could cause cytotoxicity on HepG2 cells whenever its addition made the surface tension under 41 mN/m in culture medium with or without the presence of FBS. It seems that rhamnolipids, like chemical surfactants, exhibited cytotoxicity by reducing the surface tension of culture medium rather than by changing its specific molecular structure, which had no selection on tumor cells. This study could offer helps to correct the misleading biological activity of rhamnolipids and to avoid the possible large wastes of time and expenses on developing the applications in antitumor drugs. PMID:25231070

Jiang, Lifang; Shen, Chong; Long, Xuwei; Zhang, Guoliang; Meng, Qin

2014-12-01

376

Kinetics of Expansion of Human Limbal Epithelial Progenitor Cells in Primary Culture of Explants Without Feeders  

PubMed Central

The aims of this study were to determine whether human limbal explant cultures without feeder cells result in expansion of epithelial progenitors and to estimate the optimal expansion time for progenitor cells. Limbal explants from ten human corneas were cultured for 7, 9, 11, 14, 18, and 21 days. Limbal explants from two corneas were enzymatically dissociated or directly cultured for 14 days. Progenitor cells were characterized by their ability to form colonies, by immunocytochemistry, and by quantitative real-time polymerase chain reaction. Colonies were identified after 9, 11, 14, and 18 days of culture, but not after 21 days. The number of colonies per explant was significantly higher after 14 days than after 9 and 21 days. The mean percentage of seeded cells giving rise to clones was 4.03% after 14 days of culture and 0.36% for non-cultured dissociated limbal epithelial cells. The number of cells giving rise to clones per cornea significantly increased from an average of 2275 for non-cultured cells to 24266 for cells cultured for 14 days. Immunocytochemical analysis detected positive staining for cytokeratin (CK) 3, CK5/6/8/10/13/18, CK19, vimentin, p63, and p63?, in both cultures and clones. CK3 expression increased significantly with culture time. Transcript expression was observed for CK3, CK19, vimentin, and Delta N p63? at each culture time point, both in cultures and clones. The optimal culture time for limbal explants in cholera toxin-free Green medium without feeder cells was 14 days leading to the expansion of progenitors. PMID:24312615

Ghoubay-Benallaoua, Djida; Sandali, Otman; Goldschmidt, Pablo; Borderie, Vincent

2013-01-01

377

Some Rat Sensory Neurons in Culture Express Characteristics of Differentiated Pain Sensory Cells  

NASA Astrophysics Data System (ADS)

Sensory neurons were dissociated from trigeminal ganglia or from dorsal root ganglia of rats, grown in culture, and examined for expression of properties of pain sensory cells. Many sensory neurons in culture are excited by low concentrations of capsaicin, reportedly a selective stimulus for pain sensory neurons. Many are excited by bradykinin, sensitized by prostaglandin E2, or specifically stained by an antiserum against substance P. These experiments provide a basis for the study of pain mechanisms in cell culture.

Baccaglini, Paola I.; Hogan, Patrick G.

1983-01-01

378

Cell-line Engineering of Chinese Hamster Ovary Cells for Low-temperature Culture  

E-print Network

Developments in mammalian cell culture and recombinant technology has allowed for the production of recombinant proteins for use as human therapeutics. Mammalian cell culture is typically operated at the physiological ...

Kiat, Tan Hong

379

Long-term culture of porcine bladder epithelial cells  

Microsoft Academic Search

Summary  Epithelial cells from normal pig bladders proliferated when cocultured with lethally irradiated feeder cells of the LA7 rat\\u000a mammary tumor line. When the bladder cells and feeders were plated together at a confluent density, the bladder cells proliferated\\u000a as the feeder cells died, resulting in a confluent culture of bladder cells. The bladder cells were successfully subcultured\\u000a by plating with

Ursula K. Ehmann; Martha K. Terris

2002-01-01

380

Cross-Cultural Education Study-Tour.  

ERIC Educational Resources Information Center

The report describes, in the form of transcribed interviews, cross-cultural programs in Saskatchewan, Manitoba, Montana, New Mexico and Arizona identified in a May, 1973, study-tour of cross-cultural education focusing upon people of Indian ancestry. The primary goal of these visits was to identify promising procedures and accomplishments in…

Sherk, H. G.; Callihoe, H.

381

Brushite cement additives inhibit attachment to cell culture beads.  

PubMed

Brushite-forming calcium phosphate cements are of great interest as bone replacement materials because they are resorbable in physiological conditions. Cell-attached culture beads formed from this material could be of great use for cell therapy. Despite a significant amount of work on optimizing the physicochemical properties of these materials, there are very few studies that have evaluated the capacity of the materials to facilitate cell adhesion. In this study, we have formed resorbable calcium phosphate (brushite) culture beads and for the first time we showed that cell attachment to the surface of the brushite cement (BC) could be inhibited by the presence of an intermediate dicalcium phosphate-citrate complex, formed in the cement as a result of using citric acid, a retardant and viscosity modifier used in many cement formulations. The BC beads formed from the mixture of ?-TCP/orthophosphoric acid using citric acid did not allow cell attachment without further treatment. Ageing of BC beads in serum-free Dulbecco's Modified Eagle's Medium (DMEM) solution at 37°C for 1 week greatly enhanced the cell adhesion capacity of the material. Scanning electron microscopy, X-ray diffraction (XRD), and confocal Raman microspectrometry indicated the increased capacity for cell adhesion was due to the changes in phase composition of BC. XRD patterns collected before and after ageing in aqueous solution and a high initial mass loss, suggest the formation of a dicalcium phosphate-citrate complex within the matrix. Since compacts formed from brushite powder supported cell attachment, it was hypothesized that the dicalcium phosphate-citrate complex prevented attachment to the cement surface. PMID:23242924

Jamshidi, Parastoo; Bridson, Rachel H; Wright, Adrian J; Grover, Liam M

2013-05-01

382

Bialaphos selection of stable transformants from maize cell culture  

Microsoft Academic Search

Stable transformed Black Mexican Sweet (BMS) maize callus was recovered from suspension culture cells bombarded with plasmid DNA that conferred resistance to the herbicide bialaphos. Suspension culture cells were bombarded with a mixture of two plasmids. One plasmid contained a selectable marker gene, bar, which encoded phosphinothricin acetyl transferase (PAT), and the other plasmid encoded a screenable marker for ß-glucuronidase

T. M. Spencer; W. J. Gordon-Kamm; R. J. Daines; W. G. Start; P. G. Lemaux

1990-01-01

383

Continuous cultures of fused cells secreting antibody of predefined specificity  

Microsoft Academic Search

THE manufacture of predefined specific antibodies by means of permanent tissue culture cell lines is of general interest. There are at present a considerable number of permanent cultures of myeloma cells1,2 and screening procedures have been used to reveal antibody activity in some of them. This, however, is not a satisfactory source of monoclonal antibodies of predefined specificity. We describe

G. Köhler; C. Milstein

1975-01-01

384

Cytotoxic effects of arthropod venoms on various cultured cells  

Microsoft Academic Search

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

Ephraim Cohen; Gary B. Quistad

1998-01-01

385

Ionic Interconversion of Pacemaker and Nonpacemaker Cultured Chick Heart Cells  

Microsoft Academic Search

Trypsin-dispersed cells from hearts (ventricles) of 7 to 8 day chick embryos were cultured 3 to 21 days. The cells became attached to the culture dish and assembled into monolayer communities. By means of a bridge circuit, one microelectrode was used for simultaneously passing current and recording membrane potentials (Vm). The input resistance, calculated by the measured AV,~ for a

NICK SPERELAKIS; D. LEHMKUHL

1966-01-01