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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

Human preleukaemia cell culture studies in sideroblastic anaemia  

Microsoft Academic Search

Cell structure abnormalties are found in acute leukaemia and preleukaemic states. Studies on bone marrow cells and peripheral leucocytes of 4 patients with idiopathic acquired sideroblastic anaemia showed patterns in cell culture similar to those reported in acute leukaemia: 2 of these patients later developed leukaemia. Other patients with idiopathic, secondary or congenital sideroblastosis showed no such cell culture abnormalities,

J S Senn; P H Pinkerton; G B Price; T W Mak; E A McCulloch

1976-01-01

3

A study of communication specificity between cells in culture  

PubMed Central

We have examined the specificity of communication between cells in culture by co-culturing cells derived from mammalian, avian, and arthropod organisms. Both mammalian and avian culture cells have similar gap junctional phenotypes, while the insect (arthropod) cell lines have a significantly different gap junctional structure. Electrophysiological and ultrastructural methods were used to examine ionic coupling and junctional interactions between homologous and heterologous cell types. In homologous cell systems, gap junctions and ionic coupling are present at a high incidence. Also, heterologous vertebrate cells in co-culture can communicate readily. By contrast, practically no coupling (0-8%) is detectable between heterologous insect cell lines (Homopteran or Lepidopteran) and vertebrate cells (mammalian myocardial or 3T3 cells). No gap junctions have been observed between arthropod and vertebrate cell types, even though the heterologous cells may be separated by less than 10 nm. In additional studies, a low incidence of coupling was found between heterologous insect cell lines derived from different arthropod orders. However, extensive coupling was detected between insect cell lines that are derived from the same order (Homoptera). These observations suggest that there is little or no apparent specificity for communication between vertebrate cells in culture that express the same gap junctional phenotype, while there is a definite communication specificity that exists between arthropod cells in culture. PMID:562887

1977-01-01

4

Hepatotoxicity studies with primary cultures of rat liver cells  

Microsoft Academic Search

Summary  A method for preparing primary monolayer cultures of postnatal rat hepatocytes has been developed in our laboratory. Growing\\u000a cultures in arginine-deficient medium inhibits fibroblast overgrowth, and relatively pure cultures of parenchymal hepatocytes\\u000a are obtained. This cell culture system has been used to study the cytotoxicity of two hepatotoxic agents, tetracycline and\\u000a norethindrone. Caffeine was evaluated as an agent thought to

David C. Anuforo; Daniel Acosta; Robert V. Smith

1978-01-01

5

Reply: whole-culture synchronization effective tools for cell cycle studies  

E-print Network

to study the cell cycle because they alter the size and age distributions of the cultures. We believe that whole-culture cell cycle studies work even though they alter the size and age distributions: these cells of these methods take a population of cells of varying `ages', as measured by time since last cell division

Ford, James

6

Distinguishing between linear and exponential cell growth during the division cycle: Single-cell studies, cell-culture studies, and the object of cell-cycle research  

Microsoft Academic Search

BACKGROUND: Two approaches to understanding growth during the cell cycle are single-cell studies, where growth during the cell cycle of a single cell is measured, and cell-culture studies, where growth during the cell cycle of a large number of cells as an aggregate is analyzed. Mitchison has proposed that single-cell studies, because they show variations in cell growth patterns, are

Stephen Cooper

2006-01-01

7

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

PubMed Central

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

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

2011-01-01

8

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

González, Claudia; Espinosa, Marisol; Sánchez, María Trinidad; Droguett, Karla; Ríos, Mariana; Fonseca, Ximena; Villalón, Manuel

2013-01-01

9

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

10

A phytochemical study of lignans in whole plants and cell suspension cultures of Anthriscus sylvestris.  

PubMed

In the roots of Anthriscus sylvestris 12 different lignans were detected. Arctigenin, dimethylmatairesinol, dimethylthujaplicatin, podophyllotoxin, 7-hydroxyyatein and 7-hydroxyanhydropodorhizol have not been previously reported to be present in A. sylvestris. In the cell suspension cultures, which were initiated for this study, trace amounts of deoxypodophyllotoxin could be detected. With these cell suspension cultures we carried out feeding experiments using deoxypodophyllotoxin, yatein and, anhydropodorhizol. Yatein had a toxic effect on the cell cultures and was, like anhydropodorhizol, not converted into any detectable product. Deoxypodophyllotoxin, in contrast, was converted into podophyllotoxin, yielding significantly higher concentration than measured in whole plants. PMID:14531024

Koulman, Albert; Kubbinga, Marlies E; Batterman, Sieb; Woerdenbag, Herman J; Pras, Niesko; Woolley, Jack G; Quax, Wim J

2003-08-01

11

Three-dimensional cell culture microarray for high-throughput studies of stem cell fate.  

PubMed

We have developed a novel three-dimensional (3D) cellular microarray platform to enable the rapid and efficient tracking of stem cell fate and quantification of specific stem cell markers. This platform consists of a miniaturized 3D cell culture array on a functionalized glass slide for spatially addressable high-throughput screening. A microarray spotter was used to deposit cells onto a modified glass surface to yield an array consisting of cells encapsulated in alginate gel spots with volumes as low as 60 nL. A method based on an immunofluorescence technique scaled down to function on a cellular microarray was also used to quantify specific cell marker protein levels in situ. Our results revealed that this platform is suitable for studying the expansion of mouse embryonic stem (ES) cells as they retain their pluripotent and undifferentiated state. We also examined neural commitment of mouse ES cells on the microarray and observed the generation of neuroectodermal precursor cells characterized by expression of the neural marker Sox-1, whose levels were also measured in situ using a GFP reporter system. In addition, the high-throughput capacity of the platform was tested using a dual-slide system that allowed rapid screening of the effects of tretinoin and fibroblast growth factor-4 (FGF-4) on the pluripotency of mouse ES cells. This high-throughput platform is a powerful new tool for investigating cellular mechanisms involved in stem cell expansion and differentiation and provides the basis for rapid identification of signals and conditions that can be used to direct cellular responses. PMID:20069558

Fernandes, Tiago G; Kwon, Seok-Joon; Bale, Shyam Sundhar; Lee, Moo-Yeal; Diogo, Maria Margarida; Clark, Douglas S; Cabral, Joaquim M S; Dordick, Jonathan S

2010-05-01

12

Biology on a Chip: Microfabrication for Studying the Behavior of Cultured Cells  

PubMed Central

The ability to culture cells in vitro has revolutionized hypothesis testing in basic cell and molecular biology research and has become a standard methodology in drug screening and toxicology assays. However, the traditional cell culture methodology—consisting essentially of the immersion of a large population of cells in a homogeneous fluid medium—has become increasingly limiting, both from a fundamental point of view (cells in vivo are surrounded by complex spatiotemporal microenvironments) and from a practical perspective (scaling up the number of fluid handling steps and cell manipulations for high-throughput studies in vitro is prohibitively expensive). Micro fabrication technologies have enabled researchers to design, with micrometer control, the biochemical composition and topology of the substrate, the medium composition, as well as the type of neighboring cells surrounding the microenvironment of the cell. In addition, microtechnology is conceptually well suited for the development of fast, low-cost in vitro systems that allow for high-throughput culturing and analysis of cells under large numbers of conditions. Here we review a variety of applications of microfabrication in cell culture studies, with an emphasis on the biology of various cell types. PMID:15139302

Li, Nianzhen; Tourovskaia, Anna; Folch, Albert

2013-01-01

13

Type II pneumocytes in mixed cell culture of human lung: a light and electron microscopic study.  

PubMed Central

Alveolar Type II epithelial cells dedifferentiate rapidly in vitro. Studies with animal tissue suggest that cell-cell and extracellular matrix-cell interactions are important in the retention of Type II cell morphology in vitro. Thus, in this study with human tissue, alveolar Type II cells, alveolar macrophages, and spindle cells were prepared from the same sample of lung (obtained following lobectomy for cancer, n = 3), cocultured on glass cover slips or tissue culture plastic, and studied by light microscopy with scanning (SEM) and transmission (TEM) electron microscopy for 8 days. The primary cell isolates contained approximately 45% Type II cells; the remainder were macrophages or unidentifiable cells. Clusters, made up of a single layer of cuboidal Type II cells around a central core of connective tissue (largely collagen and some elastic tissue), formed above a monolayer of spindle cells. The Type II cells were morphologically similar to those seen in vivo. The cells were still cuboidal at 8 days but had lost their lamellar bodies, which were released into the medium via the apical surface. The clusters increased in size with time (area, microns 2: day 1, 29(5-143) x 10(2); day 8, 63(10-311) x 10(2); mean(range); p less than 0.02) without changing in number per culture, suggesting Type II cell proliferation. This may have been due to factors produced by the other cells and adherence to the extracellular matrix (ECM); (free collagen fibers, present in the original preparation, spindle cells, and/or Type II cells could be responsible for presence of ECM). We propose this as a useful model for the study of human Type II epithelial cells in vitro. Images FIGURE 1. a FIGURE 1. b FIGURE 1. c FIGURE 1. d FIGURE 1. e FIGURE 1. f FIGURE 2. a FIGURE 2. b FIGURE 2. c FIGURE 2. d FIGURE 2. e FIGURE 2. f FIGURE 2. g FIGURE 3. PMID:2384069

Bingle, L; Bull, T B; Fox, B; Guz, A; Richards, R J; Tetley, T D

1990-01-01

14

Establishment of feline intestinal epithelial cell cultures for the propagation and study of feline enteric coronaviruses.  

PubMed

Feline infectious peritonitis (FIP) is the most feared infectious cause of death in cats, induced by feline infectious peritonitis virus (FIPV). This coronavirus is a virulent mutant of the harmless, ubiquitous feline enteric coronavirus (FECV). To date, feline coronavirus (FCoV) research has been hampered by the lack of susceptible cell lines for the propagation of serotype I FCoVs. In this study, long-term feline intestinal epithelial cell cultures were established from primary ileocytes and colonocytes by simian virus 40 (SV40) T-antigen- and human Telomerase Reverse Transcriptase (hTERT)-induced immortalization. Subsequently, these cultures were evaluated for their usability in FCoV research. Firstly, the replication capacity of the serotype II strains WSU 79-1683 and WSU 79-1146 was studied in the continuous cultures as was done for the primary cultures. In accordance with the results obtained in primary cultures, FCoV WSU 79-1683 still replicated significantly more efficient compared to FCoV WSU 79-1146 in both continuous cultures. In addition, the cultures were inoculated with faecal suspensions from healthy cats and with faecal or tissue suspensions from FIP cats. The cultures were susceptible to infection with different serotype I enteric strains and two of these strains were further propagated. No infection was seen in cultures inoculated with FIPV tissue homogenates. In conclusion, a new reliable model for FCoV investigation and growth of enteric field strains was established. In contrast to FIPV strains, FECVs showed a clear tropism for intestinal epithelial cells, giving an explanation for the observation that FECV is the main pathotype circulating among cats. PMID:23964891

Desmarets, Lowiese M B; Theuns, Sebastiaan; Olyslaegers, Dominique A J; Dedeurwaerder, Annelike; Vermeulen, Ben L; Roukaerts, Inge D M; Nauwynck, Hans J

2013-01-01

15

Preparation of primary cultured mesenteric artery smooth muscle cells for fluorescent imaging and physiological studies  

Microsoft Academic Search

In this protocol, we describe a method for isolation and culture of smooth muscle cells derived from the adult rat (or mouse) superior mesenteric artery. Arterial myocytes are obtained by enzymatic dissociation and established in primary culture. The cultured cells retain expression of smooth muscle-specific ?-actin and physiological responses to agonists. Cultured arterial myocytes (prepared from wild-type or transgenic animals)

Mordecai P Blaustein; Vera A Golovina

2007-01-01

16

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

PubMed

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

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

2014-01-01

17

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

18

[A pilot study on the culture and differentiation of bone marrow stromal cells from SD rats].  

PubMed

In order to observe the growth, expansion and differentiation of the cultured bone marrow stromal cells (BMSC), we isolated the BMSC from adult SD rats and cultivated them with LIF and bFGF. Then, we cultured and induced the stem cells by using retinoic acid and the culture medium confected in our lab by ourselves. We found that the BMSC could expand and generate clones when they were cultured in vitro. These cells subcultured grew rapidly and differentiated into neuron-like cells and astrocyte-like cells. The results showed that BMSC have the abilities to self renew and differentiate, thus demonstrating the culture method we used is suitable for the culture of BMSC in vitro. The bone marrow stromal cell is not difficult to obtain; it is capable of expanding and differentiating in culture. If the culture condition is appropriate, it can differentiate into neuron and astrocyte. So, it is a kind of perfect seed cells. PMID:15022454

Li, Gang; Ke, Yiquan; Jiang, Xiaodan; Xu, Ruxiang; Zhou, Yuxi; Wang, Wei; Cheng, Wenping; Liao, Keli

2004-02-01

19

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

20

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

PubMed Central

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

21

PCL-coated hydroxyapatite scaffold derived from cuttlefish bone: in vitro cell culture studies.  

PubMed

In the present study, we examined the potential of using highly porous poly(?-caprolactone) (PCL)-coated hydroxyapatite (HAp) scaffold derived from cuttlefish bone for bone tissue engineering applications. The cell culture studies were performed in vitro with preosteoblastic MC3T3-E1 cells in static culture conditions. Comparisons were made with uncoated HAp scaffold. The attachment and spreading of preosteoblasts on scaffolds were observed by Live/Dead staining Kit. The cells grown on the HAp/PCL composite scaffold exhibited greater spreading than cells grown on the HAp scaffold. DNA quantification and scanning electron microscopy (SEM) confirmed a good proliferation of cells on the scaffolds. DNA content on the HAp/PCL scaffold was significantly higher compared to porous HAp scaffolds. The amount of collagen synthesis was determined using a hydroxyproline assay. The osteoblastic differentiation of the cells was evaluated by determining alkaline phosphatase (ALP) activity and collagen type I secretion. Furthermore, cell spreading and cell proliferation within scaffolds were observed using a fluorescence microscope. PMID:25063118

Milovac, Dajana; Gamboa-Martínez, Tatiana C; Ivankovic, Marica; Gallego Ferrer, Gloria; Ivankovic, Hrvoje

2014-09-01

22

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.

23

Morphogenesis of Coronavirus HCoV-NL63 in Cell Culture: A Transmission Electron Microscopic Study  

PubMed Central

NL63 (HCoV-NL63) is a recently discovered human coronavirus that causes respiratory disease in infants and young children. NL63 productively infects LLCMK2 cells and ciliated epithelial cells of human airway cell cultures. Transmission electron microscopic (TEM) studies of NL63 infected LLCMK2 cells revealed that virions are spherical, spiked, and range from 75 to 115 nm in diameter. Virus replication predominantly occurs on the rough endoplasmic reticulum (RER), both perinuclear and cytoplasmic, and the Golgi. Plasma membrane budding was occasionally observed. As virus production increased, aberrant viral forms appeared with greater frequency. Unusual inclusions were present in infected cells including tubular and laminated structures. Pleomorphic double membrane-bound vesicles (DMV), measuring roughly 140 to 210 nm in diameter, were observed. The virus was released via exocytosis and cell lysis. In summary, we report the key morphologic characteristics of NL63 infection observed by TEM analysis. PMID:19844604

Orenstein, Jan M.; Banach, Bridget; Baker, Susan C.

2009-01-01

24

Mammalian Cell Culture Simplified.  

ERIC Educational Resources Information Center

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

Moss, Robert; Solomon, Sondra

1991-01-01

25

In vitro culturing of ciliary respiratory cells—a model for studies of genetic diseases  

PubMed Central

Primary ciliary dyskinesia (PCD) is a rare genetic disorder caused by the impaired functioning of ciliated cells. Its diagnosis is based on the analysis of the structure and functioning of cilia present in the respiratory epithelium (RE) of the patient. Abnormalities of cilia caused by hereditary mutations closely resemble and often overlap with defects induced by the environmental factors. As a result, proper diagnosis of PCD is difficult and may require repeated sampling of patients’ tissue, which is not always possible. The culturing of differentiated cells and tissues derived from the human RE seems to be the best way to diagnose PCD, to study genotype–phenotype relations of genes involved in ciliary dysfunction, as well as other aspects related to the functioning of the RE. In this review, different methods of culturing differentiated cells and tissues derived from the human RE, along with their potential and limitations, are summarized. Several considerations with respect to the factors influencing the process of in vitro differentiation (cell-to-cell interactions, medium composition, cell-support substrate) are also discussed. PMID:21125367

Zi?tkiewicz, Ewa; Witt, Micha?

2010-01-01

26

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

PubMed Central

Background: This paper describes the derivation and characterization of a novel, conditionally immortal mammary epithelial cell line named KIM-2. These cells were derived from mid-pregnant mammary glands of a mouse harbouring one to two copies of a transgene comprised of the ovine ?-lactoglobulin milk protein gene promoter, driving expression of a temperature-sensitive variant of simian virus-40 (SV40) large T antigen (T-Ag). Results KIM-2 cells have a characteristic luminal epithelial cell morphology and a stable, nontransformed phenotype at the semipermissive temperature of 37°C. In contrast, at the permissive temperature of 33°C the cells have an elongated spindle-like morphology and become transformed after prolonged culture. Differentiation of KIM-2 cells at 37°C, in response to lactogenic hormones, results in the formation of polarized dome-like structures with tight junctions. This is accompanied by expression of the milk protein genes that encode ?-casein and whey acidic protein (WAP), and activation of the prolactin signalling molecule, signal transducer and activator of transcription (STAT)5. Fully differentiated KIM-2 cultures at 37°C become dependent on lactogenic hormones for survival and undergo extensive apoptosis upon hormone withdrawal, as indicated by nuclear morphology and flow cytometric analysis. KIM-2 cells can be genetically modified by stable transfection and clonal lines isolated that retain the characteristics of untransfected cells. Conclusion KIM-2 cells are a valuable addition, therefore, to currently available lines of mammary epithelial cells. Their capacity for extensive differentiation in the absence of exogenously added basement membrane, and ability to undergo apoptosis in response to physiological signals will provide an invaluable model system for the study of signal transduction pathways and transcriptional regulatory mechanisms that control differentiation and involution in the mammary gland. PMID:11056687

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

2000-01-01

27

Cell-culture bioreactors  

SciTech Connect

When contrasted with microbial fermentation, the characteristics having a bearing on the design and operation of cell-culture bioreactors are fragility, steam sensitivity and anchorage requirements of cells, heat lability and foaming of proteins and other components of cell culture media. Design details of agitation and gas supply, bearings, seals and drives, foam control and sterilization, temperature, oxygen and pH control, water, air and gas purification, liquid feeding and level control, gas exhaust analysis and disposal, handling of liquid effluent and bioreactor installation and scale up are given.

Beck, C.; Stiefel, H.; Stinnett, T.

1987-02-16

28

Development of intestinal cell culture models for drug transport and metabolism studies  

Microsoft Academic Search

Cell culture models offer many advantageous features for the analysis of drug transport and drug metabolism. From a basic research perspective, these systems offer the potential for manipulating the environment or cellular properties as a means to address mechanistic questions. From a drug discovery perspective, cell culture models can be used to expedite identification of compounds with favorable pharmacokinetic properties,

A. Quaroni; J. Hochman

1996-01-01

29

Design Study Conducted of a Stirred and Perfused Specimen Chamber for Culturing Suspended Cells on the International Space Station  

NASA Technical Reports Server (NTRS)

A tightly knit numerical/experimental collaboration among the NASA Ames Research Center, NASA Glenn Research Center, and Payload Systems, Inc., was formed to analyze cell culturing systems for the International Space Station. The Cell Culture Unit is a facility scheduled for deployment on the space station by the Cell Culture Unit team at Ames. The facility houses multiple cell specimen chambers (CSCs), all of which have inlets and outlets to allow for replenishment of nutrients and for waste removal. For improved uniformity of nutrient and waste concentrations, each chamber has a pair of counterrotating stir bars as well. Although the CSC can be used to grow a wide variety of organic cells, the current study uses yeast as a model cell. Previous work identified groundbased protocols for perfusion and stirring to achieve yeast growth within the CSC that is comparable to that for yeast cultures grown in a shaken Ehrlenmeyer flask.

Nelson, Emily S.; Kizito, John P.

2003-01-01

30

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

31

An Ideal Selective Anti-Cancer Agent In Vitro: I - Tissue Culture Study of Human Lung Cancer Cells A549  

Microsoft Academic Search

Management of cancer is one of the challenging problems in medical practice as there are no available medical modalities that can se- lectively kill cancer cells without adverse effect on normal living cells or the functions of vital organs. Tissue culture of human lung cancer cells (A549) was used in studying the effect of agent, PM 701, to test its

FATEN A. KHORSHID; SABAH S. MUSHREF; NAGWA T. HEFFNY

2005-01-01

32

Cultured proximal cells derived from transgenic mouse provide a model to study drug toxicity.  

PubMed

The effects of gentamicin on N-acetyl-beta-D-glucosaminidase (NAG) and acid phosphatase (AcP), two lysosomal enzymes present in proximal renal tubule cells, were studied in the PKSV-PCT cell line derived from proximal convoluted tubules from the kidney of a transgenic mouse carrying SV40 large T antigen under the control of the L-type pyruvate kinase gene. Gentamicin (400 micrograms/ml for 72 hr) did not alter cell viability, but significantly reduced cell growth and favored the formation of myeloid bodies. Gentamicin (50 to 800 micrograms/ml for 72 hr) decreased in a dose-dependent manner the cellular NAG in PKSV-PCT cells and stimulated its secretion by 20 to 60%. Chloroquine (50 to 100 microns) and ammonium chloride (NH4Cl, 30mM), two lysosomotropic amines known to stimulate the secretion of lysosomal enzymes in fibroblasts and macrophages, also stimulated secreted NAG in PKSV-PCT cells. However, the effect of chloroquine was less marked in PKSV-PCT cells than in cultured mouse 3T3 fibroblasts. Gentamicin induced lysosomal alkalinization but, in contrast to chloroquine and NH4Cl, the aminoside strongly stimulated the secretion of AcP. The secretion induced by gentamicin was nonpolarized, since the percentage of secreted NAG significantly increased from both the apical and basal sides of PKSV-PCT cells grown on permeable filters. Thus, these data suggest that gentamicin alters the secretion of NAG and AcP by a non-specific pathway and indicate that the PKSV-PCT cell line is a suitable system to examine the cellular action of drugs in kidney proximal tubule cells. PMID:7474657

Riccaldi, D; Robic, D; Bens, M; Cluzeaud, F; Wu, M S; Bourbouze, R; Vandewalle, A

1995-09-01

33

Viral infection in placenta relevant cells - a morphological and immunohistochemical cell culture study.  

PubMed

Viral infections in pregnancy are known to cause fetal malformation, growth restriction, and even fetal death. Macroscopic placental examination usually shows slight and unspecific changes. Histology may show secondary, non-specific tissue reaction, i.e. villitis with lymphocytic invasion. Primary specific morphology characteristics are known for some virus, like cytomegalovirus, parvovirus, and herpes simplex, however many viral infections show non-specific changes. Placenta relevant cells as human first trimester trophoblasts HTR8/SVneo, primary human umbilical vein endothelial cells (HUVEC), and primary human embryonic fibroblasts were examined following infection with commonly occurring virus like adenovirus and enterovirus. Morphology in routine stained sections and virus-specific immunostains were studied 4, 8, 24, 48, 72 h after infection. Nuclear enlargement was seen in the infected cells. A specific diagnosis of adenovirus or enterovirus infection, however, was not possible without specific immunostains. PMID:25244625

Turowski, Gitta; Rollag, Halvor; Roald, Borghild

2015-01-01

34

Perfusion Based Cell Culture Chips  

NASA Astrophysics Data System (ADS)

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

Heiskanen, A.; Emnéus, J.; Dufva, M.

35

Application of speckle dynamics for studying metabolic activity of cell cultures with herpes virus  

NASA Astrophysics Data System (ADS)

The report considers the results of the experiments in which digital values of light intensity I and the image area correlation index ? values were recorded on a real-time basis for one or two days. Three cell cultures with viruses along with intact cultures were investigated. High correlation of dependence of ? values on time t values was demonstrated for three cultures. The ?=?(t) and I=I(t) dependences for cells with and without viruses differ considerably. It was shown that the presence of viruses could be determined as early as ten minutes after measurements were started.

Vladimirov, A. P.; Bakharev, A. A.; Malygin, A. S.; Mikhaylova, J. A.; Borodin, E. M.; Poryvayeva, A. P.; Glinskikh, N. P.

2014-05-01

36

Culture of organized cell communities  

Microsoft Academic Search

Cells cultured in vitro will tend to retain their differentiated phenotype under conditions that resemble their natural in vivo environment, for example, when cultured on polymer scaffolds in tissue culture bioreactors. In this chapter, we define organized cell communities as three-dimensional in vitro grown cell–polymer constructs that display important structural and functional features of the natural tissue. We review representative

Lisa E. Freed; Gordana Vunjak-Novakovic

1998-01-01

37

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

38

Human microvascular endothelial tissue culture cell model for studying pathogenesis of Brazilian purpuric fever.  

PubMed

Brazilian purpuric fever (BPF) is a fulminant pediatric disease characterized by fever, with rapid progression to purpura, hypotensive shock, and death. All known BPF cases have been caused by three clones of Haemophilus influenzae biogroup aegyptius and have occurred in either Brazil or Australia. Using an immortalized line of human vascular endothelial cells, we developed an in vitro assay that identifies all known BPF-causing H. influenzae biogroup aegyptius strains (R. S. Weyant, F. D. Quinn, E. A. Utt, M. Worley, V. G. George, F. J. Candal, and E. W. Ades, J. Infect. Dis. 169:430-433, 1994). With multiplicities of infection (MOIs) as low as one bacterium per 1,000 tissue culture cells, BPF-associated strains produce a unique cytotoxic effect in which the tissue culture cells detach and aggregate in large floating masses after 48 h of incubation. In this study, using a BPF-associated strain and a non-BPF-associated control, we demonstrated that strains which produce the cytotoxic phenotype were able to replicate intracellularly whereas non-BPF-associated strains, with MOIs of > or = 1,000 did not replicate and did not produce the phenotype. We also showed that this phenotype is not caused by the activity of an endotoxin or the release of some other compound from the bacterial cell, since neither gamma irradiation-killed whole BPF clone bacteria nor bacterial cell fractions at MOIs of > 1,000 produced the cytotoxic effect. Furthermore, bacteria in numbers equal to MOIs of > 1,000 treated with chloramphenicol did not produce the cytotoxic phenotype, suggesting a requirement for bacterial protein synthesis. In addition, viable bacteria separated from the tissue culture monolayer by a 0.2-micron-pore-size membrane also failed to produce the phenotype. The ability of the bacterium to invade, replicate, and produce the phenotype appears to be primarily parasite directed since phagocytosis, pinocytosis, and eukaryotic protein synthesis inhibitors, including cycloheximide, cytochalasin D, and methylamine, had no effect on the ability of the bacterium to invade and cause a cytotoxic response. Understanding the basic mechanisms involved in this tissue-destructive process should enhance our knowledge of the general pathogenesis of BPF. PMID:7768615

Quinn, F D; Weyant, R S; Worley, M J; White, E H; Utt, E A; Ades, E A

1995-06-01

39

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

NASA Technical Reports Server (NTRS)

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

Sato, Atsushige

1993-01-01

40

High CD49f expression is associated with osteosarcoma tumor progression: a study using patient-derived primary cell cultures  

PubMed Central

Overall prognosis for osteosarcoma (OS) is poor despite aggressive treatment options. Limited access to primary tumors, technical challenges in processing OS tissues, and the lack of well-characterized primary cell cultures has hindered our ability to fully understand the properties of OS tumor initiation and progression. In this study, we have isolated and characterized cell cultures derived from four central high-grade human OS samples. Furthermore, we used the cell cultures to study the role of CD49f in OS progression. Recent studies have implicated CD49f in stemness and multipotency of both cancer stem cells and mesenchymal stem cells. Therefore, we investigated the role of CD49f in osteosarcomagenesis. First, single cell suspensions of tumor biopsies were subcultured and characterized for cell surface marker expression. Next, we characterized the growth and differentiation properties, sensitivity to chemotherapy drugs, and anchorage-independent growth. Xenograft assays showed that cell populations expressing CD49fhi/CD90lo cell phenotype produced an aggressive tumor. Multiple lines of evidence demonstrated that inhibiting CD49f decreased the tumor-forming ability. Furthermore, the CD49fhi/CD90lo cell population is generating more aggressive OS tumor growth and indicating this cell surface marker could be a potential candidate for the isolation of an aggressive cell type in OSs. PMID:24802970

Penfornis, Patrice; Cai, David Z; Harris, Michael R; Walker, Ryan; Licini, David; Fernandes, Joseph D A; Orr, Griffin; Koganti, Tejaswi; Hicks, Chindo; Induru, Spandana; Meyer, Mark S; Khokha, Rama; Barr, Jennifer; Pochampally, Radhika R

2014-01-01

41

Cultured Human Renal Cortical Cells  

NASA Technical Reports Server (NTRS)

During the STS-90 shuttle flight in April 1998, cultured renal cortical cells revealed new information about genes. Timothy Hammond, an investigator in NASA's microgravity biotechnology program was interested in culturing kidney tissue to study the expression of proteins useful in the treatment of kidney diseases. Protein expression is linked to the level of differentiation of the kidney cells, and Hammond had difficulty maintaining differentiated cells in vitro. Intrigued by the improvement in cell differentiation that he observed in rat renal cells cultured in NASA's rotating wall vessel (a bioreactor that simulates some aspects of microgravity) and during an experiment performed on the Russian Space Station Mir, Hammond decided to sleuth out which genes were responsible for controlling differentiation of kidney cells. To do this, he compared the gene activity of human renal cells in a variety of gravitational environments, including the microgravity of the space shuttle and the high-gravity environment of a centrifuge. Hammond found that 1,632 genes out of 10,000 analyzed changed their activity level in microgravity, more than in any of the other environments. These results have important implications for kidney research as well as for understanding the basic mechanism for controlling cell differentiation.

1998-01-01

42

A primary chicken tracheal cell culture system for the study of infection with avian respiratory viruses  

Technology Transfer Automated Retrieval System (TEKTRAN)

A major route of infection of avian influenza virus (AIV) and Newcastle disease virus (NDV) in chickens is through cells of the airway epithelium. Here we describe the development and optimization of conditions for culture of tracheal epithelial cells from chicken embryos as well as their use in st...

43

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 to investigate the effects of ultrafast laser pulses on soft biological tissues. We characterize the physical

Marjoribanks, Robin S.

44

Principles of cancer cell culture.  

PubMed

The basics of cell culture are now relatively common, though it was not always so. The pioneers of cell culture would envy our simple access to manufactured plastics, media and equipment for such studies. The prerequisites for cell culture are a well lit and suitably ventilated laboratory with a laminar flow hood (Class II), CO(2) incubator, benchtop centrifuge, microscope, plasticware (flasks and plates) and a supply of media with or without serum supplements. Not only can all of this be ordered easily over the internet, but large numbers of well-characterised cell lines are available from libraries maintained to a very high standard allowing the researcher to commence experiments rapidly and economically. Attention to safety and disposal is important, and maintenance of equipment remains essential. This chapter should enable researchers with little prior knowledge to set up a suitable laboratory to do basic cell culture, but there is still no substitute for experience within an existing well-run laboratory. PMID:21516394

Cree, Ian A

2011-01-01

45

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

Microsoft Academic Search

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

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

1986-01-01

46

In vitro study of low molecular weight heparin effect on cell growth and cell invasion in primary cell cultures of high-grade gliomas.  

PubMed

Heparins represent the first choice for prevention and treatment of venous thromboembolism. In particular, low molecular weight heparins (LMWHs) provide pharmacokinetic advantages compared to unfractionated heparin (UFH): longer half-life, better bioavailability, and lower binding to plasma proteins. In the last years results of preclinical and clinical studies have suggested that LMWH may be able to inhibit cell growth, cell invasion, and angiogenesis, which are key mechanisms involved in tumor progression, possibly influencing favorable clinical outcome in at least a proportion of cancer patients. In this work we investigated the effect of LMWH (enoxaparin) on cell growth and cell invasion in primary cell cultures obtained from high-grade glioma specimens: 5 anaplastic astrocytoma (AA) and 13 glioblastoma multiforme (GBM). Apoptosis and expression of the thrombin receptor PAR1 were also assessed. A significant decrease in tumor cell growth was observed after treatment with 10 U/ml (-21%; p = 0.001) and 100 U/ml (-26%; p < 0.001); tumor cells from AA (grade III; WHO) were more affected by LMWH treatment compared to cell lines from GBM (grade IV; WHO). The antiproliferative effect was more pronounced in cell cultures displaying higher expression of PAR1. Glioma cell cultures were able to invade a model of basement membrane (Matrigel matrix) in standard culture conditions, but migration was not modulated significantly by LMWH treatment at any of the concentrations tested (1, 10, 100 U/ml). In conclusion, our results confirm the antineoplastic effect of LMWH, suggesting a potential direct role on tumor cell growth in high grade gliomas. PMID:17294805

Balzarotti, Marco; Fontana, Federica; Marras, Carlo; Boiardi, Amerigo; Croci, Danilo; Ciusani, Emilio; Salmaggi, Andrea

2006-01-01

47

Sedimentation of agglomerated nanoparticles under cell culture conditions studied by image based analysis  

NASA Astrophysics Data System (ADS)

Toxic effects of nanoparticles can be analyzed with alveolar macrophages in vitro. To quantify exposure of cells to particles we analyzed the sedimentation of nanoparticle agglomerates in cell culture medium (MEM) by means of phase contrast microscopy. Particles were suspended by brief ultrasonication in MEM and pipetted into a glass bottom culture dish on the stage of a Nikon-Biostation under cell culture condition. Successive images were captured from the lowermost optical plane and were converted into binary images. The number of agglomerates (N) as well as the particle-covered area (A) were determined by image analyses. Typically, N increased to a maximum value before it partially decayed due to overlapping and/or optical interference of particles, and finally became constant. In contrast, A increased in a monophasic manner. By means of mathematical modeling we identified the endpoint of sedimentation of particle agglomerates, which is an important though a largely neglected event in most cell culture experiments. This endpoint could be calculated from an approximated model function. As the method can be employed in the presence of cells, a parallel evaluation of particle sedimentation and particle uptake appears possible.

Schippritt, Darius; Wiemann, Martin; Lipinski, Hans-Gerd

2010-04-01

48

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

Microsoft Academic Search

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

Denis Gospodarowicz; Gary Greenburg; Jorge Alvarado

1979-01-01

49

Animal and cell-culture models for the study of mycobacterial infections and treatment  

Microsoft Academic Search

Emerging problems with the treatment of infections caused byMycobacterium avium andMycobacterium tuberculosis require the development of new models, both in vitro and in vivo, in which new chemotherapeutic and immunotherapeutic approaches can be tested. In this brief review, the use of cell culture models, in which drugs can be tested for their capacity to inhibit mycobacterial growth within the infected

I. M. Orme; A. D. Roberts; S. K. Furney; P. S. Skinner

1994-01-01

50

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

51

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

52

Study on application of high doses plasmodium berghei in cell culture  

NASA Astrophysics Data System (ADS)

Malaria, one of the most important infection disease problems in the world, is caused by protozoan parasites of the genus Plasmodium. This disease is responsible for hundreds of the millions of clinical cases and more than one million deaths per year, for this reason, malaria is a priority and the WHO estimates that half of the world population is at risk. In this work we study how the absorbed dose inactivates the parasite (Plasmodium berghei) in rodent model (BALB/c mice), by applying X-ray irradiation. The dose was increased from 10 to 50 Gy in parasitized red blood cells (PRBC) with merozoite stage using in vitro short cultures. Also the reduction of the irradiation effect was determined by intra-peritoneal inoculations of irradiated parasites. Afterwards, the parasitaemia was assessed daily on smears made from tail blood and stained with Giemsa's reagent. Besides, the effect of irradiation was evaluated using an immunological test as indirect immunofluorescence assay (IFA). The results of this study showed that the most effective radiation for inactivation of parasites is about 50 Gy and the immunofluorescence pattern showed a different distribution of the fluorescence on parasites. These results showed direct correlation between the effect of irradiated parasites and parasitaemia in the group of mice infected with RBC after 50 Gy irradiation. Our results indicated that the threshold is between 30 to 50 Gy to inactivate the parasites.

Spencer, L. M.; De Santis, M.; Davila, J.; Foinquinos, A.; Salcedo, E.; Sajo-Bohus, L.

2012-02-01

53

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

54

Genotoxicity studies of methyl isocyanate in Salmonella, Drosophila, and cultured Chinese hamster ovary cells  

SciTech Connect

The genotoxic effects of methyl isocyanate (MIC) were investigated using four short-term tests: the Salmonella reversion assay (Ames test), the Drosophila sex-linked recessive lethal assay, and the sister chromatic exchange (SCE) and chromosomal aberration assays in cultured Chinese hamster ovary (CHO) cells. No evidence was found for the induction of mutations in either Salmonella or Drosophila. MIC did, however, induce SCEs and chromosomal aberrations in CHO cells both in the presence and absence of Aroclor-induced rat liver S-9.

Mason, J.M.; Zeiger, E.; Haworth, S.; Ivett, J.; Valencia, R.

1987-01-01

55

Culture of Human Leukaemia Cells  

Microsoft Academic Search

THIS communication describes the culture of four additional cell lines derived from the buffy coats of patients with leukaemia. Iwakata and Grace1 provided key information for culturing leukaemia cells and reported the establishment of a cell line, R.P.M.I. No. 6410. Fifteen cell lines were subsequently derived from the buffy coats of four patients with acute and chronic myelocytic leukaemia and

G. E. Moore; E. Ito; P. Citron

1966-01-01

56

Automatic monitoring of biochemical parameters in tissue culture. Studies on synchronously growing mouse myeloma cells  

PubMed Central

An instrument is described that will maintain a population of mammalian cells at constant cell density while automatically monitoring the growth rate of the culture and the extent of precursor incorporation into a variety of cell products. The apparatus was used in an investigation of cyclic changes in the incorporation of labelled precursors into the DNA, RNA, total protein and myeloma protein synthesized in synchronous cultures of a mouse myeloma line. The incorporation of [3H]uridine into trichloroacetic acid-insoluble material reveals a slight periodicity, with maxima and minima corresponding to late S phase and the mitotic phases respectively. The incorporation of [3H]lysine into total intracellular protein also shows a slight oscillation, with maxima and minima occurring during the respective G2 and mitotic phases. Cyclical changes in the synthesis of serologically precipitable myeloma protein were found to vary somewhat according to the conditions used to synchronize the cells. In experiments conducted with 4.0mm-thymidine, maximal incorporation of label took place during S phase or early G2 phase. Experiments with 1.0mm-thymidine revealed a significantly less marked periodicity of myeloma protein synthesis. PMID:4673519

Cowan, N. J.; Milstein, C.

1972-01-01

57

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

PubMed Central

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

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

2005-01-01

58

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...

59

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

60

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

PubMed Central

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

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

2013-01-01

61

Cultured Porcine Coronary Artery Smooth Muscle Cells  

Microsoft Academic Search

Abstract—Arterial intimal thickening after endothelial injury induced in rodents has proven to be a relatively unreliable model of restenosis for testing clinically useful compounds. The same has been found for cultured rat or rabbit vascular smooth muscle cells (SMCs). To test alternative possibilities, we have studied several differentiation features of porcine coronary artery SMCs, cultured up to the 5th passage

Thomas Christen; Marie-Luce Bochaton-Piallat; Pascal Neuville; Sander Rensen; Mireille Redard; Guillaume van Eys; Giulio Gabbiani

62

Renal Tubular Cells Cultured from Genetically Modified Animals  

Microsoft Academic Search

The culture of renal tubular cells from genetically modified animals opens the opportunity of biochemical, cell biology and physiological studies under strictly controlled conditions. Either primary cultures or cell lines can be used. Through two examples of primary cultures of proximal tubular cells obtained from knock-out mice, important information about the function of proteins were obtained. Mice lacking vimentin, an

Gérard Friedlander; Isabelle Runembert; François Vrtovsnik; Fabiola Terzi

1999-01-01

63

Behaviour in culture of isolated protoplasts from “Paul's scarlet” rose suspension culture cells  

Microsoft Academic Search

Summary Protoplasts were enzymatically isolated from “Paul's scarlet” rose suspension culture cells. They were cultured in medium similar to that used to culture the cells from which they were isolated with the addition of sucrose as an osmotic stabiliser. They were studied by light and electron microscopy and their changes in size and number per culture were recorded. Expansion was

R. S. Pearce; E. C. Cocking

1973-01-01

64

A study of murine bone marrow cells cultured in bioreactors which create an environment which simulated microgravity  

NASA Technical Reports Server (NTRS)

Previous research indicated that mouse bone marrow cells could be grown in conditions of simulated microgravity. This environment was created in rotating bioreactor vessels. On three attempts mouse cells were grown successfully in the vessels. The cells reached a stage where the concentrations were doubling daily. Phenotypic analysis using a panel of monoclonal antibodies indicated that the cell were hematopoietic pluripotent stem cells. One unsuccessful attempt was made to reestablish the immune system in immunocompromised mice using these cells. Since last summer, several unsuccessful attempts were made to duplicate these results. It was determined by electron microscopy that the cells successfully grown in 1989 contained virus particles. It was suggested that these virally parasitized cells had been immortalized. The work of this summer is a continuation of efforts to grow mouse bone marrow in these vessels. A number of variations of the protocol were introduced. Certified pathogen free mice were used in the repeat experiments. In some attempts the medium of last summer was used; in others Dexture Culture Medium containing Iscove's Medium supplemented with 20 percent horse serum and 10-6 M hydrocortisone. Efforts this summer were directed solely to repeating the work of last summer. Plans were made for investigations if stem cells were isolated. Immortalization of the undifferentiated stem cell would be attempted by transfection with an oncogenic vector. Selective differentiation would be induced in the stem cell line by growing it with known growth factors and immune response modulators. Interest is in identifying any surface antigens unique to stem cells that would help in their characterization. Another goal was to search for markers on stem cells that would distinguish them from stem cells committed to a particular lineage. If the undifferentiated hematopoietic stem cell was obtained, the pathways that would terminally convert it to myeloid, lyphoid, erythroid, or other cell lines would be studied. Transfection with a known gene would be attempted and then conversion to a terminally identifiable cell.

Lawless, Brother Desales

1990-01-01

65

Prostate cell cultures as in vitro models for the study of normal stem cells and cancer stem cells  

Microsoft Academic Search

Current existing therapies for prostate cancer eradicate the majority of cells within a tumor. However, most patients with advanced cancer still progress to androgen-independent metastatic disease that remains essentially incurable by current treatment strategies. Recent evidence has shown that cancer stem cells (CSCs) are a subset of the tumor cells that are responsible for initiating and maintaining the disease. Understanding

J Miki; J S Rhim

2008-01-01

66

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...

67

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

68

Transcriptomic responses to sodium chloride-induced osmotic stress: a study of industrial fed-batch CHO cell cultures.  

PubMed

The rapidly expanding market for monoclonal antibody and Fc-fusion-protein therapeutics has increased interest in improving the productivity of mammalian cell lines, both to alleviate capacity limitations and control the cost of goods. In this study, we evaluated the responses of an industrial CHO cell line producing an Fc-fusion-protein to hyperosmotic stress, a well-known productivity enhancer, and compared them with our previous studies of murine hybridomas (Shen and Sharfstein, Biotechnol Bioeng. 2006;93:132-145). In batch culture studies, cells showed substantially increased specific productivity in response to increased osmolarity as well as significant metabolic changes. However, the final titer showed no substantial increase due to the decrease in viable cell density. In fed batch cultures, hyperosmolarity slightly repressed the cellular growth rate, but no significant change in productivity or final titer was detected. To understand the transcriptional responses to increased osmolarity and relate changes in gene expression to increased productivity and repressed growth, proprietary CHO microarrays were used to monitor the transcription profile changes in response to osmotic stress. A set of osmotically regulated genes was generated and classified by extracting their annotations and functionalities from online databases. The gene list was compared with results previously obtained from similar studies of murine-hybridoma cells. The overall transcriptomic responses of the two cell lines were rather different, although many functional groups were commonly perturbed between them. Building on this study, we anticipate that further analysis will establish connections between productivity and the expression of specific gene(s), thus allowing rational engineering of mammalian cells for higher recombinant-protein productivity. PMID:20306541

Shen, Duan; Kiehl, Thomas R; Khattak, Sarwat F; Li, Zheng Jian; He, Aiqing; Kayne, Paul S; Patel, Vishal; Neuhaus, Isaac M; Sharfstein, Susan T

2010-01-01

69

Cell culture processes for monoclonal antibody production  

PubMed Central

Animal cell culture technology has advanced significantly over the last few decades and is now generally considered a reliable, robust and relatively mature technology. A range of biotherapeutics are currently synthesized using cell culture methods in large scale manufacturing facilities that produce products for both commercial use and clinical studies. The robust implementation of this technology requires optimization of a number of variables, including (1) cell lines capable of synthesizing the required molecules at high productivities that ensure low operating cost; (2) culture media and bioreactor culture conditions that achieve both the requisite productivity and meet product quality specifications; (3) appropriate on-line and off-line sensors capable of providing information that enhances process control; and (4) good understanding of culture performance at different scales to ensure smooth scale-up. Successful implementation also requires appropriate strategies for process development, scale-up and process characterization and validation that enable robust operation and ensure compliance with current regulations. This review provides an overview of the state-of-the art technology in key aspects of cell culture, e.g., generation of highly productive cell lines and optimization of cell culture process conditions. We also summarize the current thinking on appropriate process development strategies and process advances that might affect process development. PMID:20622510

Li, Feng; Vijayasankaran, Natarajan; Shen, Amy (Yijuan); Kiss, Robert

2010-01-01

70

156 Literary and Cultural Studies Literary and Cultural Studies  

E-print Network

156 · Literary and Cultural Studies Literary and Cultural Studies Advisory Committee: Knight Center), Joyce (English), Kennedy (English ), Lowry (English), MacGovern (History/American Studies), Mac). The program in Literary and Cultural Studies brings an interdis- ciplinary perspective to the study of culture

Lewis, Robert Michael

71

USF Graduate Studies Cultural Studies  

E-print Network

Humanities USF Graduate Studies Cultural Studies and The Master of Arts in American Studies offers) is an interdisciplinary, interdepartmental program that offers students an opportunity to study the ideas and works that have - The MLA in Humanities track offers an interdisciplinary approach to the study of European

Meyers, Steven D.

72

Comparison of Artery Organ Culture and Co-culture Models for Studying Endothelial Cell Migration and Its Effect on Smooth Muscle Cell Proliferation and Migration  

Microsoft Academic Search

Arterial restenosis associated with intimal hyperplasia is the major cause of long-term failure of vascular interventions.\\u000a Endothelium injury and the proliferation and migration of smooth muscle cells (SMC) are key events in the development of intimal\\u000a hyperplasia. The objectives of this study were to develop an ex vivo artery injury model for studying endothelial cell (EC) migration and to compare

Yong-Ung Lee; Jian Luo; Eugene Sprague; Hai-Chao Han

2010-01-01

73

Advances in cell culture: anchorage dependence.  

PubMed

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

Merten, Otto-Wilhelm

2015-02-01

74

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

75

Studies on fenestral contraction in rat liver endothelial cells in culture.  

PubMed Central

Liver endothelial cells possess fenestrae, which are pores supported by a cytoskeleton ring composed of actin and myosin. Fenestrae are dynamic structures that can contract or dilate, although the mechanism for this phenomenon remains to be elucidated. Staining of actin and/or of myosin permitted measurement of fenestral diameter and area in cultured rat liver endothelial cells using digitized video-intensified fluorescence microscopy with image analysis. Within 1 minute of incubation with 0.1 micromol/L serotonin, fenestral diameter and area decreased by 24 +/- 5% and 56 +/- 7%, respectively. Contraction of fenestrae by serotonin was inhibited by chelation of extracellular Ca2+ with EGTA and by addition of Ca2+ channel blockers, such as dilthiazem and verapamil. The response of fenestrae to serotonin was mimicked by addition of a Ca2+ ionophore, A23187. Serotonin inhibited cAMP production, had no effect on inositol phosphate production, and activated phospholipase A2, causing release of arachidonic acid. These results suggest that contraction of fenestrae is associated with Ca2+ influx. In response to 0.1 micromol/serotonin, intracellular Ca2+ levels increased within 3 to 5 seconds from 150 nmol/L to >400 nmol/l followed by rapid phosphorylation of the 20-kd subunit of myosin light chain; both events dependent on extracellular Ca2+. Images Figure 1 Figure 2 Figure 11 PMID:8669487

Gatmaitan, Z.; Varticovski, L.; Ling, L.; Mikkelsen, R.; Steffan, A. M.; Arias, I. M.

1996-01-01

76

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

77

An Immunohistochemical Method to Study Breast Cancer Cell Subpopulations and Their Growth Regulation by Hormones in Three-Dimensional Cultures  

PubMed Central

The development of in vitro three-dimensional cell culture matrices offers physiologically relevant alternatives to traditional culture on plastic surfaces. However methods to analyze cell subpopulations therein are poor. Here we present a simple and inexpensive method to analyze cell subpopulations in mixed-cell colonies using standard immunohistochemical (IHC) techniques. Briefly, Matrigel™ blocks are sandwiched between two layers of HistoGel™, hardened by rapid cooling then processed for routine fixation, paraffin embedding, and IHC. We demonstrate the assay using mono- and co-cultured normal human breast, human breast cancer, and transformed mouse stromal cells along with hormone treated breast cancer cells. Judicious selection of specific antibodies allows different cell types within heterotypic colonies to be identified. A brief pulse of bromodeoxyuridine in living colonies allows proliferation of cell subpopulations to be quantified. This simple assay is useful for multiple cell types, species, and conditions. PMID:22649363

Pinto, Mauricio P.; Jacobsen, Britta M.; Horwitz, Kathryn B.

2011-01-01

78

An immunohistochemical method to study breast cancer cell subpopulations and their growth regulation by hormones in three-dimensional cultures.  

PubMed

The development of in vitro three-dimensional cell culture matrices offers physiologically relevant alternatives to traditional culture on plastic surfaces. However methods to analyze cell subpopulations therein are poor. Here we present a simple and inexpensive method to analyze cell subpopulations in mixed-cell colonies using standard immunohistochemical (IHC) techniques. Briefly, Matrigel™ blocks are sandwiched between two layers of HistoGel™, hardened by rapid cooling then processed for routine fixation, paraffin embedding, and IHC. We demonstrate the assay using mono- and co-cultured normal human breast, human breast cancer, and transformed mouse stromal cells along with hormone treated breast cancer cells. Judicious selection of specific antibodies allows different cell types within heterotypic colonies to be identified. A brief pulse of bromodeoxyuridine in living colonies allows proliferation of cell subpopulations to be quantified. This simple assay is useful for multiple cell types, species, and conditions. PMID:22649363

Pinto, Mauricio P; Jacobsen, Britta M; Horwitz, Kathryn B

2011-01-01

79

In vitro culture studies of Sutherlandia frutescens on human tumor cell lines.  

PubMed

Sutherlandia frutescens is a South African herb used traditionally by the natives to treat cancer, and more recently to improve the overall health in HIV/AIDS patients. Gas chromatography/mass spectrometer profiling and liquid chromatographic/mass spectral investigation confirmed and quantified the presence of canavanine, GABA and arginine in the herbal preparation used in this study. In vitro study demonstrated a concentration dependent effect of Sutherlandia on several tumor cell lines, with 50% inhibition (IC50) of proliferation of MCF7, MDA-MB-468, Jurkat and HL60 cells at 1/250, 1/200, 1/150 and 1/200 dilutions, respectively. Sutherlandia treatment did not induce HL60 differentiation along the macrophage/monocyte or granulocyte lineage. It demonstrated antioxidant activity in reducing free radical cations with an estimated activity of 0.5 microl of Sutherlandia extract equivalent to that of 10 microM of Trolox. However, it did not significantly suppress lipopolysaccharide stimulated nitric oxide production by murine macrophage/monocyte RAW 264.7 cells, nor did it significantly inhibit IL-1beta and TNF-alpha mRNA expression in RAW 264.7 cells. In conclusion, Sutherlandia ethanolic extract showed a concentration dependent antiproliferative effect on several human tumor cell lines but did not show significant antioxidant effects. Further studies are needed to explore the activities of this multipurpose South African herbal preparation. PMID:15182898

Tai, Joseph; Cheung, Susan; Chan, Edwin; Hasman, David

2004-07-01

80

Aseptic technique for cell culture.  

PubMed

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

Coté, R J

2001-05-01

81

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

Microsoft Academic Search

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

JERRY S. SUTTON; LEON WEISS

1966-01-01

82

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. PMID:25206215

Al-Jundi, Suhad; Mhaidat, Nizar

2013-01-01

83

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

PubMed

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

Halama, Anna

2014-12-15

84

Photoactivable sphingosine as a tool to study membrane microenvironments in cultured cells[S  

PubMed Central

Human fibroblasts from normal subjects and Niemann-Pick A (NPA) disease patients were fed with two labeled metabolic precursors of sphingomyelin (SM), [3H]choline and photoactivable sphingosine, that entered into the biosynthetic pathway allowing the synthesis of radioactive phosphatidylcholine and SM, and of radioactive and photoactivable SM ([3H]SM-N3). Detergent resistant membrane (DRM) fractions prepared from normal and NPA fibroblasts resulted as highly enriched in [3H]SM-N3. However, lipid and protein analysis showed strong differences between the two cell types. After cross-linking, different patterns of SM-protein complexes were found, mainly associated with the detergent soluble fraction of the gradient containing most cell proteins. After cell surface biotinylation, DRMs were immunoprecipitated using streptavidin. In conditions that maintain the integrity of domain, SM-protein complexes were detectable only in normal fibroblasts, whereas disrupting the membrane organization, these complexes were not recovered in the immunoprecipitate, suggesting that they involve proteins belonging to the inner membrane layer. These data suggest that differences in lipid and protein compositions of these cell lines determine specific lipid-protein interactions and different clustering within plasma membrane. In addition, our experiments show that photoactivable sphingolipids metabolically synthesized in cells can be used to study sphingolipid protein environments and sphingolipid-protein interactions. PMID:19820263

Aureli, Massimo; Prioni, Simona; Mauri, Laura; Loberto, Nicoletta; Casellato, Riccardo; Ciampa, Maria Grazia; Chigorno, Vanna; Prinetti, Alessandro; Sonnino, Sandro

2010-01-01

85

Photoactivable sphingosine as a tool to study membrane microenvironments in cultured cells.  

PubMed

Human fibroblasts from normal subjects and Niemann-Pick A (NPA) disease patients were fed with two labeled metabolic precursors of sphingomyelin (SM), [(3)H]choline and photoactivable sphingosine, that entered into the biosynthetic pathway allowing the synthesis of radioactive phosphatidylcholine and SM, and of radioactive and photoactivable SM ([(3)H]SM-N(3)). Detergent resistant membrane (DRM) fractions prepared from normal and NPA fibroblasts resulted as highly enriched in [(3)H]SM-N(3). However, lipid and protein analysis showed strong differences between the two cell types. After cross-linking, different patterns of SM-protein complexes were found, mainly associated with the detergent soluble fraction of the gradient containing most cell proteins. After cell surface biotinylation, DRMs were immunoprecipitated using streptavidin. In conditions that maintain the integrity of domain, SM-protein complexes were detectable only in normal fibroblasts, whereas disrupting the membrane organization, these complexes were not recovered in the immunoprecipitate, suggesting that they involve proteins belonging to the inner membrane layer. These data suggest that differences in lipid and protein compositions of these cell lines determine specific lipid-protein interactions and different clustering within plasma membrane. In addition, our experiments show that photoactivable sphingolipids metabolically synthesized in cells can be used to study sphingolipid protein environments and sphingolipid-protein interactions. PMID:19820263

Aureli, Massimo; Prioni, Simona; Mauri, Laura; Loberto, Nicoletta; Casellato, Riccardo; Ciampa, Maria Grazia; Chigorno, Vanna; Prinetti, Alessandro; Sonnino, Sandro

2010-04-01

86

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

PubMed Central

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

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

2014-01-01

87

Circadian gene expression in cultured cells.  

PubMed

In mammals, circadian oscillators not only exist in specialized neurons of the suprachiasmatic nucleus, but in almost all peripheral cell types. These oscillators are operative even in established fibroblast cell lines, such as Rat-1 cells or NIH3T3 cells, and in primary fibroblasts from mouse embryos or adult animals. This can be demonstrated by treating such cells for a short time period with high concentrations of serum or chemicals that activate a large number of known signaling pathways. The possibility of studying circadian rhythms in cultured cells should facilitate the biochemical and genetic dissection of the circadian clockwork and should promote the discovery of new clock components. PMID:15817311

Nagoshi, Emi; Brown, Steven A; Dibner, Charna; Kornmann, Benoît; Schibler, Ueli

2005-01-01

88

Cell culture compositions  

DOEpatents

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

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

2014-03-18

89

Versatile, Fully Automated, Microfluidic Cell Culture System  

E-print Network

Versatile, 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 and by the emergence of systems biology. We built a fully automated cell culture screening system based

Chen, Christopher S.

90

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

E-print Network

(WAP), and activation of the prolactin signalling molecule, signal transducer and activator of transcription (STAT)5. Fully differentiated KIM-2 cultures at 37°C become dependent on lactogenic hormones for survival and undergo extensive apoptosis upon...

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

91

Improvements in the primary culture of neonate rat myocardial cells by study of the mechanism of endoplasmic reticulum stress.  

PubMed

We previously found that endoplasmic reticulum stress (ERS) might be exhibited in the conventional protocol of the primary culture of neonate rat myocardial cells (NRMCs) and that the high glucose concentration (25 mmol/L) in the culture medium might be the cause. Here, we investigated if the high concentration of glucose might influence ERS in myocardial cells during culture. GRP78 expression (ERS marker) was similar in groups with tunicamycin (TM) and without TM in high glucose cultured cells (p > 0.01). Different glucose concentrations elicited different GRP78 expressions according to analyses of protein and RNA levels, which showed ERS in H/H groups. Finally, we found that GRP78 expression was higher in TM groups compared with M/M groups (p < 0.01). The conventional high-glucose culture media during primary culture of NRMCs induced ERS. We propose that medium-glucose culture media should be used and describe an improved protocol for the primary culture of NRMCs. PMID:23329407

Wang, Qiqi; Jin, Jianmei; Guo, Zhidong; Chen, Fuxu; Qiu, Yuangang; Zhu, Jianhua; Shang, Yunpeng

2013-05-01

92

Mechanism of thiamine uptake by human colonocytes: studies with cultured colonic epithelial cell line NCM460.  

PubMed

Thiamine (vitamin B(1)) is essential for normal cellular functions and growth. Mammals cannot synthesize thiamine and thus must obtain the vitamin via intestinal absorption. The intestine is exposed to a dietary thiamine source and a bacterial source in which the vitamin is synthesized by the normal microflora of the large intestine. Very little is known about thiamine uptake in the large intestine. The aim of this study was, therefore, to address this issue. Our results with human-derived colonic epithelial NCM460 cells as a model system showed thiamine uptake to be 1) temperature- and energy dependent, 2) Na(+) independent, 3) increased with increasing buffer pH from 5 to 8 and after cell acidification but inhibited by amiloride, 4) saturable as a function of concentration, 5) inhibited by thiamine structural analogs but not by unrelated organic cations, and 6) inhibited by modulators of a Ca(2+)/calmodulin-mediated pathway. NCM460 cells and native human colonic mucosa expressed the recently cloned human thiamine transporter THTR-1 (product of the SLC19A2 gene) at both mRNA and protein levels. These results demonstrate for the first time that human NCM460 colonocytes possess a specific carrier-mediated system for thiamine uptake that appears to be under the regulation of an intracellular Ca(2+)/calmodulin-mediated pathway. It is suggested that bacterially synthesized thiamine in the large intestine may contribute to thiamine nutrition of the host, especially toward cellular nutrition of the local colonocytes. PMID:11408266

Said, H M; Ortiz, A; Subramanian, V S; Neufeld, E J; Moyer, M P; Dudeja, P K

2001-07-01

93

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

94

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

Microsoft Academic Search

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

GEORGE J. TODARO; HOWARD GREEN

1963-01-01

95

Cellular and molecular aspects of thiamin uptake by human liver cells: studies with cultured HepG2 cells.  

PubMed

The liver is an important site for thiamin metabolism, utilization, and storage. Little is known about the mechanism of thiamin uptake by the human liver. In this study, we examined cellular and molecular aspects of the human liver thiamin uptake process using the human-derived liver HepG2 cells as a model system. Our studies showed that the initial rate of thiamin uptake to be: (1) Na(+)-independent and occurs with no detectable metabolic alterations in the transported substrate, (2) highly pH-dependent with diminished uptake upon decreasing incubation buffer pH from 8.0 to 5.0, (3) higher following cell acidification compared to unacidified control cells, (4) saturable as a function of concentration with an apparent K(m) of 7.7+/-1.6 microM, (5) inhibited by the thiamin structural analogues oxythiamin and amprolium but not by the unrelated organic cations tetraethylammonium (TEA) and N-methylnicotinamide (NMN), and (6) inhibited in a concentration-dependent manner by the membrane transport inhibitor amiloride. Both of the recently cloned human thiamin transporters, i.e., SLC19A2 and SLC19A3, were found to be expressed in liver HepG2 cells with the former being the predominant form. High promoter activity of the predominant form, i.e., SLC19A2, was detected in HepG2 cells, and the minimal region of the SLC19A2 promoter required for its basal activity in these cells was found to be encoded in a sequence between -356 and -36 and has multiple putative cis-regulatory elements. Mutation of a number of these putative cis-elements diminished promoter activity of the SLC19A2 minimal region. These results show the involvement of a specialized carrier-mediated mechanism for thiamin uptake by human liver HepG2 cells. In addition, SLC19A2 was found to be the predominant thiamin uptake carrier expressed in these cells and its promoter displays a high level of activity in them. PMID:12488043

Said, Hamid M; Reidling, Jack C; Ortiz, Alvaro

2002-12-23

96

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

97

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

98

9 CFR 101.6 - Cell cultures.  

Code of Federal Regulations, 2014 CFR

...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

99

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

100

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

101

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

102

Density gradient electrophoresis of cultured human embryonic kidney cells  

NASA Technical Reports Server (NTRS)

Ground based confirmation of the electrophoretic heterogeneity of human embryonic kidney cell cultures, the general characterization of their electrophoretic migration, and observations on the general properties of cultures derived from electrophoretic subpopulations were studied. Cell migration in a density gradient electrophoresis column and cell electrophoretic mobility was determined. The mobility and heterogeneity of cultured human embryonic kidney cells with those of fixed rat erythrocytes as model test particle was compared. Electrophoretically separated cell subpopulations with respect to size, viability, and culture characteristics were examined.

Plank, L. D.; Kunze, M. E.; Giranda, V.; Todd, P. W.

1985-01-01

103

Studies of a human isolate of Anaplasma phagocytophilum in cultured tick cells, ticks, and sheep.  

E-print Network

??The proteome of the Ixodes scapularis-derived cell line, ISE6, was characterized after infection with the tick-borne pathogen, Anaplasma phagocytophilum. Proteomics were used to identify proteins… (more)

Busby, Ann Taylor

2011-01-01

104

Progress Towards Drosophila Epithelial Cell Culture  

PubMed Central

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

Simcox, Amanda

2015-01-01

105

Plastid transformation of tobacco suspension cell cultures.  

PubMed

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

Staub, Jeffrey M

2014-01-01

106

Use of chemically patterned substrate to study directional effect of damaging electrical stimulation on cultured neuroblastoma cells.  

PubMed

We used ordered arrangements of neuroblastoma cells in culture on chemically patterned substrates to direct the orientation of electrical stimulation with respect to cell alignment. Chemically patterned parallel lines of self-assembled monolayer films were fabricated on glass substrates via a deep UV lithographic procedure. Cultured neuroblastoma cells deposited on these substrates formed long (approximately 300 microns in length) neuritic processes along the patterned lines in the presence of retinoic acid. Cells attached to the surface of these substrates were placed in a stimulation chamber so that an electric field (1.4-1.9 V/cm) could be applied in the direction parallel or perpendicular to neuritic or orientation. A majority of cells aligned parallel to the orientation of electrical stimulation exhibited a variety of cellular responses including neuritic tip damage, reductions in neuritic length and varicosity formation. These effects were observed to a lesser degree on the cells when electrical stimulation with the same magnitude was applied perpendicularly to the cell alignment. This work supports earlier findings that geometry is a crucial factor in determining cellular response to applied electric fields and goes on to show that cellular orientation is a key factor in determining cellular damage in culture. PMID:7895082

Matsuzawa, M; Potember, R S; Krauthamer, V

1994-12-19

107

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

108

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

109

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

110

Selection of Suitable Prodrug Candidates for in vivo Studies via in vitro Studies; The Correlation of Prodrug Stability in Between Cell Culture Homogenates and Human Tissue Homogenates  

PubMed Central

Purpose To determine the correlations/discrepancies of drug stabilities between in the homogenates of human culture cells and of human tissues. Methods Amino acid/dipeptide monoester prodrugs of floxuridine were chosen as the model drugs. The stabilities (half-lives) of floxuridine prodrugs in human tissues (pancreas, liver, and small intestine) homogenates were obtained and compared with ones in cell culture homogenates (AcPC-1, Capan-2, and Caco-2 cells) as well as human liver microsomes. The correlations of prodrug stability in human small bowel tissue homogenate vs. Caco-2 cell homogenate, human liver tissue homogenate vs. human liver microsomes, and human pancreatic tissue homogenate vs. pancreatic cell, AsPC-1 and Capan-2, homogenates were examined. Results The stabilities of floxuridine prodrugs in human small bowel homogenate exhibited the great correlation to ones in Caco-2 cell homogenate (slope = 1.0–1.3, r2 = 0.79–0.98). The stability of those prodrugs in human pancreas tissue homogenate also exhibited the good correlations to ones in AsPC-1 and Capan-2 cells homogenates (slope = 0.5–0.8, r2 = 0.58–0.79). However, the correlations of prodrug stabilities between in human liver tissue homogenates and in human liver microsomes were weaker than others (slope = 1.3–1.9, r2 = 0.07–0.24). Conclusions The correlations of drug stabilities in cultured cell homogenates and in human tissue homogenates were compared. Those results exhibited wide range of correlations between in cell homogenate and in human tissue homogenate (r2 = 0.07 – 0.98). Those in vitro studies in cell homogenates would be good tools to predict drug stabilities in vivo and to select drug candidates for further developments. In the series of experiments, 5?-O-D-valyl-floxuridine and 5?-O-L-phenylalanyl-L-tyrosyl-floxuridine would be selected as candidates of oral drug targeting delivery for cancer chemotherapy due to their relatively good stabilities compared to other tested prodrugs. PMID:22974791

Tsume, Yasuhiro; Amidon, Gordon L.

2013-01-01

111

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

112

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

113

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

114

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

115

Free radical damage to cultured porcine aortic endothelial cells and lung fibroblasts: Modulation by culture conditions  

Microsoft Academic Search

Summary  Culture conditions modulating cell damage from xanthine plus xanthine oxidase-derived partially reduced oxygen species were\\u000a studied. Porcine thoracic aorta endothelial cells and porcine lung fibroblasts were maintained in monolayer culture. Cells\\u000a were prelabeled with51Cr before xanthine plus xanthine oxidase exposure. Endothelial cells showed 30 to 100% more lysis than fibroblasts and thus\\u000a seemed more sensitive to this oxidant stress. The

Clark T. Bishop; Zermeena Mirza; James D. Crapo; Bruce A. Freeman

1985-01-01

116

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

117

Cell culture techniques in honey bee research  

Technology Transfer Automated Retrieval System (TEKTRAN)

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

118

Cell Culture Models of Transmissible Spongiform Encephalopathies  

Microsoft Academic Search

In this review, we describe the generation and use of cell culture models of transmissible spongiform encephalopathies, also known as prion diseases. These models include chronically prion-infected cell lines, as well as cultures expressing variable amounts of wild-type, mutated, or chimeric prion proteins. These cell lines have been widely used to investigate the biology of both the normal and the

Florence Béranger; Alain Mangé; Jérôme Solassol; Sylvain Lehmann

2001-01-01

119

Three-dimensional tissue culture based on magnetic cell levitation  

NASA Astrophysics Data System (ADS)

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

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

2010-04-01

120

[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

121

In vitro culture studies of Sutherlandia frutescens on human tumor cell lines  

Microsoft Academic Search

Sutherlandia frutescens is a South African herb used traditionally by the natives to treat cancer, and more recently to improve the overall health in HIV\\/AIDS patients. Gas chromatography\\/mass spectrometer profiling and liquid chromatographic\\/mass spectral investigation confirmed and quantified the presence of canavanine, GABA and arginine in the herbal preparation used in this study. In vitro study demonstrated a concentration dependent

Joseph Tai; Susan Cheung; Edwin Chan; David Hasman

2004-01-01

122

Genomics in mammalian cell culture bioprocessing  

PubMed Central

Explicitly identifying the genome of a host organism including sequencing, mapping, and annotating its genetic code has become a priority in the field of biotechnology with aims at improving the efficiency and understanding of cell culture bioprocessing. Recombinant protein therapeutics, primarily produced in mammalian cells, constitute a $108 billion global market. The most common mammalian cell line used in biologic production processes is the Chinese hamster ovary (CHO) cell line, and although great improvements have been made in titer production over the past 25 years, the underlying molecular and physiological factors are not well understood. Confident understanding of CHO bioprocessing elements (e.g. cell line selection, protein production, and reproducibility of process performance and product specifications) would significantly improve with a well understood genome. This review describes mammalian cell culture use in bioprocessing, the importance of obtaining CHO cell line genetic sequences, and the current status of sequencing efforts. Furthermore, transcriptomic techniques and gene expression tools are presented, and case studies exploring genomic techniques and applications aimed to improve mammalian bioprocess performance are reviewed. Finally, future implications of genomic advances are surmised. PMID:22079893

Wuest, Diane M.; Harcum, Sarah W.; Lee, Kelvin H.

2013-01-01

123

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

Microsoft Academic Search

BackgroundIntestinal barrier dysfunction and translocation of endotoxins are involved in the pathogenesis of alcoholic liver disease. Exposure to ethanol and its metabolite, acetaldehyde at relatively high concentrations have been shown to disrupt intestinal epithelial tight junctions in the conventional two dimensional cell culture models. The present study investigated quantitatively and qualitatively the effects of ethanol at concentrations detected in the

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

2012-01-01

124

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

125

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

126

Purification and partial characterization of a peroxidase from plant cell cultures of Cassia didymobotrya and biotransformation studies.  

PubMed Central

An acidic peroxidase (EC 1.11.1.7) produced by cell suspension cultures of Cassia didymobotrya (wild senna) was purified from culture medium collected on the 29th day. The enzyme was shown to be a glycoprotein with a pI of 3.5, a molecular mass of approx. 43 kDa by SDS/PAGE and 50 kDa by gel filtration. The N-terminal sequence was very similar to those of other plant peroxidases. The peroxidase was characterized by a high specificity towards coniferyl alcohol and other natural phenolics such as guaiacol and ferulic and caffeic acids. These findings suggest that the enzyme is involved in lignification processes of the cell wall. Moreover, the enzyme was able to catalyse the oxidation of 4,3',4'-trihydroxychalcone and 4, 3',4'-trihydroxy-3-methoxychalcone to the corresponding 3, 3'-biflavanones, as mixtures of racemic and meso forms. PMID:9531492

Vitali, A; Botta, B; Delle Monache, G; Zappitelli, S; Ricciardi, P; Melino, S; Petruzzelli, R; Giardina, B

1998-01-01

127

[Stem cell factor production from cultured nasal epithelial cells--effect on SCF production by drugs].  

PubMed

We studied whether epithelial cells cultured in serum-free medium contained other cells or not, there were differences in SCF production from cultured nasal epithelial cells between groups of nonallergic and allergic patients, and among degrees of serum mite-CAP RAST classes of allergic patients, and how drugs inhibited SCF production. As a result, no other contaminating cells except mast cell existed in cultured cells. There was a significant difference in SCF production of cultured cells between nonallergic and class 1-2, 3-4, 5-6, and between class 1-2 and 3-4, 5-6 of mite CAP-RAST class. Cyclosporin, prednisolone, fluticasone, ketotifen, and clemastine inhibited SCF production from cultured epithelial cells, but cromoglicate and suplatast did not. Inhibition means the reduction of SCF from cells, not the growth of cultured nasal epithelial cells. PMID:11905054

Koyama, Mamoru; Otsuka, Hirokuni; Kusumi, Taeko; Yamauchi, Yoko

2002-02-01

128

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

PubMed Central

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

2014-01-01

129

Organotypic 3D cell culture models: using the rotating wall vessel to study host–pathogen interactions  

Microsoft Academic Search

Appropriately simulating the three-dimensional (3D) environment in which tissues normally develop and function is crucial for engineering in vitro models that can be used for the meaningful dissection of host–pathogen interactions. This Review highlights how the rotating wall vessel bioreactor has been used to establish 3D hierarchical models that range in complexity from a single cell type to multicellular co-culture

Jennifer Barrila; Andrea L. Radtke; Aurélie Crabbé; Shameema F. Sarker; Melissa M. Herbst-Kralovetz; C. Mark Ott; Cheryl A. Nickerson

2010-01-01

130

Patterned polymer surfaces for cell culture applications.  

PubMed

We studied the physico/chemical effects of deep UV irradiation of polystyrene, PMMA and polycarbonate with respect to cell adhesion and protein immobilization. Photochemical modifications of the polymer surfaces yielded unstable peroxides and carboxylic acid groups. Patterned enzyme and antibody adsorbates were realized by coupling via carbodiimid activation of the COOH-moities. Hepatoma cells (HepG2) and fibroblasts (L929) adhered in the presence of serum proteins in the culture medium on the irradiated regions of the substrate without any further treatment. PMID:12451876

Welle, A; Gottwald, E; Weibezahn, K F

2002-01-01

131

Studies on vacuolar membrane microdomains isolated from Arabidopsis suspension-cultured cells: local distribution of vacuolar membrane proteins.  

PubMed

The local distribution of both the vacuolar-type proton ATPase (V-ATPase) and the vacuolar-type proton pyrophosphatase (V-PPase), the main vacuolar proton pumps, was investigated in intact vacuoles isolated from Arabidopsis suspension-cultured cells. Fluorescent immunostaining showed that V-PPase was distributed evenly on the vacuolar membrane (VM), but V-ATPase localized to specific regions of the VM. We hypothesize that there may be membrane microdomains on the VM. To confirm this hypothesis, we prepared detergent-resistant membranes (DRMs) from the VM in accordance with well established conventional methods. Analyses of fatty acid composition suggested that DRMs had more saturated fatty acids compared with the whole VM in phosphatidylcholine and phosphatidylethanolamine. In the proteomic analyses of both DRMs and detergent-soluble mebranes (DSMs), we confirmed the different local distributions of V-ATPase and V-PPase. The observations of DRMs with an electron microscope supported the existence of different areas on the VM. Moreover, it was observed using total internal reflection fluorescent microscopy (TIRFM) that proton pumps were frequently immobilized at specific sites on the VM. In the proteomic analyses, we also found that many other vacuolar membrane proteins are distributed differently in DRMs and DSMs. Based on the results of this study, we discuss the possibility that VM microdomains might contribute to vacuolar dynamics. PMID:23903016

Yoshida, Katsuhisa; Ohnishi, Miwa; Fukao, Yoichiro; Okazaki, Yozo; Fujiwara, Masayuki; Song, Chihong; Nakanishi, Yoichi; Saito, Kazuki; Shimmen, Teruo; Suzaki, Toshinobu; Hayashi, Fumio; Fukaki, Hidehiro; Maeshima, Masayoshi; Mimura, Tetsuro

2013-10-01

132

On Studying Organizational Cultures.  

ERIC Educational Resources Information Center

Examines the values of the concepts of symbol, language, ideology, belief, ritual, and myth in understanding the creation of new cultures and in unraveling the related processes by which entrepreneurs give energy, purpose, and commitment to the organizations they are bringing into being. (Author/IRT)

Pettigrew, Andrew M.

1979-01-01

133

Plant Tissue Culture Studies.  

ERIC Educational Resources Information Center

Plant tissue culture has developed into a valid botanical discipline and is considered a key area of biotechnology, but it has not been a key component of the science curriculum because of the expensive and technical nature of research in this area. This manual presents a number of activities that are relatively easy to prepare and perform. The…

Smith, Robert Alan

134

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

135

Culture of Cells from Amphibian Embryos.  

ERIC Educational Resources Information Center

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

Stanisstreet, Martin

1983-01-01

136

Skeletal muscle satellite cells cultured in simulated microgravity  

Microsoft Academic Search

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

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

1997-01-01

137

Embryonic Stem Cells: Isolation, Characterization and Culture  

NASA Astrophysics Data System (ADS)

Embryonic stem cells are pluripotent cells isolated from the mammalian blastocyst. Traditionally, these cells have been derived and cultured with mouse embryonic fibroblast (MEF) supportive layers, which allow their continuous growth in an undifferentiated state. However, for any future industrial or clinical application hESCs should be cultured in reproducible, defined, and xeno-free culture system, where exposure to animal pathogens is prevented. From their derivation in 1998 the methods for culturing hESCs were significantly improved. This chapter wills discuss hESC characterization and the basic methods for their derivation and maintenance.

Amit, Michal; Itskovitz-Eldor, Joseph

138

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...

139

Primary cell-culture of the digestive gland of the marine mussel Mytilus edulis: a time-course study of antioxidant- and biotransformation-enzyme activity and ultrastructural changes  

Microsoft Academic Search

A primary culture of a mixed-cell population from the digestive gland of the marine mussel Mytilus edulis L. was developed for potential use in toxicological studies. Cells were maintained for up to 2 wk in a suspension culture,\\u000a and their survival rate was monitored with regard to different cell types. No cell proliferation was observed. Smaller cell\\u000a types survived with

C. Birmelin; R. K. Pipe; P. S. Goldfarb; D. R. Livingstone

1999-01-01

140

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

E-print Network

occurs 1n the salivary gland of the larval tick, then sporozoites are injected into the host when the larva feeds (2, 6, 7). Once the paras1te reaches the host circulatory system, trophozoites, the free living form of the parasite, penetrate host... in sal1vary tissue were single nucleated. Upon enter1ng salivary cells, these merozo1tes became more spherical; and, The Journal used as a patter n for style and format is In Vitro, as nuclear material d1sintegrated, they formed large sch1zonts...

Droleskey, Robert Edward

1981-01-01

141

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

142

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

Code of Federal Regulations, 2011 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...

2011-04-01

143

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

Code of Federal Regulations, 2013 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...

2013-04-01

144

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

Code of Federal Regulations, 2012 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...

2012-04-01

145

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

Code of Federal Regulations, 2014 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...

2014-04-01

146

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

147

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

148

3D cell culture systems: advantages and applications.  

PubMed

Cell cultures are important material of study for the variety of advantages that they offer. Both established continuous cell lines and primary cell cultures continue to be invaluable for basic research and for direct applications. Technological advancements are necessary to address emerging complex challenges and the way cells are cultured in vitro is an area of intense activity. One important advancement in cell culture techniques has been the introduction of three dimensional culture systems. This area is one of the fastest growing experimental approaches in life sciences. Augmented with advancements in cell imaging and analytical systems, as well as the applications of new scaffolds and matrices, cells have been increasingly grown as three dimensional models. Such cultures have proven to be closer to in vivo natural systems, thus proving to be useful material for many applications. Here, we review the three dimensional way of culturing cells, their advantages, the scaffolds and matrices currently available, and the applications of such cultures in major areas of life sciences. PMID:24912145

Ravi, Maddaly; Paramesh, V; Kaviya, S R; Anuradha, E; Solomon, F D Paul

2015-01-01

149

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

150

In vitro cell culture model for toxic inhaled chemical testing.  

PubMed

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

151

Fibroblast-like synovial cells from normal and inflamed knee joints differently affect the expression of pain-related receptors in sensory neurones: a co-culture study  

PubMed Central

Innervation of the joint with thinly myelinated and unmyelinated sensory nerve fibres is crucial for the occurrence of joint pain. During inflammation in the joint, sensory fibres show changes in the expression of receptors that are important for the activation and sensitization of the neurones and the generation of joint pain. We recently reported that both neurokinin 1 receptors and bradykinin 2 receptors are upregulated in dorsal root ganglion (DRG) neurones (the cell bodies of sensory fibres) in the course of acute and chronic antigen-induced arthritis in the rat. In this study, we begin to address mechanisms of the interaction between fibroblast-like synovial (FLS) cells and sensory neurones by establishing a co-culture system of FLS cells and DRG neurones. The proportion of DRG neurones expressing neurokinin 1 receptor-like immunoreactivity was not altered in the co-culture with FLS cells from normal joints but was significantly upregulated using FLS cells from knee joints of rats with antigen-induced arthritis. The proportion of DRG neurones expressing bradykinin 2 receptors was slightly upregulated in the presence of FLS cells from normal joints but upregulation was more pronounced in DRG neurones co-cultured with FLS cells from acutely inflamed joints. In addition, the expression of the transient receptor potential V1 (TRPV1) receptor, which is involved in inflammation-evoked thermal hyperalgesia, was mainly upregulated by co-culturing DRG neurones with FLS cells from chronically inflamed joints. Upregulation of neurokinin 1 receptors but not of bradykinin 2 and TRPV1 receptors was also observed when only the supernatant of FLS cells from acutely inflamed joint was added to DRG neurones. Addition of indomethacin to co-cultures inhibited the effect of FLS cells from acutely inflamed joints on neurokinin 1 receptor expression, suggesting an important role for prostaglandins. Collectively, these data show that FLS cells are able to induce an upregulation of pain-related receptors in sensory neurones and, thus, they could contribute to the generation of joint pain. Importantly, the influence of FLS cells on DRG neurones is dependent on their state of activity, and soluble factors as well as direct cellular contacts are crucial for their interaction with neurones. PMID:17254343

von Banchet, Gisela Segond; Richter, Jonny; Hückel, Marion; Rose, Christina; Bräuer, Rolf; Schaible, Hans-Georg

2007-01-01

152

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

153

Differentiation of islet cells in long-term culture.  

PubMed

Our previous studies in the hamster pancreatic cancer model have shown that exocrine pancreatic cancer arises from ductal/ductular cells, as well as from within the islets, most probably from islet precursor (stem) cells. To identify and characterize these cells, we established a long-term culture from isolated hamster islets and investigated their growth, differentiation, and expression of biomarkers. Islets maintained their original form and structure within the first 14 days in culture. However, beginning at day 7, ductular structures began to form within the islets. At day 21 in culture, acinar cells, intermediary cells, oncocytes, and cells comparable to pancreatic hepatocytes also appeared between ductular and endocrine cells. The number of duct-like cells gradually increased, whereas the number of hormone-producing cells decreased. After 35 days in culture, the exocrine cells disappeared, and undifferentiated cells formed a monolayer. These cells expressed cytokeratins, alpha1-antitrypsin, transforming growth factor-alpha, epidermal growth factor receptor, carbonic anhydrase II, vimentin, laminin, and showed binding to tomato lectin and Phaseolus vulgaris leukoagglutinin. They did not express the regulatory transcriptional factors, insulin-promoting factor 1, NKx6.1, Pax6, and NeuroD. The results thus indicate that islet cells have potential to form exocrine cells. At present, it is not clear whether these cells originate from preexisting stem cells or from transdifferentiated islet cells. PMID:10824687

Schmied, B M; Liu, G; Matsuzaki, H; Ulrich, A; Hernberg, S; Moyer, M P; Weide, L; Murphy, L; Batra, S K; Pour, P M

2000-05-01

154

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

155

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

PubMed Central

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

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

2012-01-01

156

A Non-Contact Suspension Culture Approach to the Culture of Osteogenic Cells Derived from a  

E-print Network

: mesenchymal stem cells; suspension culture; osteogenesis Introduction Adult bone marrow (BM) is the most common tissue used as a source of mesenchymal stromal cells (MSCs) and multiple studies have identified markers that can be used to identify BM-derived MSCs as mesenchymal stem cells, as described by Dominici

Zandstra, Peter W.

157

21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2013 CFR

...2013-04-01 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....

2013-04-01

158

21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2012 CFR

...2012-04-01 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....

2012-04-01

159

21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2011 CFR

...2011-04-01 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....

2011-04-01

160

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

161

21 CFR 864.2280 - Cultured animal and human cells.  

Code of Federal Regulations, 2014 CFR

...2014-04-01 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....

2014-04-01

162

Human nasal and tracheo-bronchial respiratory epithelial cell culture.  

PubMed

Human airway epithelial (hAE) cell cultures are instrumental for studying basic and applied aspects of respiratory tract biology, disease, and therapy. When primary epithelial cells from the human nasal passages or tracheo-bronchial airways are grown on porous supports at an air-liquid interface (ALI) they undergo mucociliary differentiation, reproducing both the in vivo morphology and key physiologic processes. These cultures are useful for studying basic biology, disease pathogenesis, gene therapy and aerosol administration of drugs. This chapter gives detailed protocols for tissue procurement, cell isolation, production of complex media, and cell culture initiation and maintenance needed for hAE cell ALI cultures with non-proprietary reagents. PMID:23097104

Fulcher, M Leslie; Randell, Scott H

2013-01-01

163

Establishment and characterisation of a rapidly dividing diploid cell suspension culture of Arabidopsis thaliana suitable for cell cycle synchronisation  

Microsoft Academic Search

Polyploid plants often have altered gene expression, biochemistry, and metabolism compared to their diploid predecessors. Therefore cultured diploid cells have distinct benefits over cultured polyploid cells for the study of gene regulation and metabolism of the parent plant. Here we report methods for establishing and maintaining a rapidly dividing diploid Arabidopsis thaliana cell suspension culture, and subsequent cell cycle synchronisation.

Ranjith Pathirana; Jocelyn R. Eason

2006-01-01

164

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

165

Optimizing stem cell culture. Boudewijn van der Sanden, Mehdi Dhobb, Franois Berger  

E-print Network

in optimizing stem cell culture, not only because cell culture is widely used in basic research for studyingOptimizing stem cell culture. Boudewijn van der Sanden, Mehdi Dhobb, François Berger¶ , Didier Wion, France. Email : didier.wion@ujf-grenoble.fr Abstract. Stem cells always balance between self

Boyer, Edmond

166

Cell Cultures and Retroviral Particles From a Tumor of a Moray Eel  

Microsoft Academic Search

Until recently, fish cell culture primarily has been useful only in the propagation and study of epidemic viruses significant to the fishing industry. Such fish cell lines derived were developed by appropriating classical techniques of mammalian cell culture, with serum as the major growth supplement. Using an approach in which culture medium is formulated in a cell-type-specific manner with minimal

Charles Buck; Charles Walsh; Raymond Davis; Araz Toumadje; Kenichi Kusamoto; Angela Helmrich; Christine Chapline; Patricia Mericko; David Barnes

2001-01-01

167

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? 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

2014-10-01

168

Culture and Manipulation of Embryonic Cells  

PubMed Central

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

169

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

170

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

171

Magnesium variability of lymphocytes from cell culture.  

PubMed

We determined the magnesium content for two different Burkitt's lymphoma cell lines (EW 36 and CA 46) after transfer to new media for 7 consecutive days. Aliquots of the cell culture were washed and the cell pellet was obtained by centrifugation and lysed with distilled water with and without the addition of 0.25% lanthanum oxide. Magnesium was determined by atomic absorption spectrophotometry. The addition of lanthanum permitted the detection of between 8 and 40% more magnesium. The increased magnesium liberated by the addition of lanthanum was calculated as the "bound" magnesium. The results show that the total magnesium is inversely related and the bound magnesium directly related to the age of the cell culture. Thus, there is a decrease of lymphocyte magnesium content and an increase in the percentage of magnesium bound for these two cell lines with increasing age of the cell culture. PMID:4078199

Hosseini, J M; Elin, R J

1985-01-01

172

Mammosphere culture of cancer stem cells in a microfluidic device  

NASA Astrophysics Data System (ADS)

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

Saadin, Katayoon; White, Ian M.

2012-03-01

173

Characterization of Human Fungiform Papillae Cells in Culture  

PubMed Central

The ability to maintain human fungiform papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Here we describe a method for enzymatically isolating human cells from fungiform papillae obtained by biopsy and maintaining them in culture for more than 7 passages (7 months) without loss of viability and while retaining many of the functional properties of acutely isolated taste cells. Cells in these cultures exhibited increases in intracellular calcium when stimulated with perceptually appropriate concentrations of several taste stimuli, indicating that at least some of the native signaling pathways were present. This system can provide a useful model for molecular studies of the proliferation, differentiation, and physiological function of human fungiform papillae cells. PMID:21471186

Brand, Joseph G.; Spielman, Andrew I.; Lischka, Fritz W.; Teeter, John H.; Breslin, Paul A.S.; Rawson, Nancy E.

2011-01-01

174

Intracellular protein degradation in cultured rat muscle cells  

E-print Network

in homeo- stasis, serving as a source of energy which can be freed by degrada- tion of muscle protein to precursor substrates for glucose synthesis although the mass of skeletal muscle protein remains relatively stable during the early stages... to their constituent amino acids (1). Elucidation of mechanisms and regulation of intracellular proteolysis has been aided by the use of cell culture studies from many diverse tissue types. Mammalian cells in culture have proven an especially useful mo...

Miller, Gwendolyn Beth

2012-06-07

175

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

176

Isolation and Culture of Epithelial Stem Cells  

PubMed Central

In the skin, epithelial stem cells in the hair follicle contribute not only to the generation of a new hair follicle with each hair cycle, but also to the repair of the epidermis during wound healing. When these stem cells are isolated and expanded in culture, they can give rise to hair follicles, sebaceous glands, and epidermis when combined with dermis and grafted back onto Nude mice. In this chapter, we provide a method for isolating hair follicle epithelial stem cells from the skin of adult mice using immunofluorescent labeling to allow for the specific purification of epithelial stem cells by fluorescence-activated cell sorting (FACS). Notably, this method relies exclusively on cell surface markers, making it suitable for use with any strain of mouse and at various stages of the hair cycle. We also provide a detailed protocol for culturing epithelial stem cells isolated by FACS, allowing for analysis using a wide variety of culture assays. Additionally, we provide notes on using cultured cells for specific applications, such as viral manipulation and grafting. These techniques should be useful for directly evaluating stem cell function in normal mice and in mice with skin defects. PMID:19089359

Nowak, Jonathan A.; Fuchs, Elaine

2009-01-01

177

Comparative assessment of the stability of nonfouling poly(2-methyl-2-oxazoline) and poly(ethylene glycol) surface films: an in vitro cell culture study.  

PubMed

Poly(ethylene glycol) (PEG) has been the most frequently reported and commercially used polymer for surface coatings to convey nonfouling properties. PEGylated surfaces are known to exhibit limited chemical stability, particularly due to oxidative degradation, which limits long-term applications. In view of excellent anti-adhesive properties in the brush conformation and resistance to oxidative degradation, poly(2-methyl-2-oxazoline) (PMOXA) has been proposed recently as an alternative to PEG. In this study, the authors systematically compare the (bio)chemical stability of PEG- and PMOXA-based polymer brush monolayer thin films when exposed to cultures of human umbilical vein endothelial cells (HUVECs) and human foreskin fibroblasts (HFFs). To this end, the authors used cell-adhesive protein micropatterns in a background of the nonfouling PEG and PMOXA brushes, respectively, and monitored the outgrowth of HUVECs and HFFs for up to 21 days and 1.5 months. Our results demonstrate that cellular micropatterns spaced by PMOXA brushes are significantly more stable under serum containing cell culture conditions in terms of confinement of cells to the adhesive patterns, when compared to corresponding micropatterns generated by PEG brushes. Moreover, homogeneous PEG and PMOXA-based brush monolayers on Nb2O5 surfaces were investigated after immersion in endothelial cell medium using ellipsometry and x-ray photoelectron spectroscopy. PMID:25280844

Chen, Yin; Pidhatika, Bidhari; von Erlach, Thomas; Konradi, Rupert; Textor, Marcus; Hall, Heike; Lühmann, Tessa

2014-09-01

178

Isolation and culture of pulmonary endothelial cells.  

PubMed

Methods for isolation, identification and culture of pulmonary endothelial cells are now routine. In the past, methods of isolation have used proteolytic enzymes to detach cells; thereafter, traditional methods for cell passaging have used trypsin/EDTA mixtures. Cells isolated and passaged using proteolytic enzymes have been useful in establishing the field and in verifying certain endothelial properties. However, there is a growing awareness of the role of endothelial cells in processing vasoactive substances, in responding to hormones and other agonists and in cell-cell interactions with other cell types of the vascular wall, with blood cells and with cellular products. Consequently, a new requirement has arisen for cells in vitro that maintain the differentiated properties of their counterparts in vivo. The deleterious effects of trypsin and other proteolytic enzymes commonly used in cell culture on surface structures of endothelial cells such as enzymes, receptors and junctional proteins, as well as on extracellular layers such as the glycocalyx or "endothelial fuzz," have led to the development of methods that avoid use of proteolytic enzymes at both the isolation step and during subsequent subculture. This chapter describes traditional methods for isolating pulmonary endothelial cells but emphasizes newer approaches using mechanical harvest and scale-up using microcarriers. The new methods allow maintenance of long-term, large-scale cultures of cells that retain the full complement of surface properties and that maintain the cobblestone monolayer morphology and differentiated functional properties. Methods for identification of isolated cells are therefore also considered as methods for validation of cultures during their in vitro lifespan. PMID:6090112

Ryan, U S

1984-06-01

179

Immunodissection and culture of rabbit cortical collecting tubule cells  

SciTech Connect

A mouse monoclonal antibody designated IgG3 (rct-30) has been prepared that reacts specifically with an antigen on the surface of all cells comprising the cortical and medullary rabbit renal collecting tubule including the arcades. Plastic culture dishes coated with IgG3 (rct-30) were used to isolate collecting tubule cells from collagenase dispersions of rabbit renal cortical cells by immunoadsorption. Typically, 10W rabbit cortical collecting tubule (RCCT) cells were obtained from 5 g of renal cortex (2 kidneys). Between 20 and 30% of the RCCT cells were reactive with peanut lectin suggesting that RCCT cells are a mixture of principal and intercalated cells. Approximately 10X RCCT cells were obtained after 4 to 5 days in primary culture. Moreover, RCCT cells continued to proliferate after passaging with a doubling time of approx.32 h. RCCT cells passaged once and then cultured 4-5 days were found 1) to synthesize cAMP in response to arginine vasopressin (AVP), prostaglandin E2 (PGE2), isoproterenol, and parathyroid hormone, but not calcitonin, prostaglandin D2, or prostaglandin I, and 2) to release PGE2 in response to bradykinin but not arginine vasopressin or isoproterenol. The results indicate that cultured RCCT cells retain many of the hormonal, histochemical, and morphological properties expected for a mixture of principal and intercalated rabbit cortical collecting tubule epithelia. RCCT cells should prove useful both for studying hormonal interactions in the cortical collecting tubule and as a starting population for isolating intercalated collecting tubule epithelia.

Spielman, W.S.; Sonnenburg, W.K.; Allen, M.L.; Arend, L.J.; Gerozissis, K.; Smith, W.L.

1986-08-01

180

Human cell culture in a space bioreactor  

NASA Technical Reports Server (NTRS)

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

Morrison, Dennis R.

1988-01-01

181

Transferring isolated mitochondria into tissue culture cells  

PubMed Central

We have developed a new method for introducing large numbers of isolated mitochondria into tissue culture cells. Direct microinjection of mitochondria into typical mammalian cells has been found to be impractical due to the large size of mitochondria relative to microinjection needles. To circumvent this problem, we inject isolated mitochondria through appropriately sized microinjection needles into rodent oocytes or single-cell embryos, which are much larger than tissue culture cells, and then withdraw a ‘mitocytoplast’ cell fragment containing the injected mitochondria using a modified holding needle. These mitocytoplasts are then fused to recipient cells through viral-mediated membrane fusion and the injected mitochondria are transferred into the cytoplasm of the tissue culture cell. Since mouse oocytes contain large numbers of mouse mitochondria that repopulate recipient mouse cells along with the injected mitochondria, we used either gerbil single-cell embryos or rat oocytes to package injected mouse mitochondria. We found that the gerbil mitochondrial DNA (mtDNA) is not maintained in recipient rho0 mouse cells and that rat mtDNA initially replicated but was soon completely replaced by the injected mouse mtDNA, and so with both procedures mouse cells homoplasmic for the mouse mtDNA in the injected mitochondria were obtained. PMID:22753025

Yang, Yi-Wei; Koob, Michael D.

2012-01-01

182

Calculator programmed assistance in making cell cultures  

Microsoft Academic Search

Summary A calculator program and instructions are presented for facilitating the preparation of single cell suspensions to be used in cell culture systems. The program has been written for the Texas Instruments Inc. TI-59 programmable calculator with printer. The instructions provided are intended for the novice with no previous programming experience and only a basic understanding of the calculator operation.

J. C. Bullaro

1980-01-01

183

Three dimensional culture of pineal cell aggregates: a model of cell-cell co-operation.  

PubMed

Three dimensional (3-D) cultures of pineal cell aggregates were obtained by constant gyratory shaking the heterogenous cell populations, obtained from the rat pineals, in the DMEM (Dulbecco's modified Eagle's medium). Within 4 days, the pineal cells became organized into a tissue like configuration appearing as a compact ball, evidenced by the scanning electron microscopy. The 3-D aggregates seemed to be mainly composed of pinealocytes (round-oval cells), glial (elongated cells) and other unknown cells. The heterogenous cells were separated by intercellular spaces. The ultrastructural characteristics revealed by transmission electron microscopy exhibited the presence of granular lysosomes, typical of pinealocytes actively involved in the secretion. These pineal cell aggregates secreted melatonin and other indole amines i.e. 5-methoxytryptamine (5-MT), indole acetic acid (IAA), 5-methoxy-3-indole acetic acid (5-MIAA), tryptophol (TOL) and 5-methoxytryptophol (5-MTL) in the culture medium, indicating the functional aspect of pinealocytes. The 3-D aggregates cultures had advantages over the pineal monolayer cultures as, after 4 days of culture, the amounts of indole amines secreted by 3-D aggregates were higher than those secreted by monolayer cultures. Besides, the 3-D aggregates remained functional till 24 days in the gyratory culture conditions. In the continuous perifusion system, the 3-D aggregates secreted melatonin while challanged with isoproterenol. This 3-D model of pineal cell aggregates might be useful, in future, to perform other kinetic studies of the release of indole amines in perifusion experiments as this system allows the maintenance of pineal cells for a long period of time. PMID:7550281

Khan, N A; Shacoori, V; Havouis, R; Querné, D; Moulinoux, J P; Rault, B

1995-05-01

184

Isolating phagosomes from tissue culture cells.  

PubMed

Phagocytosis is the process by which receptors at the plasma membrane are used to engulf a particle such as a bacterium, parasite, or dead cell. Phagosomes can be isolated from tissue culture cells by various centrifugation methods, including the use of differential density gradients or sucrose step gradients, but these methods are time-consuming or otherwise difficult. We describe here a protocol that avoids centrifugation and relies instead on the uptake of magnetic beads to rapidly isolate the phagosomal compartment from tissue culture cells. PMID:25447278

Pryor, Paul R; Rofe, Adam P

2014-12-01

185

Effect of cell substrate on antioxidant enzyme activities in cultured renal glomerular epithelium.  

PubMed Central

The activities of three antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase, were monitored in isolated guinea pig glomeruli and primary or subcultured glomerular epithelial cells. Cell injury was assessed by morphologic studies and by measurement of cellular lipid peroxidation (levels of malondialdehyde). Antioxidant enzyme activities were very different in cultured cells than in parent glomeruli. The possible effect of culture substrates (tissue culture plastic, bovine corneal endothelial [BCE] cell basement membrane, and PF-HR-9 endodermal cell basement membrane) on antioxidant enzyme status, cell morphology, and lipid peroxidation was also assessed. Glomerular epithelial cells cultured on the BCE cell basement membrane substrate survived longer and showed less lipid peroxidation than cells cultured on plastic or the HR-9 substrate. Cells cultured on a plastic substrate had substantially less glutathione peroxidase activity than cells cultured on either BCE or HR-9 basement membranes. Images Figure 3 Figure 1 Figure 2 Figure 5 PMID:3348362

Yang, A. H.; Oberley, T. D.; Oberley, L. W.; Ramanathan, R.

1988-01-01

186

Effect of LLLT on endothelial cells culture.  

PubMed

Growth factors as vascular endothelial growth factor (VEGF), produced by the endothelial cells, take an essential part in pathological and physiological angiogenesis. The possibility of angiogenesis modulation by application of laser radiation may contribute to the improvement of its use in this process. Thus, the aim of the study was to investigate the influence of low-level laser therapy (LLLT) on the proliferation of endothelial cells, secretion of VEGF-A and presence of soluble VEGF receptors (sVEGFR-1 and sVEGFR-2) in the medium after in vitro culture. Isolated human umbilical vein endothelial cells (HUVECs) were irradiated using a diode laser at a wavelength of 635 nm and power density of 1,875 mW/cm(2). Depending on radiation energy density, the experiment was conducted in four groups: I 0 J/cm(2) (control group), II 2 J/cm(2), III 4 J/cm(2), and IV 8 J/cm(2). The use of laser radiation wavelength of 635 nm, was associated with a statistically significant increase in proliferation of endothelial cells (p?=?0.0041). Moreover, at 635-nm wavelength, all doses of radiation significantly reduced the concentration of sVEGFR-1 (p?=?0.0197). PMID:25231826

Góralczyk, Krzysztof; Szyma?ska, Justyna; ?ukowicz, Ma?gorzata; Drela, Ewelina; Kotzbach, Roman; Dubiel, Mariusz; Michalska, Ma?gorzata; Góralczyk, Barbara; Zaj?c, Andrzej; Ro??, Danuta

2015-01-01

187

Characteristics of cardiac cell cultures derived from human myocardial explants.  

PubMed

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

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

2013-11-01

188

Tissue culture studies of retinal development.  

PubMed

Because of a limited number of cell types, a series of well-described cell-type-specific markers and a stereotyped sequence of cell development, the retina has been a valuable model of CNS development. Dissociated and explant cultures have been used to help define some of the requirements for differentiation of each major cell class. In addition to mixed-cell cultures it is now possible to use cell purification or selective growth methods to give cultures of single cell types. Alteration of gene expression by viral infection has proved to be a valuable method to help elucidate developmental pathways. PMID:12507462

Zhang, Samuel Shao-Min; Fu, Xin Yuan; Barnstable, Colin J

2002-12-01

189

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

190

Modulation of cyclin transcript levels in cultured cells of Arabidopsis thaliana  

Microsoft Academic Search

Previous studies on the cell cycle of Arabidopsis thaliana have been hindered by the lack of synchronous cell culture systems. We have used liquid callus cultures and a cycloheximide-synchronized suspension culture of Arabidopsis to investigate changes in cyclin transcript levels in response to exogenous auxin, cytokinin, and nutrients, and during the cell cycle. CYCD7 (67) transcript was virtually undetectable in

Roderic A. U. A. Fuerst; Rajeev Soni; James A. H. Murray; Keith Lindsey

1996-01-01

191

Canine coronavirus induces apoptosis in cultured cells  

Microsoft Academic Search

Canine coronavirus (CCoV) is widespread in dogs in several countries and causes mild enteric illness evolving to severe enteritis in young pups.In in vitro cultures canine coronaviruses generally induce extensive cell death, however nature of the events leading to cell death remains largely unknown.We analysed the induction of cytopathic effect by CCoV in a canine fibrosarcoma cell line (A-72) in

A. Ruggieri; L. Di Trani; I. Gatto; M. Franco; E. Vignolo; B. Bedini; G. Elia; C. Buonavoglia

2007-01-01

192

Comparative study on the cytotoxicity of different Myrtaceae essential oils on cultured vero and RC-37 cells.  

PubMed

Medicinally and commercially important essential oils from the family Myrtaceae, i.e. cajuput, clove, kanuka and manuka were phytochemically analysed by GC-MS. Cytotoxicity of these essential oils was evaluated in a standard neutral red assay. Maximum noncytotoxic concentrations for cajuput oil and clove oil were determined at 0.006%, kanuka oil and manuka oil were more cytotoxic with a maximum noncytotoxic concentration of 0.001%. The compounds alpha-pinene, eugenol and leptospermone demonstrated maximum noncytotoxic concentrations at dilutions of 0.001%, 0.003% and 0.001%, respectively. However, the terpene 1,8-cineole was about 100 times less toxic to cultured cells with a maximum noncytotoxic concentration of 0.1% and a TC50 value of 0.44%. Manuka essential oil exhibited high levels of virucidal activity against HSV-1 as well against drug-resistant HSV-1 isolates in viral suspension tests. Determination of cytotoxicity of natural products is an important prerequisite for application in cosmetic and health care products and in antiviral tests. PMID:19069246

Schnitzler, P; Wiesenhofer, K; Reichling, J

2008-11-01

193

Biocompatibility assessment of perfluorochemical oils in microbial and plant cell cultures  

Microsoft Academic Search

The effects of various perfluorochemical (PFC) oils on growth and structure of microbial and plant cell cultures have been studied. Growth of microbial cells was unaffected by culture with PFCs and no obvious deleterious effects on cell structure or alterations in polypeptide profiles of cell extracts were observed. Incubation of S. dulcamara cells with FDC oil produced an increase in

Alastair T. King; John Bray; Bernard J. Mulligan; Kenneth C. Lowe

1990-01-01

194

Nuclear magnetic resonance studies of biological systems: Applications to liver preservation and metabolism in cultured pituitary tumor cells  

SciTech Connect

This study centers on applications of both {sup 31}P and {sup 13}C nuclear magnetic resonance spectroscopy to two different biological systems. The first application utilizes {sup 31}P NMR to study mobile phospholipids in the MMQ cell line, a pituitary tumor cell line. These measurements characterize membrane phospholipids thought to be part of a RNA-proteolipid complex unique to cellular transformation. The second application utilizes both {sup 31}P and {sup 13}C spectroscopy to study liver preservation and transplantation an a rat model. In this work, several questions were addressed: (1) to what extent do successful preservation solutions slow ATP breakdown (2) can clinically successful preservation conditions ameliorate total nucleotide breakdown (3) to what extent is energy reconstitution following cold storage correlated with transport success and (4) can any spectroscopic parameter be used as a diagnostic indicator of tissue viability

Fralix, T.A.

1989-01-01

195

Fluorous solvent for cell culture  

Microsoft Academic Search

Incubation of mouse melanoma B16 cells in fluorous solvents with low boiling point such as perfluoromethylcyclohexane, 1,1,1,3,3,3-hexafluoro-2-propanol, ethylpentafluoropropionate resulted in cell death. However, cells lived up to 2 days in fluorous alcohols such as 2,2,3,3,4,4,5,5-octafluoro-1-pentanol and 3,3,4,4,5,5,6,6,6-nonafluoro-1-hexanol with relatively higher fluorine content. Remarkably, cells survived deprived of nutrition up to 4 days when incubated in 2,2,3,3,4,4,5,5,6,6,6-undecafluoro-1-hexanol or in 2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptanol that

Maria Carmelita Z. Kasuya; Xiaonan Wen; Kenichi Hatanaka; Kageyasu Akashi

2011-01-01

196

Differential effects of selective frankincense (Ru Xiang) essential oil versus non-selective sandalwood (Tan Xiang) essential oil on cultured bladder cancer cells: a microarray and bioinformatics study  

PubMed Central

Background Frankincense (Boswellia carterii, known as Ru Xiang in Chinese) and sandalwood (Santalum album, known as Tan Xiang in Chinese) are cancer preventive and therapeutic agents in Chinese medicine. Their biologically active ingredients are usually extracted from frankincense by hydrodistillation and sandalwood by distillation. This study aims to investigate the anti-proliferative and pro-apoptotic activities of frankincense and sandalwood essential oils in cultured human bladder cancer cells. Methods The effects of frankincense (1,400–600 dilutions) (v/v) and sandalwood (16,000–7,000 dilutions) (v/v) essential oils on cell viability were studied in established human bladder cancer J82 cells and immortalized normal human bladder urothelial UROtsa cells using a colorimetric XTT cell viability assay. Genes that responded to essential oil treatments in human bladder cancer J82 cells were identified using the Illumina Expression BeadChip platform and analyzed for enriched functions and pathways. The chemical compositions of the essential oils were determined by gas chromatography–mass spectrometry. Results Human bladder cancer J82 cells were more sensitive to the pro-apoptotic effects of frankincense essential oil than the immortalized normal bladder UROtsa cells. In contrast, sandalwood essential oil exhibited a similar potency in suppressing the viability of both J82 and UROtsa cells. Although frankincense and sandalwood essential oils activated common pathways such as inflammatory interleukins (IL-6 signaling), each essential oil had a unique molecular action on the bladder cancer cells. Heat shock proteins and histone core proteins were activated by frankincense essential oil, whereas negative regulation of protein kinase activity and G protein-coupled receptors were activated by sandalwood essential oil treatment. Conclusion The effects of frankincense and sandalwood essential oils on J82 cells and UROtsa cells involved different mechanisms leading to cancer cell death. While frankincense essential oil elicited selective cancer cell death via NRF-2-mediated oxidative stress, sandalwood essential oil induced non-selective cell death via DNA damage and cell cycle arrest. PMID:25006348

2014-01-01

197

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

198

Tissue culture studies of retinal development  

Microsoft Academic Search

Because of a limited number of cell types, a series of well-described cell-type-specific markers and a stereotyped sequence of cell development, the retina has been a valuable model of CNS development. Dissociated and explant cultures have been used to help define some of the requirements for differentiation of each major cell class. In addition to mixed-cell cultures it is now

Samuel Shao-Min Zhang; Xin-Yuan Fu; Colin J Barnstable

2002-01-01

199

[Presence of Mycoplasma in laboratory cell cultures from Cordoba, Argentina].  

PubMed

In this paper we determined the prevalence of mycoplasma contamination in 17 cell lines. Eighty per cent of the laboratories that currently use cell culture techniques participated in this study. Hoechst 33258 dye was used to detect mycoplasma contamination. The relationship between culture maintenance conditions and the presence of mycoplasma were analyzed, considering the use of antibiotics in the culture media, fetal calf serum (FCS) quality, culture media processing, use of disponsable labware, type of laminar flow cabinet, quantity of operators, and cell culture system. Thirty-five per cent of the analyzed cell lines showed mycoplasma contamination. Those lines belonged to 2 of the 8 surveyed laboratories. When confronting the working conditions versus mycoplasma contamination, 66% of the laboratories that employ non-certified FCS or reuse their labware, show mycoplasma contamination. Mycoplasma presence was found in 50% of the laboratories that use closed culture system, or more than one operator. Laboratories that process their culture media or that include antibiotic in the growing media, show a 40% contamination. The results obtained help to establish working conditions necessary to avoid introducing or spreading the microorganism. PMID:9793145

Cumino, A C; Córdoba, P; Zapata, T M

1998-01-01

200

Recombinant spider silk matrices for neural stem cell cultures.  

PubMed

Neural stem cells (NSCs) have the capacity to differentiate into neurons, astrocytes, and oligodendrocytes. Accordingly, NSCs hold great promise in drug screening and treatment of several common diseases. However, a major obstacle in applied stem cell research is the limitation of synthetic matrices for culturing stem cells. The objective of this study was to evaluate the suitability of recombinant spider silk (4RepCT) matrices for growth of NSCs. NSCs isolated from the cerebral cortices of mid-gestation rat embryos were cultured on either 4RepCT matrices or conventional poly-L-ornithine and fibronectin (P + F) coated polystyrene plates. From 48 h of culture, no significant differences in cell proliferation or viability were detected in NSC cultures on 4RepCT compared to control matrices (polystyrene plates coated with P + F). The NSCs retained an undifferentiated state, displaying low or no staining for markers of differentiated cells. Upon stimulation NSCs grown on 4RepCT differentiated efficiently into neuronal and astrocytic cells to virtually the same degree as control cultures, but a slightly less efficient oligodendrocyte differentiation was noted. We suggest that recombinant spider silk matrices provide a functional microenvironment and represent a useful tool for the development of new strategies in neural stem cell research. PMID:22863380

Lewicka, Michalina; Hermanson, Ola; Rising, Anna U

2012-11-01

201

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.

202

Advanced Cell Culture Techniques for Cancer Drug Discovery  

PubMed Central

Human cancer cell lines are an integral part of drug discovery practices. However, modeling the complexity of cancer utilizing these cell lines on standard plastic substrata, does not accurately represent the tumor microenvironment. Research into developing advanced tumor cell culture models in a three-dimensional (3D) architecture that more prescisely characterizes the disease state have been undertaken by a number of laboratories around the world. These 3D cell culture models are particularly beneficial for investigating mechanistic processes and drug resistance in tumor cells. In addition, a range of molecular mechanisms deconstructed by studying cancer cells in 3D models suggest that tumor cells cultured in two-dimensional monolayer conditions do not respond to cancer therapeutics/compounds in a similar manner. Recent studies have demonstrated the potential of utilizing 3D cell culture models in drug discovery programs; however, it is evident that further research is required for the development of more complex models that incorporate the majority of the cellular and physical properties of a tumor. PMID:24887773

Lovitt, Carrie J.; Shelper, Todd B.; Avery, Vicky M.

2014-01-01

203

Automation of 3-dimensional cell culture in arrayed microfluidic devices  

PubMed Central

The increasing interest in studying the interactions between cells and the extracellular matrix (ECM) has created a need for high throughput low cost three-dimensional (3D) culture systems. The recent development of tubeless microfluidics via passive pumping provides a high throughput microchannel culture platform compatible with existing high throughput infrastructures (e.g. automated liquid handlers). Here we build on a previously reported high throughput two-dimensional (2D) system to create a robust automated system for 3D culture. Operational controls including temperature and sample handling have been characterized and automated. Human mammary fibroblasts (HMFs) suspended in type-I collagen are loaded and cultured in microchannel arrays, and used to optimize the system operational parameters. A Peltier cooler maintains the collagen as a liquid at 4°C during cell seeding, followed by polymerization at 37°C. Optimization of this platform is discussed (e.g. controlling collagen contraction, increasing cell viability, preventing the removal of microchannel contents), and 3D distribution of HMFs is examined by fluorescent microscopy. Finally, we validate the platform by automating a previously developed 3D breast carcinoma co-culture assay. The platform allows more efficient 3D culture experiments and lays the foundation for high throughput studies of cell-ECM interactions. PMID:21609700

Montanez-Sauri, Sara I.; Sung, Kyung Eun; Puccinelli, John P.; Pehlke, Carolyn; Beebe, David J.

2011-01-01

204

Prion protein interactions and TSE infections in cell culture models  

Microsoft Academic Search

The process by which transmissible spongiform encephalopathy (TSE) agents, or prions, infect cells is unknown. There are also\\u000a no effective treatments available for TSE diseases. Studies of cultured cells persistently infected with TSE agents have greatly\\u000a contributed to understanding these and many other aspects of TSE disease. New cell lines have been developed to increase the\\u000a repertoire of TSE strains

Gerald S. Baron

205

Shape Memory Polymers for Active Cell Culture  

PubMed Central

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape. Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture. Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions. PMID:21750496

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

2011-01-01

206

Shape memory polymers for active cell culture.  

PubMed

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

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

2011-01-01

207

Bacterial Cellulose as a Substrate for Microbial Cell Culture  

PubMed Central

Bacterial cellulose (BC) has a range of structural and physicochemical properties that make it a particularly useful material for the culture of bacteria. We studied the growth of 14 genera of bacteria on BC substrates produced by Acetobacter xylinum and compared the results to growth on the commercially available biopolymers agar, gellan, and xanthan. We demonstrate that BC produces rates of bacterial cell growth that typically exceed those on the commercial biopolymers and yields cultures with higher titers of cells at stationary phase. The morphology of the cells did not change during growth on BC. The rates of nutrient diffusion in BC being higher than those in other biopolymers is likely a primary factor that leads to higher growth rates. Collectively, our results suggest that the use of BC may open new avenues in microbiology by facilitating bacterial cell culture and isolation. PMID:24441155

Yin, Na; Santos, Thiago M. A.; Auer, George K.; Crooks, John A.; Oliver, Piercen M.

2014-01-01

208

Bacterial cellulose as a substrate for microbial cell culture.  

PubMed

Bacterial cellulose (BC) has a range of structural and physicochemical properties that make it a particularly useful material for the culture of bacteria. We studied the growth of 14 genera of bacteria on BC substrates produced by Acetobacter xylinum and compared the results to growth on the commercially available biopolymers agar, gellan, and xanthan. We demonstrate that BC produces rates of bacterial cell growth that typically exceed those on the commercial biopolymers and yields cultures with higher titers of cells at stationary phase. The morphology of the cells did not change during growth on BC. The rates of nutrient diffusion in BC being higher than those in other biopolymers is likely a primary factor that leads to higher growth rates. Collectively, our results suggest that the use of BC may open new avenues in microbiology by facilitating bacterial cell culture and isolation. PMID:24441155

Yin, Na; Santos, Thiago M A; Auer, George K; Crooks, John A; Oliver, Piercen M; Weibel, Douglas B

2014-03-01

209

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates  

PubMed Central

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates. The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival. The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens. PMID:19066533

Frank, Lauren E.; Caldera-Siu, Angela D.; Pothos, Emmanuel N.

2008-01-01

210

Cell-cell coupling in cultures of striatal and cortical astrocytes of the monkey Cebus apella.  

PubMed

Astrocytes were cultured from striatum and neocortex of fetal (embryonic day 90) monkeys (Cebus apella). The cultures grew well, and the cells retained viability after freeze-storage and thawing. The cells displayed depolarized membrane potentials (-19 and -33 mV, for striatal and cortical cells, respectively) and the vast majority of cells were dye-coupled to a mean of 7 (1-18) neighbouring cells. Cell coupling was blocked by octanol (0.25 and 0.5 mM) but was independent of high K+ (10 and 50 mM) and glutamate (500 microm). Thus, cultures of fetal primate astrocytic cells are established as a model system for studies on astroglial cell-cell coupling. PMID:10841445

Gayol, S; Pannicke, T; Reichenbach, A; Colombo, J A

1999-01-01

211

THREE CROSS-CULTURAL STUDIES OF SUBJECTIVE CULTURE  

Microsoft Academic Search

3 INTERRELATED CROSS CULTURAL STUDIES, USING AMERICAN AND GREEK SS, CONCERNED THE (1) ROLE PERCEPTIONS, (2) BEHAVIORAL INTENTIONS, AND (3) PERCEPTIONS OF SOCIAL BEHAVIOR IN THE SS. IN 1, 100 ROLES WERE JUDGED ON A SAMPLE OF 120 BEHAVIOR DESCRIPTIVE SCALES. BEHAVIORS WERE OBTAINED FROM POOLS OF SEVERAL THOUSAND BEHAVIORS ELICITED INDEPENDENTLY IN EACH CULTURE. SS JUDGED THE APPROPRIATENESS OF

HARRY C. TRIANDIS; VASSO VASSILIOU; MARIA NASSIAKOU

1968-01-01

212

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

213

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

214

Crucial factor causing collapse and aggregation of cultured cells in epon resin.  

PubMed

Ultrastructural artifacts regarding collapse and aggregation of cultured cells have been problematic, especially when investigated apoptotic cells. The infiltration process during sample preparation is considered to be the most crucial factor for this problem. This study was conducted using two culture systems: a suspension culture system of human T-lymphocyte Jurkat cells and rabbit mature dendritic cells and a monolayer culture system of human lung macrophages, human breast cancer cells (A-546 cells) and cat bone-invasive gingival cancer cells (sccf3 cells). Fixation was conducted prior to removing or detaching the cells from the culture dishes. Initial infiltration with a 1 : 3 volume ratio of epon resin : propylene oxide was found to be the most crucial step among these cultured cells. The improved epon-resin infiltration method could eliminate the artifacts. Thus, differentiation between artifactual images and true images is highly possible. PMID:25274402

Tangkawattana, Prasarn; Yamaguchi, Mamoru; Klomkleaw, Wuthichai; Niu, Hua; Minaguchi, Jun A; Takehana, Kazushige

2014-01-01

215

Using Haworthia Cultured Cells as an Aid in Teaching Botany  

ERIC Educational Resources Information Center

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

Majumdar, Shyamal K.; Castellano, John M.

1977-01-01

216

Neonatal rat heart cells cultured in simulated microgravity  

Microsoft Academic Search

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

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

1997-01-01

217

Maintaining dendritic cell viability in culture.  

PubMed

When mouse dendritic cells (DCs) are isolated from tissues, purified and placed in a nutritive culture they die more rapidly than would be expected from their normal turnover in vivo. This can distort culture assays of DC function. We therefore tested several approaches to prolonging DC survival in culture. Of several cytokines tested granulocyte-macrophage colony stimulating factor was most effective at preserving the viability of conventional DCs (cDCs) but was ineffective for plasmacytoid DCs (pDCs). Surprisingly, Fms-like tyrosine kinase 3 ligand, crucial for DC development, produced only a marginal improvement in DC survival in culture, and interleukin-3, reported to prevent apoptosis of human pDCs, produced only a minor improvement in survival of mouse DCs. Genetic manipulation of cell death pathways was also tested, to avoid activation effects exerted by cytokine signalling. The isolation of DCs from mice overexpressing Bcl-2 was especially effective in maintaining pDC viability but gave a lesser improvement in cDC viability. DCs isolated from Bim(-/-)Noxa(-/-) mice also showed improved culture survival, but in this case with pDCs showing the least improvement. PMID:25081090

Vremec, David; Hansen, Jacinta; Strasser, Andreas; Acha-Orbea, Hans; Zhan, Yifan; O'Keeffe, Meredith; Shortman, Ken

2015-02-01

218

Development of a bovine luteal cell in vitro culture system suitable for co-culture with early embryos.  

PubMed

The cross talk between the corpus luteum (CL) and the early embryo, potentially relevant to pregnancy establishment, is difficult to evaluate in the in vivo bovine model. In vitro co-culture of bovine luteal cells and early embryos (days 2-8 post in vitro fertilization) may allow the deciphering of this poorly understood cross talk. However, early embryos and somatic cells require different in vitro culture conditions. The objective of this study was to develop a bovine luteal cell in vitro culture system suitable for co-culture with early embryos in order to evaluate their putative steroidogenic and prostanoid interactions. The corpora lutea of the different stages of the estrous cycle (early, mid, and late) were recovered postmortem and enriched luteal cell populations were obtained. In experiments 1 and 2, the effects of CL stage, culture medium (TCM, DMEM-F12, or SOF), serum concentration (5 or 10%), atmosphere oxygen tension (5 or 20%), and refreshment of the medium on the ability of luteal cells to produce progesterone (P(4)) were evaluated. The production of P(4) was significantly increased in early CL cultures, and luteal cells adapted well to simple media (SOF), low serum concentrations (5%), and oxygen tensions (5%). In experiment 3, previous luteal cell cryopreservation did not affect the production of P(4), PGF(2?), and PGE(2) compared to fresh cell cultures. This enables the use of pools of frozen-thawed cells to decrease the variation in cell function associated with primary cell cultures. In experiment 4, mineral oil overlaying culture wells resulted in a 50-fold decrease of the P(4) quantified in the medium, but had no effect on PGF(2?) and PGE(2) quantification. In conclusion, a luteal cell in vitro culture system suitable for the 5-d-long co-culture with early embryos was developed. PMID:23054443

Batista, M; Torres, A; Diniz, P; Mateus, L; Lopes-da-Costa, L

2012-10-01

219

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; Käppeli, Patrick; Shamsollahi, Mohammad Javad; Ferguson, Stephen J.; Gantenbein-Ritter, Benjamin

2013-01-01

220

Rosmarinic acid production in Coleus cell cultures.  

PubMed

Cell suspension cultures of Coleus blumei Benth. have been found to accumulate 8-11% of their dry weight as rosmarinic acid (?-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 rosmarinic acid failed to enhance the latter's production and in some cases inhibited it. PMID:24420667

Razzaque, A; Ellis, B E

1977-01-01

221

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

SciTech Connect

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

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

2011-01-21

222

Co-culture system of human salivary gland epithelial cells and immune cells from primary Sjögren's syndrome patients: an in vitro approach to study the effects of Rituximab on the activation of the Raf-1/ERK1/2 pathway.  

PubMed

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disorder of the exocrine glands with associated lymphocytic infiltrates in the affected glands. Dryness of the mouth and eyes results from involvement of the salivary and lacrimal glands. The efficacy of Rituximab (RTX) in pSS is still open to debate. This study delineates the signaling pathway involved in RTX-mediated down-regulation of pro-inflammatory factors in a co-culture system of pSS salivary gland epithelial cells (SGEC) with syngeneic pSS B-lymphocytes. In addition, the effects of RTX on the activation of the Raf-1/ERK1/2 pathway in pSS SGEC co-cultured with syngeneic pSS T-lymphocytes were also investigated. This study demonstrated that RTX may interfere with the ERK1/2 pathway in a syngeneic co-culture of pSS SGEC with pSS B-lymphocytes, leading to decreased cytokine production by SGEC. These novel findings reveal that syngeneic co-culture of pSS SGEC with pSS B-lymphocytes leads to a down-regulation of Raf-1 in epithelial cells that adversely regulates the activity of the ERK1/2 pathway and determines a subsequent reduction of the release of pro-inflammatory factors. PMID:25381666

Lisi, Sabrina; Sisto, Margherita; D'Amore, Massimo; Lofrumento, Dario Domenico

2014-11-01

223

Chemically modified tetracyclines induce apoptosis in cultured mast cells  

Microsoft Academic Search

Chemically modified tetracyclines are a group of non-antimicrobial tetracycline derivatives, which possess antiinflammatory, anticollagenolytic and antiproliferative properties. Here we studied the effects of four different chemically modified tetracyclines (CMT-1, CMT-3, CMT-8 and CMT-308) on proliferation and viability of cultured mouse and human mast cells. All studied CMTs (25 ?M) effectively inhibited the viability and proliferation of human mast cell line

Charlotta Sandler; Katariina Nurmi; Ken A. Lindstedt; Timo Sorsa; Lorne M. Golub; Petri T. Kovanen; Kari K. Eklund

2005-01-01

224

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

225

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

PubMed Central

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories. PMID:20186512

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

2010-01-01

226

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

227

Method of determining the number of cells in cell culture  

SciTech Connect

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

Connolly, D.T.

1990-06-12

228

Cultural Heritage in the Crosshairs: Protecting Cultural Property during Conflict provides case studies of Cultural  

E-print Network

by participating nations. This volume provides a series of case studies and "lessons learned" to assess the current" by Michael Hallett Chapter 9: "A Case Study in Cultural Heritage Protection in a Time of War" by CPT BenjaminCultural Heritage in the Crosshairs: Protecting Cultural Property during Conflict provides case

229

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

230

Aragonite Precipitation by “Proto-Polyps” in Coral Cell Cultures  

PubMed Central

The mechanisms of coral calcification at the molecular, cellular and tissue levels are poorly understood. In this study, we examine calcium carbonate precipitation using novel coral tissue cultures that aggregate to form “proto-polyps”. Our goal is to establish an experimental system in which calcification is facilitated at the cellular level, while simultaneously allowing in vitro manipulations of the calcifying fluid. This novel coral culturing technique enables us to study the mechanisms of biomineralization and their implications for geochemical proxies. Viable cell cultures of the hermatypic, zooxanthellate coral, Stylophora pistillata, have been maintained for 6 to 8 weeks. Using an enriched seawater medium with aragonite saturation state similar to open ocean surface waters (?arag?4), the primary cell cultures assemble into “proto-polyps” which form an extracellular organic matrix (ECM) and precipitate aragonite crystals. These extracellular aragonite crystals, about 10 µm in length, are formed on the external face of the proto-polyps and are identified by their distinctive elongated crystallography and X-ray diffraction pattern. The precipitation of aragonite is independent of photosynthesis by the zooxanthellae, and does not occur in control experiments lacking coral cells or when the coral cells are poisoned with sodium azide. Our results demonstrate that proto-polyps, aggregated from primary coral tissue culture, function (from a biomineralization perspective) similarly to whole corals. This approach provides a novel tool for investigating the biophysical mechanism of calcification in these organisms. PMID:22514707

Mass, Tali; Drake, Jeana L.; Haramaty, Liti; Rosenthal, Yair; Schofield, Oscar M. E.; Sherrell, Robert M.; Falkowski, Paul G.

2012-01-01

231

Aragonite precipitation by "proto-polyps" in coral cell cultures.  

PubMed

The mechanisms of coral calcification at the molecular, cellular and tissue levels are poorly understood. In this study, we examine calcium carbonate precipitation using novel coral tissue cultures that aggregate to form "proto-polyps". Our goal is to establish an experimental system in which calcification is facilitated at the cellular level, while simultaneously allowing in vitro manipulations of the calcifying fluid. This novel coral culturing technique enables us to study the mechanisms of biomineralization and their implications for geochemical proxies. Viable cell cultures of the hermatypic, zooxanthellate coral, Stylophora pistillata, have been maintained for 6 to 8 weeks. Using an enriched seawater medium with aragonite saturation state similar to open ocean surface waters (?(arag)~4), the primary cell cultures assemble into "proto-polyps" which form an extracellular organic matrix (ECM) and precipitate aragonite crystals. These extracellular aragonite crystals, about 10 µm in length, are formed on the external face of the proto-polyps and are identified by their distinctive elongated crystallography and X-ray diffraction pattern. The precipitation of aragonite is independent of photosynthesis by the zooxanthellae, and does not occur in control experiments lacking coral cells or when the coral cells are poisoned with sodium azide. Our results demonstrate that proto-polyps, aggregated from primary coral tissue culture, function (from a biomineralization perspective) similarly to whole corals. This approach provides a novel tool for investigating the biophysical mechanism of calcification in these organisms. PMID:22514707

Mass, Tali; Drake, Jeana L; Haramaty, Liti; Rosenthal, Yair; Schofield, Oscar M E; Sherrell, Robert M; Falkowski, Paul G

2012-01-01

232

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

233

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

234

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

235

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

236

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...

237

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

238

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

239

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

240

Sequencing technologies for animal cell culture research.  

PubMed

Over the last 10 years, 2nd and 3rd generation sequencing technologies have made the use of genomic sequencing within the animal cell culture community increasingly commonplace. Each technology's defining characteristics are unique, including the cost, time, sequence read length, daily throughput, and occurrence of sequence errors. Given each sequencing technology's intrinsic advantages and disadvantages, the optimal technology for a given experiment depends on the particular experiment's objective. This review discusses the current characteristics of six next-generation sequencing technologies, compares the differences between them, and characterizes their relevance to the animal cell culture community. These technologies are continually improving, as evidenced by the recent achievement of the field's benchmark goal: sequencing a human genome for less than $1,000. PMID:25214225

Kremkow, Benjamin G; Lee, Kelvin H

2015-01-01

241

Alloresponses of cord blood cells in primary mixed lymphocyte cultures.  

PubMed

The aim of this study was to compare the alloreactive responses against HLA antigens of cord blood cells with those of adult peripheral blood cells. In primary mixed lymphocyte cultures and bulk cell-mediated lympholysis experiments cord blood cells demonstrated significantly decreased proliferation and cytotoxicity. Experiments analyzing the specificity of anti-HLA cytotoxic T lymphocytes (CTL) revealed that cord blood (CB) CTL reacted only partially with third-party cells expressing the stimulating HLA antigens. Lower frequencies of IL-2 producing helper, cytotoxic T-cell precursors and IL-4 producing CB cells were found, whereas the frequencies of IFN-gamma producing cells, as determined by ELISpot experiments, were equivalent to the frequencies of adult IFN-gamma producing cells. Our results imply that, although CB cells have significantly decreased proliferative and cytotoxic alloresponses in bulk mixed lymphocyte cultures, their IFN-gamma production is comparable with that of adult mononuclear cells. Preserved production of IFN-gamma may be a risk factor for the development of graft-versus-host disease and should be taken into consideration when evaluating the possibility for stem cell transplantation with HLA-mismatched CB. PMID:11872233

Slavcev, A; Stríz, I; Ivasková, E; Breur-Vriesendorp, B S

2002-03-01

242

Optimization of human corneal endothelial cell culture: density dependency of successful cultures in vitro  

PubMed Central

Background Global shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. Limited ex vivo expansion of primary human corneal endothelial cells is possible, and a considerable clinical interest exists for development of tissue-engineered constructs using cultivated corneal endothelial cells. The objective of this study was to investigate the density-dependent growth of human corneal endothelial cells isolated from paired donor corneas and to elucidate an optimal seeding density for their extended expansion in vitro whilst maintaining their unique cellular morphology. Results Established primary human corneal endothelial cells were propagated to the second passage (P2) before they were utilized for this study. Confluent P2 cells were dissociated and seeded at four seeding densities: 2,500 cells per cm2 (‘LOW’); 5,000 cells per cm2 (‘MID’); 10,000 cells per cm2 (‘HIGH’); and 20,000 cells per cm2 (‘HIGH×2’), and subsequently analyzed for their propensity to proliferate. They were also subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when each culture became confluent. At the two lower densities, proliferation rates were higher than cells seeded at higher densities, though not statistically significant. However, corneal endothelial cells seeded at lower densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like), and remained hypertrophic after one month in culture. Comparatively, cells seeded at higher densities were significantly homogeneous, compact and circular at confluence. Potentially, at an optimal seeding density of 10,000 cells per cm2, it is possible to obtain between 10 million to 25 million cells at the third passage. More importantly, these expanded human corneal endothelial cells retained their unique cellular morphology. Conclusions Our results demonstrated a density dependency in the culture of primary human corneal endothelial cells. Sub-optimal seeding density results in a decrease in cell saturation density, as well as a loss in their proliferative potential. As such, we propose a seeding density of not less than 10,000 cells per cm2 for regular passage of primary human corneal endothelial cells. PMID:23641909

2013-01-01

243

Recycling cultured cells for immunofluorescent labeling.  

PubMed

A method to use sequential rounds of immunofluorescent labeling in cell cultures is presented. The method is based on the utilization of a non-liquid reducing agent, sodium dithionite, in conjunction with ionic or non-ionic detergents (SDS or TX100, respectively) at room temperature. This method preserves cell morphology and substrate antigenicity, and operates through the complete extraction of most primary and secondary antibodies. Using this protocol, the sequential immunolocalization of different proteins is possible, without signal interference with previous immunolabeling rounds. In addition, the method is also useful to recycle blotted membranes in immunoblots. PMID:11479721

Espada, J; Juarranz, A; Villanueva, A; Cañete, M; Andrés, I; Stockert, J C

2001-07-01

244

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

245

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

246

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

247

German Language and Cultural Studies Major and German Studies Minor  

E-print Network

German Language and Cultural Studies Major and German Studies Minor www.german.pitt.edu Revised: 12 for the German Language and Cultural Studies major The German Language and Cultural Studies major requires Advanced German 2: Structures GER 1104 German for Social Scientists 1 GER 1106 German Cultural History

Jiang, Huiqiang

248

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

PubMed

Human umbilical cord mesenchymal stem cells (hUCMSCs) have been recently used in transplant therapy. The proliferation and migration of MSCs are the determinants of the efficiency of MSC transplant therapy. Sertoli cells are a kind of "nurse" cells that support the development of sperm cells. Recent studies show that Sertoli cells promote proliferation of endothelial cells and neural stem cells in co-culture. We hypothesized that co-culture of UCMSCs with Sertoli cells may also promote proliferation and migration of UCMSCs. To examine this hypothesis, we isolated UCMSCs from human cords and Sertoli cells from mouse testes, and co-cultured them using a Transwell system. We found that UCMSCs exhibited strong proliferation ability and potential to differentiate to other cell lineages such as osteocytes and adipocytes. The presence of Sertoli cells in co-culture significantly enhanced the proliferation and migration potential of UCMSCs (P<0.01). Moreover, these phenotypic changes were accompanied with upregulation of multiple genes involved in cell proliferation and migration including phospho-Akt, Mdm2, phospho-CDC2, Cyclin D1, Cyclin D3 as well as CXCR4, phospho-p44 MAPK and phospho-p38 MAPK. These findings indicate that Sertoli cells boost UCMSC proliferation and migration potential. PMID:22975347

Zhang, Fenxi; Hong, Yan; Liang, Wenmei; Ren, Tongming; Jing, Suhua; Lin, Juntang

2012-10-12

249

Clearance of Human-Pathogenic Viruses from Sludge: Study of Four Stabilization Processes by Real-Time Reverse Transcription-PCR and Cell Culture  

PubMed Central

Sludges derived from wastewater treatment are foul-smelling, biologically unstable substances. As well as containing numerous pathogenic microorganisms, they also consist of organic matter that can be used as agricultural fertilizer. Legislation nevertheless requires sludges to be virologically tested prior to spreading by the counting of infectious enterovirus particles. This method, based on culture of enterovirus on BGM cells, is lengthy and not very sensitive. The aim of this study was to propose an alternative method of genome quantification for all enteroviruses that is applicable to verifying the elimination of viruses in complex samples such as sludges. Our complete protocol was compared to the official method, consisting of enterovirus enumeration with the most probable number of cythopathic unit (MPNCU) assay through the study of four stabilization procedures: liming, composting, heat treatment, and mesophile anaerobic digestion. Enterovirus quantities at the start of the stabilization procedures were between 37 and 288 MPNCU/g on the one scale and between 4 and 5 log genome copies/g on the other. It was shown that all procedures except mesophile anaerobic digestion were highly effective in the elimination of enterovirus particles and genomes in wastewater sludges. Reduction of viruses by mesophile anaerobic digestion was by only 1 log (infectious particles and genomes). In conclusion, stabilization processes can indeed be checked by virological quality control of sludges with gene amplification. However, the infectivity of genomes needs to be confirmed with cell culture or a correlation model if the virological risk inherent in the agricultural use of such sludges is to be fully addressed. PMID:15345430

Monpoeho, S.; Maul, A.; Bonnin, C.; Patria, L.; Ranarijaona, S.; Billaudel, S.; Ferré, V.

2004-01-01

250

Isolation, culture, and transplantation of muscle satellite cells  

PubMed Central

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

Motohashi, Norio; Asakura, Yoko; Asakura, Atsushi

2014-01-01

251

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

252

Development of a culture system to induce microglia-like cells from haematopoietic cells  

PubMed Central

Aims Microglia are the resident immune cells in the central nervous system, originating from haematopoietic-derived myeloid cells. A microglial cell is a double-edged sword, which has both pro-inflammatory and anti-inflammatory functions. Although understanding the role of microglia in pathological conditions has become increasingly important, histopathology has been the only way to investigate microglia in human diseases. Methods To enable the study of microglial cells in vitro, we here establish a culture system to induce microglia-like cells from haematopoietic cells by coculture with astrocytes. The characteristics of microglia-like cells were analysed by flow cytometry and functional assay. Results We show that triggering receptor expressing on myeloid cells-2-expressing microglia-like cells could be induced from lineage negative cells or monocytes by coculture with astrocytes. Microglia-like cells exhibited lower expression of CD45 and MHC class II than macrophages, a characteristic similar to brain microglia. When introduced into brain slice cultures, these microglia-like cells changed their morphology to a ramified shape on the first day of the culture. Moreover, we demonstrated that microglia-like cells could be induced from human monocytes by coculture with astrocytes. Finally, we showed that interleukin 34 was an important factor in the induction of microglia-like cells from haematopoietic cells in addition to cell–cell contact with astrocytes. Purified microglia-like cells were suitable for further culture and functional analyses. Conclusion Development of in vitro induction system for microglia will further promote the study of human microglial cells under pathological conditions as well as aid in the screening of drugs to target microglial cells. PMID:24016036

Noto, D; Sakuma, H; Takahashi, K; Saika, R; Saga, R; Yamada, M; Yamamura, T; Miyake, S

2014-01-01

253

Contamination of cell cultures with bovine viral diarrhea virus (BVDV).  

PubMed

The incidence of contamination of cell strains used in biological and virological studies and of fetal calf sera (FCS) manufactured by Russian and foreign companies used for cell culturing with noncytocidal bovine viral diarrhea virus (BVDV; Pestivirus, Flaviviridae) was analyzed. The virus was detected by reverse transcription PCR and indirect immunofluorescence with monoclonal antibodies to BVDV virion envelope glycoprotein in 25% of 117 cell strains and 45% of 35 tested FCS lots. The virus multiplied and persisted in a wide spectrum of human cell strains and in monkey, swine, sheep, rabbit, dog, cat, and other animal cells. The levels of BVDV genome RNA in contaminated cell cultures reached 10(2)-10(3) g-eq/cell and in serum samples 10(3)-10(7) g-eq/ml. These facts necessitate testing of cells and FCS for BVDV reproduced in cells without signs of infection detectable by light microscopy. The molecular mechanisms of long-term virus persistence in cells without manifestation of cell destruction are unknown. PMID:22808499

Uryvaev, L V; Dedova, A V; Dedova, L V; Ionova, K S; Parasjuk, N A; Selivanova, T K; Bunkova, N I; Gushina, E A; Grebennikova, T V; Podchernjaeva, R J

2012-05-01

254

Dinitrosyl iron complexes with thiol-containing ligands and apoptosis: studies with HeLa cell cultures.  

PubMed

No pro-apoptotic effect of dinitrosyl iron complexes (DNIC) with glutathione, cysteine or thiosulfate was established after incubation of HeLa cells in Eagle's medium. However, DNIC with thiosulfate manifested pro-apoptotic activity during incubation of HeLa cells in Versene's solution supplemented with ethylene diamine tetraacetate (EDTA) known to induce the decomposition of these DNIC. The water-soluble ?-phenanthroline derivative bathophenanthroline disulfonate (BPDS) had a similar effect on DNIC with glutathione during incubation of HeLa cells in Eagle's medium. It was assumed that EDTA- or BPDS-induced pro-apoptotic effect of DNIC with thiosulfate or glutathione is coupled with the ability of decomposing DNIC to initiate S-nitrosylation of proteins localized on the surface of HeLa cells. Presumably, the pro-apoptotic effect of S-nitrosoglutathione (GS-NO) on HeLa cells preincubated in Eagle's medium is mediated by the same mechanism, although the pro-apoptotic effect based on the ability of GS-NO to initiate the release of significant amounts of NO and its oxidation to cytotoxic peroxynitrite in a reaction with superoxide should not be ruled out either. No apoptotic activity was found in the presence of bivalent iron and glutathione favoring the conversion of GS-NO into DNIC with glutathione. It is suggested that interaction of HeLa cells with intact DNIC with glutathione or thiosulfate results in the formation of DNIC bound to cell surface proteins. PMID:21354319

Giliano, Nadezhda Ya; Konevega, Leonid V; Noskin, Leonid A; Serezhenkov, Vladimir A; Poltorakov, Alexander P; Vanin, Anatoly F

2011-04-30

255

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

256

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

257

Cultural studies of Morchella elata.  

PubMed

The in vitro growth of Morchella elata was characterized with respect to the effects of a variety of substrates, isolates, developmental status of the parental ascoma, temperature, and pH. Optimal substrates for growth included sucrose, mannose and lactose, but the growth of some isolates was substantially reduced in some composite media. Maltose and potato-dextrose media limited growth and caused changes in colony morphology; mycelial pigmentation was black in the case of maltose, and mycelial margins were plumose in potato-dextrose cultures. Rapid growth was most reliably achieved in a composite medium containing 1:1 sucrose:mannose. Isolates derived from single ascospores shortly after ejection from ascomata varied in ability to grow in the various substrates. This may be related to variable maturity or dormancy; increasing growth rates correlated with pileus length in the parental ascomata, and ascomata that initially produced slower-growing or abortive colonies produced faster-growing colonies after storage at 20 degrees C for 96 wk. The growth of M. elata derived from recently ejected ascospores was optimal at 16-24 degrees C or above for a faster-growing isolate, and 20-24 degrees C or above for a slow-growing isolate. Although neither isolate grew at 8 degrees C or below in an initial experiment, spawn cultured on puffed wheat at 28 degrees C produced mycelia that proliferated when transferred to soil media and incubated at 8 degrees C. Growth of M. elata in liquid cultures adjusted with potassium hydroxide was optimal at pH 7.0, and was relatively sensitive to more acidic or alkaline pH. When calcium carbonate was used to adjust pH, optimal growth shifted to pH 7.7 or above, suggesting that wood ash and other calcium compounds may not only stimulate growth in natural settings, but also alter the optimal pH for proliferation of M. elata. Further studies with other substrate combinations and incubation conditions will be necessary to fully understand the connections between in vitro growth and the ecological behaviour of the fungus. PMID:16769512

Winder, Richard S

2006-05-01

258

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

259

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

260

Isolation, culture, and characterization of human pancreatic duct cells.  

PubMed

To establish a suitable control for pancreatic tumor cell lines, we have isolated and cultured primary human pancreatic duct cells from transplant donors. Duct cells were isolated by dissecting the main pancreatic duct and first-degree branches and enzymatic digestion. Aggregates of cells were cultured for 1 up to 5 weeks and monitored for changes in morphology and growth by phase contrast microscopy. Contaminating fibroblasts were mechanically removed from day 4 on and by cloning of epithelial cells. Cultured cells were characterized by phase contrast microscopy, electron microscopy, and immunofluorescence with antibodies against intermediate filaments (cytokeratins, vimentin, desmin), mucins (Du-Pan-2, CA 19-9), carbonic anhydrase II, acinar cell enzymes (amylase, lipase, trypsin), and islet cells. About 90% of the cultured cells could be identified as ductal epithelial cells by their expression of cytokeratins, mucins, and carbonic anhydrase II. These cells showed the ultrastructural features of duct cells. After 3-5 weeks of culture, most of the cultured cells showed co-expression of cytokeratins and vimentin in addition to duct cell markers. About 10% of cells were contaminating fibroblasts (vimentin positive, cytokeratin negative). The cultured normal human duct cells as the postulated cells of origin of the pancreatic adenocarcinoma may serve as a useful control for cultured pancreatic tumor cell lines. PMID:8460098

Trautmann, B; Schlitt, H J; Hahn, E G; Löhr, M

1993-03-01

261

Heritability and Control of Differentiated Function in Cultured Cells  

PubMed Central

An established tissue culture cell line which retains a differentiated function in vitro is described. The cell line is of connective tissue origin, and its characteristic property is the synthesis and secretion of acid mucopolysaccharides, mainly hyaluronic acid. This differentiated cell function, the activity of which depends on continuous gene action, was found to be possessed by each of eleven clonal substrains, and is therefore a genetically heritable cell character. Rate of acid mucopolysaccharide biosynthesis falls sharply under the influence of the environmental conditions existing in crowded cultures, and this rate also declines if protein synthesis is directly inhibited with puromycin. Environmental modification of a differentiated product of gene action is thus illustrated in this study. PMID:14025353

Davidson, E. H.

1963-01-01

262

Isolation and cell culture propagation of rotaviruses from turkeys and chickens  

Microsoft Academic Search

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

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

1979-01-01

263

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

264

Arsenic exposure induces the Warburg effect in cultured human cells  

SciTech Connect

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

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

2013-08-15

265

Culture and characterization of oral mucosal epithelial cells on human amniotic membrane for ocular surface reconstruction  

PubMed Central

Purpose To culture oral mucosal epithelial cells on deepithelialized human amniotic membrane without the use of feeder cells and to compare the characteristics of cultured oral cells with cultured limbal and conjunctival epithelial cells for use in ocular surface reconstruction. Methods Oral biopsies were obtained from healthy volunteers after informed consent and were cultured on deepithelialized amniotic membrane for three to four weeks. Confluent cultures of limbal, oral, and conjunctival cells were subjected to characterization of markers of stem cells and of epithelial differentiation by reverse-transcription polymerase chain reaction (RT–PCR) and by immunohistochemistry. Ultrastructural studies were also performed using electron microscopy. Results A sheet of healthy, stratified oral epithelial cells was obtained within three to four weeks of culture. Electron microscopy demonstrated that the cells formed gap junctions and desmosomes. RT–PCR analysis showed that cultured oral epithelial cells expressed markers of epithelial differentiation such as cytokeratin K3, K4, K13, K15 and connexin 43. The cells also expressed stem cell markers of epithelial cells such as ?N isoforms of p63 as well as p75, a marker for stem cells of oral epithelium. The cells did not express cytokeratin K12 or Pax-6, an eye-specific transcription factor. Conclusions Oral epithelial cells can be cultured as explants on deepithelialized amniotic membrane without using feeder cells. Characterization showed that these cells maintain the phenotypic characteristics of oral epithelial cells and that the culture is a heterogeneous population of differentiated cells and stem cells. We find the cultured oral epithelial cells usable for ocular surface reconstruction in patients having bilateral ocular surface diseases. PMID:18334934

Madhira, Soundarya Lakshmi; Vemuganti, Geeta; Bhaduri, Anirban; Gaddipati, Subhash; Sangwan, Virender Singh

2008-01-01

266

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

PubMed

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

Hunter, Wayne B

2010-02-01

267

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

268

Equipment for large-scale mammalian cell culture.  

PubMed

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

Ozturk, Sadettin S

2014-01-01

269

A Microfluidic Localized, Multiple Cell Culture Array using Vacuum Actuated Cell Seeding: Integrated Anticancer Drug Testing  

PubMed Central

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 cells 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

2013-01-01

270

Cannabinoids induce incomplete maturation of cultured human leukemia cells  

SciTech Connect

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

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

1987-08-01

271

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

272

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 non-myocyte 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 which 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 in cultured in HARV's 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 HARV's 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 tissue-like organizations of cardiac cells in simulated microgravity.

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

1994-01-01

273

Comparative study of radical scavenger and antioxidant properties of phenolic compounds from Vitis vinifera cell cultures using in vitro tests  

Microsoft Academic Search

Vitis vinifera cell suspensions were used to isolate and characterize the flavonoids (anthocyanins, catechins) and non-flavonoids (stilbenes) found in red wine. Furthermore, we showed that astringin is produced although this stilbene has not previously been reported to be a constituent of V. vinifera or wine. The ability of these compounds to act as radical scavengers was investigated using 1,1-diphenyl-2-picryl-hydrazyl (DPPH),

Bernard Fauconneau; Pierre Waffo-Teguo; François Huguet; Laurence Barrier; Alain Decendit; Jean-Michel Merillon

1997-01-01

274

Heat-Transfer-Method-Based Cell Culture Quality Assay through Cell Detection by Surface Imprinted Polymers.  

PubMed

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

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

2015-02-17

275

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

SciTech Connect

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

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

2012-10-12

276

Radiosensitivity of cultured insect cells: II. Diptera  

SciTech Connect

The radiosensitivity of five dipteran cell lines representing three mosquito genera and one fruit fly genus were examined. These lines are: (1) ATC-10, Aedes aegypti; (2) RU-TAE-14, Toxorhynchites amboinensis; (3) RU-ASE-2A, Anopheles stephensi; (4) WR69-DM-1, Drosophila melanogaster; and (5) WR69-DM-2, Drosophila melanogaster. Population doubling times for these lines range from approximately 16 to 48 hr. Diploid chromosome numbers are six for the mosquito cells and eight for the fruit fly cells D/sub 0/ values are 5.1 and 6.5 Gy for the Drosophila cell lines and 3.6, 6.2, and 10.2 Gy for the mosquito cell lines. The results of this study demonstrate that dipteran insect cells are a few times more resistant to radiation than mammalian cells, but not nearly as radioresistant as lepidopteran cells.

Koval, T.M.

1983-10-01

277

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; Rodríguez-Moyá, María; Gonzalez, Ramon; Altamirano, Claudia

2014-01-01

278

Importance of NF-?B in rheumatoid synovial tissues: in situ NF-?B expression and in vitro study using cultured synovial cells  

PubMed Central

OBJECTIVES—To examine whether inhibition of NF-?B induces apoptosis of human synovial cells stimulated by tumour necrosis factor ? (TNF?), interleukin 1? (IL1?), and anti-Fas monoclonal antibody (mAb).?METHODS—The expression of proliferating cell nuclear antigen (PCNA), NF-?B, and the presence of apoptotic synovial cells were determined in synovial tissues. Apoptosis of cultured synovial cells was induced by inhibition of NF-?B nuclear translocation by Z-Leu-Leu-Leu-aldehyde (LLL-CHO). The activation of caspase-3 and expression of XIAP and cIAP2 in synovial cells in LLL-CHO induced apoptosis was also examined.?RESULTS—Abundant PCNA+ synovial cells were found in rheumatoid arthritis (RA) synovial tissue, though a few apoptotic synovial cells were also detected in the RA synovial tissues. Nuclear NF-?B was expressed in RA synovial cells. Electrophoretic mobility shift assay showed that treatment of cells with TNF? or IL1? significantly stimulated nuclear NF-?B activity. A small number of apoptotic synovial cells expressing intracellular active caspase-3 were found after treatment of cells with LLL-CHO. Although treatment of RA synovial cells with TNF? or IL1? alone did not induce apoptosis, apoptosis induced by LLL-CHO and caspase-3 activation were clearly enhanced in TNF? or IL1? stimulated synovial cells compared with unstimulated synovial cells. Furthermore, induction of apoptosis of synovial cells with caspase-3 activation by anti-Fas mAb was clearly increased by LLL-CHO. The expression of cIAP2 and XIAP in synovial cells may not directly influence the sensitivity of synovial cells to apoptosis induced by LLL-CHO.?CONCLUSION—The results suggest that NF-?B inhibition may be a potentially important therapeutic approach for RA by correcting the imbalance between apoptosis and proliferation of synovial cells in RA synovial tissue.?? PMID:11406522

Yamasaki, S; Kawakami, A; Nakashima, T; Nakamura, H; Kamachi, M; Honda, S; Hirai, Y; Hida, A; Ida, H; Migita, K; Kawabe, Y; Koji, T; Furuichi, I; Aoyagi, T; Eguchi, K

2001-01-01

279

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

280

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

PubMed Central

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

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

2015-01-01

281

Oxygenation of intensive cell-culture system.  

PubMed

The abilities of various methods of oxygenation to meet the demands of high-cell-density culture were investigated using a spin filter perfusion system in a bench-top bioreactor. Oxygen demand at high cell density could not be met by sparging with air inside a spin filter (oxygen transfer values in this condition were comparable with those for surface aeration). Sparging with air outside a spin filter gave adequate oxygen transfer for the support of cell concentrations above 10(7) ml-1 in fully aerobic conditions but the addition of antifoam to control foaming caused blockage of the spinfilter mesh. Bubble-free aeration through immersed silicone tubing with pure oxygen gave similar oxygen transfer rates to that of sparging with air but without the problems of bubble damage and fouling of the spin filter. A supra-optimal level of dissolved oxygen (478% air saturation) inhibited cell growth. However, cells could recover from this stress and reach high density after reduction of the dissolved oxygen level to 50% air saturation. PMID:8590652

Emery, A N; Jan, D C; al-Rubeai, M

1995-11-01

282

Increased osmolarity and cell clustering preserve canine notochordal cell phenotype in culture.  

PubMed

Degeneration of the intervertebral disc (IVD) is associated with a loss of notochordal cells (NCs) from the nucleus pulposus (NP) and their replacement by chondrocyte-like cells. NCs are known to maintain extracellular matrix quality and stimulate the chondrocyte-like NP cells, making NCs attractive for designing new tissue engineering approaches for IVD regeneration. However, optimal conditions, such as osmolarity and other characteristics of the culture media, for long-term culture of NCs are not known. The purpose of this study was to investigate the effects of different culture media and osmolarity on the physiology of NCs in vitro. NC clusters isolated from canine IVDs were suspended in alginate beads and cultured at 37°C under normoxic conditions for 28 days. Three different culture conditions were investigated; (1) Dulbecco's modified Eagle's medium (DMEM)/F12 (300 mOsm/L), (2) ?-MEM (300 mOsm/L), and (3) ?-MEM adjusted to 400 mOsm/L to mimic a hyperosmolar environment. NC morphology, expression of genes related to NC markers, matrix production and remodeling, and DNA- and glycosaminoglycan (GAG) analyses were performed on 1, 7, 14, and 28 days in culture. Large, vesicle-containing cells organized in clusters, characterized as NCs, remained present during 28 days for all culture conditions. However, the proportion of the NC clusters decreased over time, whereas the proportion of spindle-shaped cells increased. Gene expression profiling at 7, 14, and 28 days in culture compared to day 1 indicated a initial loss of NC phenotype followed by some recovery of brachyury and aggrecan gene expression after 28 days of culture supporting a potential recovery of NC phenotype. NCs cultured in ?-MEM adjusted to 400 mOsm/L showed the highest gene expression of brachyury, cytokeratin 18, and aggrecan, the highest GAG production, and the lowest collagen 1?1 gene expression. In conclusion, NCs cultured in alginate in native cell clusters, partially retained their characteristic morphology and recovered their phenotype in long-term culture. The type of culture medium and medium osmolarity appear to be important factors for culturing NC clusters. These findings provide additional information concerning the maintenance of NCs in vitro that may aid further mechanistic inquiry into the biology of NCs. PMID:24304309

Spillekom, Sandra; Smolders, Lucas A; Grinwis, Guy C M; Arkesteijn, Irene T M; Ito, Keita; Meij, Björn P; Tryfonidou, Marianna A

2014-08-01

283

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

284

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

285

Cyclic AMP Signaling Functions as a Bimodal Switch in Sympathoadrenal Cell Development in Cultured Primary Neural Crest Cells  

Microsoft Academic Search

Cells of the vertebrate neural crest (crest cells) are an invaluable model system to address cell fate specification. Crest cells are amenable to tissue culture, and they differentiate to a variety of neuronal and nonneuronal cell types. Earlier studies have determined that bone morphogenetic proteins (BMP-2, -4, and -7) and agents that elevate intracellular cyclic AMP (cAMP) stimulate the development

MATTHEW L. BILODEAU; THERESA BOULINEAU; RONALD L. HULLINGER; OURANIA M. ANDRISANI

2000-01-01

286

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

287

Extracellular DNA in culture of primary and transformed cells, infected and not infected with mycoplasma.  

PubMed

The composition and kinetics of accumulation of extracellular DNA in cultures of primary human endotheliocytes, cervical adenocarcinoma, and mycoplasma-infected cervical adenocarcinoma cells were studied. The content of DNA bound to cell surface did not change during culturing. The concentration of extracellular DNA in culture medium increased during the lag phase and at the beginning of the exponential growth phase, which probably attests to active secretion of DNA by cells. Spontaneous extracellular DNA synthesis was observed only in cell culture infected with mycoplasma. PMID:19526133

Morozkin, E S; Sil'nikov, V N; Rykova, E Yu; Vlassov, V V; Laktionov, P P

2009-01-01

288

Inducing hepatic differentiation of human mesenchymal stem cells in pellet culture  

Microsoft Academic Search

Extensive cell–cell or cell–matrix interaction in three-dimensional (3D) culture is important for the maintenance of adult hepatocyte function and the maturation of hepatic progenitors. However, although there is significant interest in inducing the transdifferentiation of adult stem cells into the hepatic lineage, very few studies have been conducted in a 3D culture configuration. The aim of this study is to

Shin-Yeu Ong; Hui Dai; Kam W. Leong

2006-01-01

289

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

290

Mycoplasma Removal from Cell Culture Using Antimicrobial Photodynamic Therapy  

PubMed Central

Abstract Objective: The objective of this research was to determine the effectiveness of antimicrobial photodynamic therapy (aPDT) in the removal of mycoplasmas from contaminated cells. Background data: Mycoplasmas often contaminate cell cultures. The cell-contaminating mycoplasmas are removed by antibiotics, but the use of antibiotics usually induces antibiotic-resistant bacteria. aPDT is expected to be a possible alternative to antibiotic treatments for suppressing infections. Materials and Methods: Mycoplasma salivarium (Ms)-infected human embryonic kidney (HEK) 293 cells were irradiated using a red light-emitting diode (LED) in the presence of methylene blue (MB) as a photosensitizer. The Ms viable count was determined using culture on agar plates or using a mycoplasma detection kit. Results: aPDT performed using red LED irradiation was effective in decreasing live Ms in the presence of MB without damaging the HEK293 cells. aPDT removed live Ms from the infected cells after washing the cells with sterilized phosphate-buffered saline (PBS) to decrease the initial number of live Ms before aPDT. Conclusions: This study suggests that aPDT could remove mycoplasmas from contaminated cells. PMID:23402393

Hasebe, Akira; Ishikawa, Isao; Shamsul, Haque M.; Ohtani, Makoto; Segawa, Taku; Saeki, Ayumi; Tanizume, Naoho; Oouchi, Manabu; Okagami, Yoshihide; Okano, Teruo

2013-01-01

291

Prostacyclin synthesis in irradiated endothelial cells cultured from bovine aorta.  

PubMed

Confluent monolayers of bovine aortic endothelial cells were examined 2-72 h after exposure to 0.5-5.0 Gy of 60Co gamma-rays. Accumulation of prostacyclin [PGI2, measured as 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha)] in the culture media and PGI2 production stimulated by exogenous arachidonate were correlated with cell detachment and release of lactate dehydrogenase (LDH) activity. Platelet adherence to irradiated and control monolayers also was studied. There were simultaneous time- and dose-dependent increases in cell detachment and in the titers of 6-keto-PGF1 alpha and LDH activity in the culture medium. These changes were evident between 4 and 8 h after 5 Gy or at 24 h after 0.5 Gy. Four hours after 5 Gy, both adherent and detached endothelial cells showed a twofold increase in PGI2 production during a 15-min incubation with arachidonate (10 microM). However, by 72 h this increase was less significant. The accumulation of 6-keto-PGF1 alpha appeared to be related to cell destruction, but radiation also stimulated PGI2 synthesis independent of cell detachment. There was an increased platelet interaction with irradiated monolayers, as a result of platelet adherence to subendothelial matrix exposed after cell detachment. However, irradiation did not alter the nonadherent property of the endothelial cell surface toward platelets. PMID:3920190

Rubin, D B; Drab, E A; Ts'ao, C H; Gardner, D; Ward, W F

1985-02-01

292

Polyglycolic acid-polylactic acid scaffold response to different progenitor cell in vitro cultures: a demonstrative and comparative X-ray synchrotron radiation phase-contrast microtomography study.  

PubMed

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

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

2014-04-01

293

In vitro Spermatogenesis – Optimal Culture Conditions for Testicular Cell Survival, Germ Cell Differentiation, and Steroidogenesis in Rats  

PubMed Central

Although three-dimensional testicular cell cultures have been demonstrated to mimic the organization of the testis in vivo and support spermatogenesis, the optimal culture conditions and requirements remain unknown. Therefore, utilizing an established three-dimensional cell culture system that promotes differentiation of pre-meiotic murine male germ cells as far as elongated spermatids, the present study was designed to test the influence of different culture media on germ cell differentiation, Leydig cell functionality, and overall cell survival. Single-cell suspensions prepared from 7-day-old rat testes and containing all the different types of testicular cells were cultured for as long as 31?days, with or without stimulation by gonadotropins. Leydig cell functionality was assessed on the basis of testosterone production and the expression of steroidogenic genes. Gonadotropins promoted overall cell survival regardless of the culture medium employed. Of the various media examined, the most pronounced expression of Star and Tspo, genes related to steroidogenesis, as well as the greatest production of testosterone was attained with Dulbecco’s modified eagle medium?+?glutamine. Although direct promotion of germ cell maturation by the cell culture medium could not be observed, morphological evaluation in combination with immunohistochemical staining revealed unfavorable organization of tubules formed de novo in the three-dimensional culture, allowing differentiation to the stage of pachytene spermatocytes. Further differentiation could not be observed, probably due to migration of germ cells out of the cell colonies and the consequent lack of support from Sertoli cells. In conclusion, the observations reported here show that in three-dimensional cultures, containing all types of rat testicular cells, the nature of the medium per se exerts a direct influence on the functionality of the rat Leydig cells, but not on germ cell differentiation, due to the lack of proper organization of the Sertoli cells. PMID:24616715

Reda, Ahmed; Hou, Mi; Landreh, Luise; Kjartansdóttir, Kristín Rós; Svechnikov, Konstantin; Söder, Olle; Stukenborg, Jan-Bernd

2014-01-01

294

High-affinity binding of fibronectin to cultured Kupffer cells  

SciTech Connect

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

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

1990-11-01

295

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

296

Cell biologic properties in explant culture and heterotransplantation of malignant uterine cervical cells.  

PubMed

The cell of origin of uterine cervical cancer was studied by using culture, enzyme histochemistry and heterotransplantation. Twenty-seven epidermoid carcinomas (8 large cell keratinizing squamous, 12 large cell nonkeratinizing squamous and 7 small cell nonkeratinizing squamous) and 2 adenocarcinomas of the uterine cervix were placed in culture. An outgrowth of carcinoma cells in vitro was observed in 22 of 29 cases: 6 keratinizing, 8 large cell nonkeratinizing and 6 small cell nonkeratinizing carcinomas and 2 adenocarcinomas. The squamous carcinomas showed a squamous-cell outgrowth pattern, except for one large cell nonkeratinizing and three small cell nonkeratinizing carcinomas that showed a glandular-cell outgrowth pattern. One of three keratinizing carcinomas was transplantable into the subcutis of BALB/c nude mice, producing keratinizing tumors; three of six large cell and one of three small cell nonkeratinizing carcinomas reproduced themselves, while the other two small cell carcinomas produced poorly differentiated adenocarcinomas in mice. The transplanted adenocarcinoma produced a well-differentiated adenocarcinoma resembling the original tumor. Small cell carcinomas and adenocarcinomas contained a heat-stable, L-phenylalanine-sensitive alkaline phosphatase. These results suggest that many uterine cervical cancers originate from the reserve cell. PMID:3425154

Ishiwata, I; Ishiwata, C; Soma, M; Nozawa, S; Ishikawa, H

1987-01-01

297

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

298

Studies on Rat Hair Cultures  

Microsoft Academic Search

By means of two different quantification techniques the Authors were able to show that the addition of increasing concentrations of testosterone to the medium of hair-cultures simultaneously provokes an increased volume of epidermis as well as of hair-follicles and a better differentiation of hair follicles. The addition of dihydrotestosterone, on the contrary, does not induce any change.

Marcella Guarrera; P. Cardo; G. Moretti; E. Rampini; Carla Divano

1976-01-01

299

Ilex paraguariensis cell suspension culture characterization and response against ethanol  

Microsoft Academic Search

Cell suspension cultures of Ilex paraguariensis, a South American native tree known as the maté plant, were initiated in order to investigate plant defense. Cultures were characterized for their cell growth, chemical composition and sugar consumption. The present work quantified some effects of salicylic acid, methyl jasmonate, cellulase and ethanol on cell growth and sugar metabolism. Results suggest that salicylic

Kátia H. Kraemer; Eloir P. Schenkel; Robert Verpoorte

2002-01-01

300

Biolistic transformation of cotton embryogenic cell suspension cultures  

Technology Transfer Automated Retrieval System (TEKTRAN)

Genetic transformation of cotton is highly dependent on the ability to regenerate fertile plants from transgenic cells through somatic embryogenesis. Induction of embryogenic cell cultures is genotype-dependant. However, once embryogenic cell cultures are available, they can be effectively used fo...

301

Cholera toxin stimulation of human mammary epithelial cells in culture  

SciTech Connect

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

Stampfer, M.R.

1982-06-01

302

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

303

Determination of queuosine modification system deficiencies in cultured human cells.  

PubMed

Queuosine-deficient tRNAs are often observed in neoplastic cells. In order to determine possible sites for malfunction of the multistep queuosine modification system, comprehensive studies were performed on two human neoplastic cell lines, the HxGC(3) colon adenocarcinoma and the MCF-7 breast adenocarcinoma, which are 100 and 50-60% queuosine deficient, respectively. These results were compared with data obtained from normal human fibroblast (HFF) cultures which maintain 100% queuosine-modified tRNA populations. Queuine uptake in all three cell types was similar and each demonstrated activation by protein kinase C (PKC). However, incorporation of queuine into tRNA by tRNA:guanine ribosyltransferase (TGRase; E.C. 2.4.2.24) and PKC-catalyzed activation of this enzyme occurred only in HFF and MCF-7 cells. The HxGC(3) cell line exhibited no TGRase activity as was expected. Treatment with 5-azacytidine (5-azaC) induced TGRase activity to a level 20% of that in HFF and MCF-7 cells; however, this 5-azaC-induced TGRase activity was not regulated by PKC. Salvage of the queuine base from tRNA degradation products has been shown in mammalian cells and was measured in the HFF cells. However, salvage activity in the MCF-7 cell line was deficient. Therefore, it was shown by direct measurements that the HxGC(3) cell line is completely lacking in queuosine-modified tRNA due to loss of functional TGRase, while the MCF-7 cell line has an inefficient queuine salvage mechanism resulting in a significant deficiency of queuosine-modified tRNA. These techniques can be applied to any cultured cell types to determine specific lesions of the queuosine modification system, which have been suggested to be associated with neoplastic progression. PMID:10479483

Morris, R C; Galicia, M C; Clase, K L; Elliott, M S

1999-09-01

304

Variation of T-type calcium channel protein expression affects cell division of cultured tumor cells  

Microsoft Academic Search

In this study we investigated the T-type calcium channel and its involvement in the cell division of U87MG cultured glioma cells and N1E-115 neuroblastoma cells. Using Western blot analysis, we found that expression of both ?1G and ?1H subunits of the T-type calcium channel decreased during conditions associated with a decrease in proliferation as evidenced by increased expression of cyclin

Amith Panner; Leanne L. Cribbs; Gina M. Zainelli; Thomas C. Origitano; Sanjay Singh; Robert D. Wurster

2005-01-01

305

Stochastic synchronization analysis of cultured human glial cells  

NASA Astrophysics Data System (ADS)

The production of calcium waves is a property of a healthy astrocyte culture when exposed to the neurotransmitter kainate [Jung et al, J. Neurophys, 79, 1098 (1998)]. Healthy and epileptic tissues differ to a great extent in their dynamics: while a healthy cell culture shows much pattern formation, and wave propagation, the epileptic tissue shows spatially irregular flickering activity or global oscillation. Developing statistical tools to describe healthy versus epileptic tissue dynamics could be very important in order to study the effects of specific drugs, or to identify oscillation centers in the epileptic brain. We perform a statistical analysis in terms of phase synchronization. We show that hyper active epileptic astrocyte cultures are characterized by synchronization between different regions of the network taken from the uncus part of the brain.

Balazsi, Gabor; Cornell-Bell, Ann; Simonotto, Enrico; Neiman, Alexander; Moss, Frank

2000-03-01

306

Characterization of biomaterial-free cell sheets cultured from human oral mucosal epithelial cells.  

PubMed

The purpose of this study was to report the characteristics of biomaterial-free sheets cultured from human oral mucosal epithelial cells without fibrin support, in vitro and after transplantation to limbal-deficient models. Human oral mucosal epithelial cells and limbal epithelial cells were cultured for 2 weeks, and the colony-forming efficiency (CFE) rates were compared. Markers of stem cells (p63), cell proliferation (Ki-67) and epithelial differentiation (cytokeratin; K1, K3, K4, K13) were observed in colonies and in biomaterial-free sheets. Biomaterial-free sheets which had been detached with 1% dispase or biomaterial-free sheets generated by fibrin support were transplanted to 12 limbal-deficient rabbit models. In vitro cell viability, in vivo stability and cytokeratin characteristics of biomaterial-free sheets were compared with those of sheets formed by fibrin-coated culture 1 week after transplantation. Mean CFE rate was significantly higher in human oral mucosal epithelial cells (44.8%) than in human limbal epithelial cells(17.7%). K3 and K4 were well expressed in both colonies and sheets. Biomaterial-free sheets had two to six layers of stratified cells and showed an average of 79.8% viable cells in the sheets after detachment. Cytokeratin expressions of biomaterial-free sheets were comparable to those of sheets cultured by fibrin support, in limbal-deficient models. Both p63 and Ki-67 were well expressed in colonies, isolated sheets and sheets transplanted to limbal-deficient models. Our results suggest that biomaterial-free sheets cultured from human oral mucosal epithelial cells without fibrin support can be an alternative option for cell therapy in use for the treatment of limbal-deficient diseases. Copyright © 2014 John Wiley & Sons, Ltd. PMID:25407749

Hyun, Dong Won; Kim, Yun Hee; Koh, Ah Young; Lee, Hyun Ju; Wee, Won Ryang; Jeon, Saewha; Kim, Mee Kum

2014-11-19

307

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

308

Establishment and validation of an in vitro co-culture model to study the interactions between bone and prostate cancer cells  

Microsoft Academic Search

Bone is the preferred site for prostate cancer (PCa) metastases. Once the tumor has established itself within the bone there\\u000a is virtually no cure. To better understand the interactions between the PCa cells and bone environment in the metastatic process\\u000a new model systems are needed. We have established a two-compartment in vitro co-culturing model that can be used to follow

Annika Nordstrand; Jonas Nilsson; Åse Tieva; Pernilla Wikström; Ulf H. Lerner; Anders Widmark

2009-01-01

309

COMMUNICATION TO THE EDITOR High Cell Density Culture of  

E-print Network

COMMUNICATION TO THE EDITOR High Cell Density Culture of Metabolically Engineered Escherichia coli- tation processes employing several different bacteria have been developed to improve PHB productivity

310

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

ERIC Educational Resources Information Center

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

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

2002-01-01

311

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

312

Development of a gastrointestinal tract microscale cell culture analog to predict drug transport  

Technology Transfer Automated Retrieval System (TEKTRAN)

Microscale cell culture analogs (uCCAs) are used to study the metabolism and toxicity of a chemical or drug. These in vitro devices are physical replicas of physiologically based pharmacokinetic models that combine microfabrication and cell culture. The goal of this project is to add an independent ...

313

The use of mouse embryo cell cultures in primary isolation of chikungunya virus  

PubMed Central

Successful primary isolation of chikungunya virus from clinical specimens was carried out using mouse embryo cell cultures. Parallel isolation studies using the classical intracerebral inoculation of suckling mice showed that both systems are about equally sensitive. However, the mouse embryo cultures seem to have certain advantages over the use of mice; these include economy of materials and time, precision, and a greater uniformity of reaction. Furthermore, the infective tissue-culture fluid provides a more convenient source of complement-fixing antigen. Compared with other cell cultures, mouse embryo cultures have the advantage of being readily available in all arbovirus laboratories. The method should be particularly useful in epidemic situations. PMID:5317083

David-West, Tam S.

1971-01-01

314

Cell and tissue culture of Miscanthus Sacchariflorus  

SciTech Connect

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

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

1995-11-01

315

AIDS IN AFRICA, CULTURAL STUDIES IN GEORGIA  

Microsoft Academic Search

This essay discusses the challenges of teaching a unit on AIDS in Africa in the conservative context of Georgia Institute of Technology. According to the author, the students’ dominant orientation as applied scientists and Christians disputed many of the methodological goals of cultural studies, including geopolitical and institutional critique as well as the presence of cultural biases in ostensibly objective

Tabitha Sparks

2005-01-01

316

Automated and online characterization of adherent cell culture growth in a microfabricated bioreactor.  

PubMed

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; Szita, Nicolas

2014-10-01

317

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

318

Mechanisms of platelet activation by cultured human cancer cells and cells freshly isolated from tumor tissues.  

PubMed

We studied the effects on platelet function of cells isolated from freshly dissociated human tumor tissues (11 breast carcinomas, 9 colon carcinomas and 1 lymph node metastasis from melanoma) obtained at surgery as compared with cultured human tumor cells: namely, human melanoma 1402 cell line derived from a primary tumor and two lines derived from lymph node metastases (ME 7110/2 and Me 665/1) as well as a human hepatoma cell line (Hep G2). The three melanoma cell lines activated platelets by producing ADP, as evidenced by the inhibitory effect of apyrase and by the direct measurement of the agonist in the supernatants of tumor cell suspensions; this production was much greater by the cells derived from metastases than by the cells derived from the primary tumor. On the other hand, aggregation induced by Hep G2 hepatoma cells was unaffected by apyrase and was inhibited by hirudin or concanavalin A, suggesting that the cells aggregate platelets by producing thrombin, probably through tissue factor activity of the cells themselves. Cells isolated from 16 of the 21 human tumor tissues possessed a potent platelet-aggregating effect, which was not inhibited by apyrase, hirudin or concanavalin A, but was virtually abolished by the cysteine protease inhibitors iodoacetic acid or p-hydroxymercuri-phenylsulfonate. Collectively, our data demonstrate that cells isolated from freshly dissociated tumor tissues activate platelets through tumor-associated cysteine proteinases rather than by the ADP- or thrombin-dependent mechanisms characteristic of cultured human tumor cell lines. PMID:2767927

Grignani, G; Pacchiarini, L; Ricetti, M M; Dionigi, P; Jemos, V; Zucchella, M; Fratino, P

1989-01-01

319

A Rat Primary Hepatocyte Culture Model for Aging Studies  

PubMed Central

The purpose of this protocol is to establish a primary hepatocyte culture system as a suitable model to examine age-related changes in Phase II detoxication gene expression. Hepatocytes are isolated using a two-step collagenase perfusion technique from young (3 to 6 months) and old (24 to 28 months) rats and placed in primary culture using collagen (Type I)-coated plates as the extracellular matrix. A supplemented William’s E Medium is used as the medium. This culture system maintains hepatocyte viability from both young and old rats for ~60 hr, as measured by lactate dehydrogenase activity, while also maintaining their respective phenotypes relative to Phase II detoxification. We thus conclude that a collagen-based cell culture system is suitable to study age-associated deficits in Nrf2/ARE-mediated Phase II gene regulation provided that experiments can be conducted within 60 hr after cell isolation. PMID:23045003

Shenvi, Swapna V.; Dixon, Brian M.; Shay, Kate Petersen; Hagen, Tory M.

2014-01-01

320

Optimization of Storage Temperature for Cultured ARPE-19 Cells  

PubMed Central

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

321

Methylmercury disrupts the balance between phosphorylated and non-phosphorylated cofilin in primary cultures of mice cerebellar granule cells A proteomic study  

SciTech Connect

Methylmercury is an environmental contaminant that is particularly toxic to the developing central nervous system; cerebellar granule neurons are especially vulnerable. Here, primary cultures of cerebellar granule cells (CGCs) were continuously exposed to methylmercury for up to 16 days in vitro (div). LC50 values were 508 +- 199, 345 +- 47, and 243 +- 45 nM after exposure for 6, 11, and 16 div, respectively. Proteins from cultured mouse CGCs were separated by 2DE. Seventy-one protein spots were identified by MALDI-TOF PMF and MALDI-TOF/TOF sequencing. Prolonged exposure to a subcytotoxic concentration of methylmercury significantly increased non-phosphorylated cofilin both in cell protein extracts (1.4-fold; p < 0.01) and in mitochondrial-enriched fractions (1.7-fold; p < 0.01). The decrease in P-cofilin induced by methylmercury was concentration-dependent and occurred after different exposure times. The percentage of P-cofilin relative to total cofilin significantly decreased to 49 +- 13% vs. control cells after exposure to 300 nM methylmercury for 5 div. The balance between the phosphorylated and non-phosphorylated form of cofilin regulates actin dynamics and facilitates actin filament turnover. Filamentous actin dynamics and reorganization are responsible of neuron shape change, migration, polarity formation, regulation of synaptic structures and function, and cell apoptosis. An alteration of the complex regulation of the cofilin phosphorylation/dephosphorylation pathway could be envisaged as an underlying mechanism compatible with reported signs of methylmercury-induced neurotoxicity.

Vendrell, Iolanda [Department of Neurochemistry and Neuropharmacology, Institut d'Investigacions Biomediques de Barcelona, Consejo Superior de Investigaciones Cientificas, CSIC - IDIBAPS, Barcelona (Spain); CIBER Epidemiologia y Salud Publica (CIBERESP) (Spain); Carrascal, Montserrat [CSIC/UAB Proteomics Laboratory, Institut d'Investigacions Biomediques de Barcelona, Consejo Superior de Investigaciones Cientificas, CSIC - IDIBAPS, Barcelona (Spain); Campos, Francisco [Department of Neurochemistry and Neuropharmacology, Institut d'Investigacions Biomediques de Barcelona, Consejo Superior de Investigaciones Cientificas, CSIC - IDIBAPS, Barcelona (Spain); CIBER Epidemiologia y Salud Publica (CIBERESP) (Spain); Abian, Joaquin [CSIC/UAB Proteomics Laboratory, Institut d'Investigacions Biomediques de Barcelona, Consejo Superior de Investigaciones Cientificas, CSIC - IDIBAPS, Barcelona (Spain); Sunol, Cristina, E-mail: csenqi@iibb.csic.e [Department of Neurochemistry and Neuropharmacology, Institut d'Investigacions Biomediques de Barcelona, Consejo Superior de Investigaciones Cientificas, CSIC - IDIBAPS, Barcelona (Spain); CIBER Epidemiologia y Salud Publica (CIBERESP) (Spain)

2010-01-01

322

Comparison of Various Methods for Preparation of Viral Serological Antigens from Infected Cell Cultures  

PubMed Central

In efforts to prepare more potent and sensitive viral serological antigens, several aspects of the production of antigens from infected cell cultures were studied. Antigens derived from whole, infected culture material and from the cellular and fluid phases were compared. Freezing and thawing, sonication, and alkaline buffer extraction were compared for effectiveness in releasing antigen from host cells. The effect of the multiplicity of infection on titers of viral antigens produced in cell cultures was studied. Generally, higher titered antigens were derived from the infected cells than from the culture fluids, but for certain viruses complement-fixing (CF) antigens derived from the culture fluids gave higher antibody titers than did cell-associated antigens. With each virus-host cell system studied, treatment with alkaline buffers extracted appreciable amounts of CF antigen from the host cells, but in some instances more antigen was released by freezing and thawing or by sonication. Extraction of infected cells with alkaline buffers was not a satisfactory method for preparation of hemagglutinating (HA) antigens for any of the viruses studied. The highest-titered HA antigens were produced from infected cells disrupted by freezing and thawing or sonication. The highest titered CF and HA antigens were produced from cell cultures infected at multiplicities of one or greater. Complement-fixing antigens produced by infecting cells in suspension and then planting had lower titers than antigens produced in parallel by infecting developed monolayers. Optimal methods are summarized for preparation of serological antigens to a variety of viruses of man. PMID:4994353

Schmidt, Nathalie J.; Lennette, Edwin H.

1971-01-01

323

Culture of cells from mammalian tissue cryopreserved without cryoprotection  

E-print Network

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

Charles, Lara Nicole

2009-05-15

324

Design of 3D printed insert for hanging culture of Caco-2 cells.  

PubMed

A Caco-2 cell culture on Transwell, an alternative testing to animal or human testing used in evaluating drug intestinal permeability, incorrectly estimated the absorption of actively transported drugs due to the low expression of membrane transporters. Similarly, three-dimensional (3D) cultures of Caco-2 cells, which have been recommended to be more physiological relevant, were not superior to the Transwell culture in either accuracy or convenience in drug permeability testing. Using rapid 3D printing prototyping techniques, this study proposed a hanging culture of Caco-2 cells that performed with high accuracy in predicting drug permeability in humans. As found, hanging cultured Caco-2 cells formed a confluent monolayer and maintained high cell viability on the 3D printed insert. Compared with the normal culture on Transwell, the Caco-2 cells on the 3D printed insert presented ?30-100% higher brush border enzyme activity and ?2-7 folds higher activity of P-glycoprotein/multidrug resistance-associated protein 2 during 21 days of incubation. For the eight membrane transporter substrates, the predictive curve of the 3D printing culture exhibited better linearity (R(2) = 0.92) to the human oral adsorption than that of the Transwell culture (R(2) = 0.84), indicating better prediction by the 3D printing culture. In this regard, the 3D printed insert for hanging culture could be potentially developed as a convenient and low-cost tool for testing drug oral absorption. PMID:25514920

Shen, Chong; Meng, Qin; Zhang, Guoliang

2014-01-01

325

Communication Studies, Cultural Studies, and Media Studies Infobase  

NSDL National Science Digital Library

Mick Underwood's extensive infobase of communication, culture, and media studies introduces various models and theories of communication and explains their social and cultural implications. In addition, the infobase includes a detailed index of terms, a bibliography of references, a list of links, a message forum, a chat room, and a series of online quizzes. The great volume of information at this site is effectively managed via several navigation tools: a site map, an index, pull-down menus, and meaningful icons.

326

Imaging and Analysis of Three-Dimensional Cell Culture Models  

PubMed Central

Three-dimensional (3D) cell cultures are important tools in cell biology research and tissue engineering because they more closely resemble the architectural microenvironment of natural tissue, compared to standard two-dimensional cultures. Microscopy techniques that function well for thin, optically transparent cultures, however, are poorly suited for imaging 3D cell cultures. Three-dimensional cultures may be thick and highly scattering, preventing light from penetrating without significant distortion. Techniques that can image thicker biological specimens at high resolution include confocal microscopy, multiphoton microscopy, and optical coherence tomography. In this chapter, these three imaging modalities are described and demonstrated in the assessment of functional and structural features of 3D chitosin scaffolds, 3D micro-topographic substrates from poly-dimethyl siloxane molds, and 3D Matrigel cultures. Using these techniques, dynamic changes to cells in 3D microenvironments can be non-destructively assessed repeatedly over time. PMID:19957133

Graf, Benedikt W.; Boppart, Stephen A.

2013-01-01

327

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

PubMed

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

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

2014-01-01

328

Effects of pantothenic acid on fibroblastic cell cultures  

Microsoft Academic Search

Summary To evaluate the effects of pantothenic acid during wound healing processes, fibroblastic cell cultures originating from foreskin were established and subcultured by trypsinization. PA (40 µg\\/ml) was added to the basal culture medium. The cell proliferation was estimated by cell count and determination of3H-thymidine incorporation. The protein synthesis and secretion were determined by dosage in the cells and in

B. Lacroix; E. Didier; J. F. Grenier

1988-01-01

329

Cerebral microvessels and derived cells in tissue culture  

Microsoft Academic Search

Summary  An endothelial cell line has been established from a primary culture of cerebral microvessels isolated from Swiss-Webster\\u000a mice. The microvessels were isolated by a mechanical dispersion and filtration technique. The cells that emerged from these\\u000a microvessels, maintained in organoid cultures, proliferated and formed plaques of a single or mixed cell type. The endothelial\\u000a cell line, designated ME-2, was isolated from

Lawrence E. DeBault; Eduardo Henriquez; Michael N. Hart; Pasquale A. Cancilla

1981-01-01

330

Clinical-scale human umbilical cord blood cell expansion in a novel automated perfusion culture system  

Microsoft Academic Search

Use of umbilical cord blood (CB) for stem cell transplantation has a number of advantages, but a major disadvantage is the relatively low cell number available. Ex vivo cell expansion has been proposed to overcome this limitation, and this study therefore evaluated the use of perfusion culture systems for CB cell expansion. CB was cryopreserved using standard methods and the

Koller; I Manchel; RJ Maher; KL Goltry; AK Smith

1998-01-01

331

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

332

Radiosensitivity of cultured insect cells: I. Lepidoptera  

SciTech Connect

The radiosensitivity of five lepidopteran insect cell lines representing five different genera has been investigated. These lines are: (1) TN-368, Trichoplusia ni; (2) IPLB-SF-1254, Spodoptera frugiperda; (3) IPLB-1075, Heliothis zea; (4) MRRL-CHl, clone GVl, Manduca sexta; and (5) IAL-PID2, Plodia interpunctella. The cell lines grew at different rates and had population doubling times that ranged from 19 to 52 hr. All of the lines are highly heteroploid and have approximate chromosome numbers near or above 100. The chromosomes are very small. All of the lines are extremely radioresistant; cell populations are able to recover from 260 kVp X-ray exposures up to and including 400 Gy, the highest dose examined. Cell survival curves were obtainable for only the TN-368 and IPLB-SF-1254 lines. The TN-368 cells displayed a biphasic survival response with D/sub 0/, d/sub q/, and n values of 65.7 and 130.2 Gy, 9.0 and -36.1 Gy, and 1.2 and 0.8, respectively, for the steep and shallow portions of the curve. The IPLB-SF-1254 cells had a D/sub 0/ of 63.9 Gy. D/sub q/ of 19.0 Gy, and n value of 1.4. These studies provide definitive evidence of the radioresistance of lepidopteran cells, and suggest that this radioresistance is a characteristic of lepidopteran insects.

Koval, T.M.

1983-10-01

333

Bovine ephemeral fever virus in cell culture and mice  

Microsoft Academic Search

Summary Light, immunofluorescent and electron microscopic observations were carried out sequentially on mice and VERO cell cultures infected with bovine ephemeral fever (BEF) virus. In early harvests from cell culture, 185×73 nm cone-shaped particles with nearly parallel sides predominated; these particles had all other features typical of the Rhabdoviruses (surface projections, envelope, axial channel, precisely coiled helical nucleocapsid with 35

Frederick A. Murphy; William P. Taylor; Cedric A. Mims; Sylvia G. Whitfield

1972-01-01

334

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

335

High-Aspect-Ratio Rotating Cell-Culture Vessel  

NASA Technical Reports Server (NTRS)

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

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

1992-01-01

336

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

Microsoft Academic Search

Cells of Chlorella fusca var. vacuolata (Cambridge 211\\/8p) resisted efforts aimed at producing naked protoplasts by enzymatic degradation of the cell wall, and a study of the development and composition of the wall was therefore undertaken.1.After cytokinesis has produced naked autospores within the mother cell wall, cell wall formation commences outside the autospore plasma membrane with the appearance of small

A. W. Atkinson; B. E. S. Gunning; P. C. L. John

1972-01-01

337

Cultural Studies' Networking Strategies in the South  

Microsoft Academic Search

In the 1990s I started to take an interest in the networking capacity for Cultural Studies when I saw how Kuan-Hsing Chen set up the Inter-Asia Cultural Studies group, an alternative intellectual knowledge-exchange which now has an eponymous (Taylor and Francis) journal and a vital and well-funded network of scholars working among Taiwan, China, Japan, Hong Kong, Singapore, India, Korea,

Stephen Muecke

338

Biona-C Cell Culture pH Monitoring System  

NASA Technical Reports Server (NTRS)

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

Friedericks, C.

1999-01-01

339

Culture phases, cytotoxicity and protein expressions of agarose hydrogel induced Sp2/0, A549, MCF-7 cell line 3D cultures.  

PubMed

Advancements in cell cultures are occurring at a rapid pace, an important direction is culturing cells in 3D conditions. We demonstrate the usefulness of agarose hydrogels in obtaining 3 dimensional aggregates of three cell lines, A549, MCF-7 and Sp2/0. The differences in culture phases, susceptibility to cisplatin-induced cytotoxicity are studied. Also, the 3D aggregates of the three cell lines were reverted into 2D cultures and the protein profile differences among the 2D, 3D and revert cultures were studied. The analysis of protein profile differences using UniProt data base further augment the usefulness of agarose hydrogels for obtaining 3D cell cultures. PMID:25371010

Ravi, Maddaly; Kaviya, S R; Paramesh, V

2014-11-01

340

Studies of alpha-granule proteins in cultured human megakaryocytes.  

PubMed

alpha-Granule protein storage is important for producing platelets with normal haemostatic function. The low to undetectable levels of several megakaryocyte-synthesized alpha-granule proteins in normal plasma suggest megakaryocytes are important to sequester these proteins in vivo. alpha-Granule protein storage in vitro has been studied using other cell types, with differences observed in how some proteins are processed compared to platelets. Human megakaryocytes, cultured from cord blood CD34(+) cells and grown in serum-free media containing thrombopoietin, were investigated to determine if they could be used as a model for studying normal alpha-granule protein processing and storage. ELISA indicated that cultured megakaryocytes contained the alpha-granule proteins multimerin, von Willebrand factor, thrombospondin-1, beta-thromboglobulin and platelet factor 4, but no detectable fibrinogen and factor V. A significant proportion of the alpha-granule protein in megakaryocyte cultures was contained within the cells (averages: 41-71 %), consistent with storage. Detailed analyses of multimerin and von Willebrand factor confirmed that alpha-granule proteins were processed to mature forms and were predominantly located in the alpha-granules of cultured megakaryocytes.Thrombopoietin-stimulated cultured megakaryocytes provide a useful model for studying alpha-granule protein processing and storage. PMID:14597980

Veljkovic, Dragoslava Kika; Cramer, Elisabeth M; Alimardani, Gulie; Fichelson, Serge; Massé, Jean-Marc; Hayward, Catherine P M

2003-11-01

341

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

PubMed

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

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

2014-02-01

342

From cells to organisms: Can we learn about aging from cells in culture?  

SciTech Connect

Can studying cultured cells inform us about the biology of aging? The idea that this may be was stimulated by the first formal description of replicative senescence. Replicative senescence limits the proliferation of normal human cells in culture, causing them to irreversibly arrest growth and adopt striking changes in cell function. We now know that telomere shortening, which occurs in most somatic cells as a consequence of DNA replication, drives replicative senescence in human cells. However, rodent cells also undergo replicative senescence, despite very long telomeres, and DNA damage,the action of certain oncogenes and changes in chromatin induce a phenotype similar to that of replicatively senescent cells. Thus,replicative senescence is an example of the more general process of cellular senescence, indicating that the telomere hypothesis of aging is a misnomer. Cellular senescence appears to be a response to potentially oncogenic insults, including oxidative stress. The growth arrest almost certainly suppresses tumorigenesis, at least in young organisms, whereas the functional changes may contribute to aging,although this has yet to be critically tested. Thus, cellular senescence may be an example of antagonistic pleiotropy.Cross-species comparisons suggest there is a relationship between the senescence of cells in culture and organismal life span, but the relationship is neither quantitative nor direct.

Campisi, Judith

2000-12-21

343

Human embryonic stem cells: Derivation, culture, and differentiation: A review  

PubMed Central

The greatest therapeutic promise of human embryonic stem cells (hESC) is to generate specialized cells to replace damaged tissue in patients suffering from various degenerative diseases. However, the signaling mechanisms involved in lineage restriction of ESC to adopt various cellular phenotypes are still under investigation. Furthermore, for progression of hESC-based therapies towards clinical applications, appropriate culture conditions must be developed to generate genetically stable homogenous populations of cells, to hinder possible adverse effects following transplantation. Other critical challenges that must be addressed for successful cell implantation include problems related to survival and functional efficacy of the grafted cells. This review initially describes the derivation of hESC and focuses on recent advances in generation, characterization, and maintenance of these cells. We also give an overview of original and emerging differentiation strategies used to convert hESC to different cell types. Finally, we will discuss transplantation studies of hESC-derived cells with respect to safety and functional recovery. PMID:20714081

Vazin, Tandis; Freed, William J.

2010-01-01

344

Human alveolar epithelial type II cells in primary culture.  

PubMed

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

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

2015-02-01

345

SITES OF NUCLEOLUS PRODUCTION IN CULTURED CHINESE HAMSTER CELLS  

PubMed Central

Chinese hamster cell strains in the early passages in culture display wide variation in number of nucleolus-like bodies per cell, though such strains are characteristically euploid. A variety of criteria indicate that the nucleolus-like bodies are true nucleoli. Their Azure B- and fast green-staining properties indicate the presence of RNA and protein; they have typical nucleolar fine structure, including both fibrous and granular components; radioautography reveals that their patterns of uptake of uridine-3H into RNA are similar to those reported for nucleoli of other cell types; actinomycin D, at a level which selectively inhibits ribosomal RNA synthesis, greatly reduces their RNA synthesis and also causes segregation of fibrous and granular nucleolar components. Colchicine was used to experimentally fragment the nuclei of these cells into a number of separate karyomeres, each presumably containing some, or only one, of the chromosomes of the complement. Almost all the karyomeres contain nucleolus-like bodies which, by the same criteria applied to the multiple nucleolus-like bodies of uninuclear cells, appear to be true nucleoli. The nucleoli of individual karyomeres of the same cell often differ from each other in fine structure while the multiple nucleoli of a uninuclear cell generally resemble each other. The evidence presented in this study indicates that Chinese hamster cells contain many nucleolus-producing sites scattered through the genome. PMID:4177660

Phillips, Stephanie Gordon; Phillips, David M.

1969-01-01

346

Cultural Studies and Media Ecology: Meyrowitz's Medium Theory and Carey's Cultural Studies.  

ERIC Educational Resources Information Center

Examines work of two communication and media studies scholars, Joshua Meyrowitz and James Carey. Suggests their studies represent media ecology with analyses of the dynamic interaction between communication, consciousness, and culture. Highlights how their work embodies a North American cultural studies approach to media studies (moving away from…

Flayhan, Donna P.

2001-01-01

347

A microwell array system for stem cell culture  

PubMed Central

Directed embryonic stem (ES) cell differentiation is a potentially powerful approach for generating a renewable source of cells for regenerative medicine. Typical in vitro ES cell differentiation protocols involve the formation of ES cell aggregate intermediates called embryoid bodies (EBs). Recently, we demonstrated the use of poly(ethylene glycol) (PEG) microwells as templates for directing the formation of these aggregates, offering control over parameters such as size, shape, and homogeneity. Despite these promising results, the previously developed technology was limited as it was difficult to reproducibly obtain cultures of homogeneous EBs with high efficiency and retrievability. In this study, we improve the platform by optimizing a number of features: material composition of the microwells, cell seeding procedures, and aggregate retrieval methods. Adopting these modifications, we demonstrate an improved degree of homogeneity of the resulting aggregate populations and establish a robust protocol for eliciting high EB formation efficiencies. The optimized microwell array system is a potentially versatile tool for ES cell differentiation studies and high-throughput stem cell experimentation. PMID:18001830

Moeller, Hannes-Christian; Mian, Matthew; Shrivastava, Shamit; Chung, Bong Geun; Khademhosseini, Ali

2008-01-01

348

Highly reproducible quantification of apoptotic cells using micropatterned culture of neurons.  

PubMed

The quantification of apoptotic cells is an integral component of many cell-based assays in biological studies. However, current methods for quantifying apoptotic cells using conventional random cultures have shown great limitations, especially for the quantification of primary neurons. Randomly distributed neurons under primary culture conditions can lead to biased estimates, and vastly different estimates of cell numbers can be produced within the same experiment. In this study, we developed a simple, accurate, and reliable technique for quantifying apoptotic neurons by means of micropatterned cell cultures. A polydimethylsiloxane (PDMS) microstencil was used as a physical mask for micropatterning cell cultures, and primary granular neurons (GNs) were successfully cultured within the micropattern-confined regions and homogeneously distributed over the entire field of each pattern. As compared with the conventional method based on random cultures, the micropatterned culture method allowed for highly reproducible quantification of apoptotic cells. These results were also confirmed by using GNs derived from mice with neurodegeneration. We hope that this micropatterning method based on the use of a PDMS microstencil can overcome the technical obstacles existing in current biological studies and will serve as a powerful tool for facilitating the study of apoptosis-involved diseases. PMID:25277814

Lee, Hyun; Kim, Gyu Man; Choi, Jin Ho; Lee, Jong Kil; Bae, Jae-Sung; Jin, Hee Kyung

2015-01-15

349

In Vitro Cell Culture Models for Evaluating Controlled Release Pulmonary Drug Delivery  

Microsoft Academic Search

\\u000a A variety of cell culture systems for modeling the pulmonary system have been developed. They offer the potential to study\\u000a various cell biology-related questions in the lung field. In this chapter primary cell cultures, continuous disease models,\\u000a and coculture models are discussed. The use of these models in biopharmaceutical research is then reviewed along with discussion\\u000a on drug permeability, transporters,

Stephen T. Buckley; Kwang-Jin Kim; Carsten Ehrhardt

350

Human dendritic cell culture and bacterial infection.  

PubMed

Dendritic cells (DC) play a key role in the development of natural immunity to microbes. The DC form a bridge between the innate and adaptive immune system by providing key instructions particularly to antigen naïve T-cells. The interaction of DC with T lymphocytes involves three signals: (1) antigen processing and presentation in context of MHC Class I and/or II, (2) expression of T cell co-stimulatory molecules, and (3) cytokine production. Studying the interactions of DCs with specific pathogens allows for better understanding of how protective immunity is generated, and may be particularly useful for assessing vaccine components. In this chapter, we describe methods to generate human monocyte-derived DCs and assess their maturation, activation, and function, using interaction with the gram-negative bacterial pathogen Neisseria meningitidis as a model. PMID:21993649

Jones, Hannah E; Klein, Nigel; Dixon, Garth L J

2012-01-01

351

In vitro Culture of Human Testicular Stem Cells on Feeder-Free Condition  

PubMed Central

Background Spermatogonial stem cells are subpopulation of spermatogonial cells in testis tissue that support beginning and maintenance of spermatogenesis. Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) could be a specific marker for identification of spermatogonial stem cells including spermatogonial sperm cells (SSCs) in testis tissue and during the culture; therefore we undertook this study to culture these human testicular stem cells (hTSCs) in vitro and approved the presence of human testicular stem cells (hTSCs) by UCHL1, also known as PGP9.5. Methods Enzymatic digestion of human testicular biopsies was done by collagenase IV (4 mg/ml) and trypsin (0.25%). Differential plating of testicular cells in DMEM/F12 and 10% FBS was applied for 16 hr. Floating cells were collected and transferred onto laminin-coated plates with Stem-Pro 34 media supplemented with growth factors of GDNF, bFGF, EGF and LIF to support self-renewal divisions; testicular stem cell clusters were passaged every 14 days for two months. Spermatogonial cells propagation was studied through Expression of UCHL1 in testis tissue and the entire testicular stem cell culture. Results Testicular stem cell clusters from 10 patients with obstructive azoospermia were cultured on laminin-coated plates and subsequently propagated for two months. The average of harvested viable cells was approximately 89.6%. UCHL1 was expressed as specific marker in testicular stem cells entire the culture. Conclusion Human testicular stem cells could be obtained from human testicular tissue by a simple digestion, culturing and propagation method for long-term in vitro conditions. Propagation of these cells approved by specific marker UCHL1, during the culture period. PMID:23926556

Piravar, Zeinab; Jeddi-Tehrani, Mahmood; Sadeghi, Mohammad Reza; Mohazzab, Arash; Eidi, Akram; Akhondi, Mohammad Mehdi

2013-01-01

352

Establishment and characterization of Xenopus oviduct cells in primary culture  

SciTech Connect

Based on previously established procedure of Xenopus hepatocytes, the authors describe tubular oviduct cells in primary culture which continue to secrete substantial quantities of egg jelly for several days, as can be visualized microscopically. Freshly isolated cells exhibited a culture shock response, from which they recovered by the third day in culture. This recovery was characterized by (a) the diminished synthesis of heat shock proteins hsp 70 and hsp 85, (b) the cessation of the drop in number of estrogen receptor, and (c) the enhanced rate of synthesis of cellular and secreted proteins. The oviduct estrogen receptor had the same characteristics as those in other estrogen target tissues and was present in the same amount as in adult female Xenopus hepatocytes. The successful establishment and characterization of primary cultures of both liver and oviduct cells now fulfill the conditions required for investigating the basis for tissue specificity of regulation by estrogen of Xenopus egg protein gene expression in primary cell culture.

Marsh, J.; Tata, J.R. (National Inst. for Medical Research, London (United Kingdom))

1987-11-01

353

Modulation of cyclin transcript levels in cultured cells of Arabidopsis thaliana.  

PubMed Central

Previous studies on the cell cycle of Arabidopsis thaliana have been hindered by the lack of synchronous cell culture systems. We have used liquid callus cultures and a cycloheximide-synchronized suspension culture of Arabidopsis to investigate changes in cyclin transcript levels in response to exogenous auxin, cytokinin, and nutrients, and during the cell cycle. CYCD1 (delta 1) transcript was virtually undetectable in liquid-cultured callus or suspension-culture cells. CYCD2 (delta 2) transcript levels were largely unaffected by the readdition of phytohormones or nitrate to the growth medium, and remained constant throughout the cell cycle in suspension-culture cells. CYCD3 (delta 3) transcript levels were strongly dependent on nitrate, and were induced at the G1/S transition following phytohormone readdition. In synchronized suspension-culture cells, CYCD3 transcript accumulated during the S phase, and remained constant thereafter. These results support the hypothesis that D cyclins function as part of the cellular machinery that integrates diverse signals impinging upon commitment to cell division. In synchronized cells transcripts of the mitotic cyclins CYC1, CYC2, and CYC3 reached a maximum with peak mitotic index, but CYC3 transcript levels increased earlier than those of CYC1 or CYC2. The kinetics of accumulation of CYC transcript levels support their classification as A-type (CYC3) and B-type (CYC1 and CYC2) cyclins, respectively. PMID:8938409

Fuerst, R A; Soni, R; Murray, J A; Lindsey, K

1996-01-01

354

Human colon cells: culture and in vitro transformation.  

PubMed

Normal human colon mucosal epithelial cells were cultured in vitro and treated with the oncogenic simian DNA virus (SV40) and the chemical carcinogen azoxymethane. Both SV40 and azoxymethane altered a number of phenotypic characteristics of the normal human colon cells, including their morphology, culture longevity, growth in soft agar, substrate adherence, and peanut agglutinin binding. The SV40 transformants synthesized intranuclear T antigen. These data indicate that normal human colon mucosal cells were transformed toward the malignant phenotype. PMID:6328655

Moyer, M P; Aust, J B

1984-06-29

355

Calcification of osteoblastlike rat osteosarcoma cells in agarose suspension cultures  

Microsoft Academic Search

Summary  Ros 17\\/2 clonal rat osteosarcoma cells calcify when cultured in the presence of 10 ?g\\/ml ?-glycerol phosphate in an agarose\\u000a gel. Culture in 1% agarose inhibited cell division while allowing cells to remain metabolically active and viable for over\\u000a 21 days. Serial photography of the same microscopic field shows a progressive deposition of calcium phosphate during the course\\u000a of the

Satoru K. Nishimoto; William F. Stryker; Marcel E. Nimni

1987-01-01

356

Metabolic events in synchronised cell cultures of Acer pseudoplatanus L  

Microsoft Academic Search

Cell division was synchronised in 4-litre batch cultures of Acer pseudoplatanus L. by starvation and regrowth. Up to five consecutive cell cycles were observed in each culture. Mitosis and cytokinesis were synchronised within 0.2 cell cycles. Accumulation of extractable DNA was discontinuous and separate from cytokinesis. Correction for the degree of synchrony in the population gave: G1=13–37 h, S=15 h,

P. J. King; B. J. Cox; M. W. Fowler

1974-01-01

357

Multizone Paper Platform for 3D Cell Cultures  

PubMed Central

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

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

2011-01-01

358

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

SciTech Connect

Normal differentiation and malignant transformation of human cells are characterized by specific changes in surface antigen phenotype. In the present study, the authors have defined six cell-surface antigens of human sarcomas and normal mesenchymal cells, by using mixed hemadsorption assays and immunochemical methods for the analysis of cultured cells and immunohistochemical staining for the analysis of normal tissues and > 200 tumor specimens. Differential patterns of F19, F24, G171, G253, S5, and Thy-1 antigen expression were found to characterize (i) subsets of cultured sarcoma cell lines, (ii) cultured fibroblasts derived from various organs, (iii) normal resting and activated mesenchymal tissues, and (iv) sarcoma and nonmesenchymal tumor tissues. These results provide a basic surface antigenic map for cultured mesenchymal cells and mesenchymal tissues and permit the classification of human sarcomas according to their antigenic phenotypes.

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

1988-05-01

359

Chemotherapy in heterogeneous cultures of cancer cells with interconversion  

NASA Astrophysics Data System (ADS)

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

Dilão, Rui

2015-02-01

360

Chemotherapy in heterogeneous cultures of cancer cells with interconversion.  

PubMed

Recently, the interconversion between differentiated and stem-like cancer cells has been observed. Here, we model the in vitro growth of heterogeneous cell cultures in the presence of interconversion from differentiated cancer cells to cancer stem cells (CSCs), showing that, by targeting only CSC with cytotoxic agents, it is not always possible to eradicate cancer. We have determined the kinetic conditions under which cytotoxic agents in in vitro heterogeneous cultures of cancer cells eradicate cancer. In particular, we have shown that the chemotherapeutic elimination of in vitro cultures of heterogeneous cancer cells is effective only if it targets all cancer cell types, and if the induced death rates for the different subpopulations of cancer cell types are large enough. The quantitative results of the model are compared and validated with experimental data. PMID:25429401

Dilão, Rui

2014-01-01

361

Cultural Policy in Poland. Studies and Documents on Cultural Policies.  

ERIC Educational Resources Information Center

A survey of cultural policy in Poland, prepared for UNESCO, is one of a series showing how cultural policies are planned and implemented in member states. The dual traditions of the ready assimilation of European elements into Polish culture and Poland's determination to maintain a national identity throughout 123 years of partition are presented…

Balicki, Stanislaw Witold; And Others

362

Cultural Policy in Israel. Studies and Documents on Cultural Policies.  

ERIC Educational Resources Information Center

A survey of cultural policy in Israel, prepared for UNESCO, is one of a series of booklets to show how cultural policies are planned and implemented in various countries. The series provides a guide to these countries which have yet to establish cultural policies to help them profit from past experiences. The historical background of Israel's…

Michman, Jozeph

363

PVP-coated, negatively charged silver nanoparticles: A multi-center study of their physicochemical characteristics, cell culture and in vivo experiments.  

PubMed

PVP-capped silver nanoparticles with a diameter of the metallic core of 70 nm, a hydrodynamic diameter of 120 nm and a zeta potential of -20 mV were prepared and investigated with regard to their biological activity. This review summarizes the physicochemical properties (dissolution, protein adsorption, dispersability) of these nanoparticles and the cellular consequences of the exposure of a broad range of biological test systems to this defined type of silver nanoparticles. Silver nanoparticles dissolve in water in the presence of oxygen. In addition, in biological media (i.e., in the presence of proteins) the surface of silver nanoparticles is rapidly coated by a protein corona that influences their physicochemical and biological properties including cellular uptake. Silver nanoparticles are taken up by cell-type specific endocytosis pathways as demonstrated for hMSC, primary T-cells, primary monocytes, and astrocytes. A visualization of particles inside cells is possible by X-ray microscopy, fluorescence microscopy, and combined FIB/SEM analysis. By staining organelles, their localization inside the cell can be additionally determined. While primary brain astrocytes are shown to be fairly tolerant toward silver nanoparticles, silver nanoparticles induce the formation of DNA double-strand-breaks (DSB) and lead to chromosomal aberrations and sister-chromatid exchanges in Chinese hamster fibroblast cell lines (CHO9, K1, V79B). An exposure of rats to silver nanoparticles in vivo induced a moderate pulmonary toxicity, however, only at rather high concentrations. The same was found in precision-cut lung slices of rats in which silver nanoparticles remained mainly at the tissue surface. In a human 3D triple-cell culture model consisting of three cell types (alveolar epithelial cells, macrophages, and dendritic cells), adverse effects were also only found at high silver concentrations. The silver ions that are released from silver nanoparticles may be harmful to skin with disrupted barrier (e.g., wounds) and induce oxidative stress in skin cells (HaCaT). In conclusion, the data obtained on the effects of this well-defined type of silver nanoparticles on various biological systems clearly demonstrate that cell-type specific properties as well as experimental conditions determine the biocompatibility of and the cellular responses to an exposure with silver nanoparticles. PMID:25383306

Ahlberg, Sebastian; Antonopulos, Alexandra; Diendorf, Jörg; Dringen, Ralf; Epple, Matthias; Flöck, Rebekka; Goedecke, Wolfgang; Graf, Christina; Haberl, Nadine; Helmlinger, Jens; Herzog, Fabian; Heuer, Frederike; Hirn, Stephanie; Johannes, Christian; Kittler, Stefanie; Köller, Manfred; Korn, Katrin; Kreyling, Wolfgang G; Krombach, Fritz; Lademann, Jürgen; Loza, Kateryna; Luther, Eva M; Malissek, Marcelina; Meinke, Martina C; Nordmeyer, Daniel; Pailliart, Anne; Raabe, Jörg; Rancan, Fiorenza; Rothen-Rutishauser, Barbara; Rühl, Eckart; Schleh, Carsten; Seibel, Andreas; Sengstock, Christina; Treuel, Lennart; Vogt, Annika; Weber, Katrin; Zellner, Reinhard

2014-01-01

364

Propagation of human germ stem cells in long-term culture  

PubMed Central

Background: Spermatogonial stem cells (SSCs), a subset of undifferentiated type A spermatogonia, are the foundation of complex process of spermatogenesis and could be propagated in vitro culture conditions for long time for germ cell transplantation and fertility preservation. Objective: The aim of this study was in vitro propagation of human spermatogonial stem cells (SSCs) and improvement of presence of human Germ Stem Cells (hGSCs) were assessed by specific markers POU domain, class 5, transcription factor 1 (POU5F1), also known as Octamer-binding transcription factor 4 (Oct-4) and PLZF (Promyelocytic leukaemia zinc finger protein). Materials and Methods: Human testicular cells were isolated by enzymatic digestion (Collagenase IV and Trypsin). Germ cells were cultured in Stem-Pro 34 media supplemented by growth factors such as glial cell line-derived neurotrophic factor, basic fibroblast growth factor, epidermal growth factor and leukemia inhibitory factor to support self-renewal divisions. Germline stem cell clusters were passaged and expanded every week. Immunofluorecent study was accomplished by Anti-Oct4 antibody through the culture. The spermatogonial stem cells genes expression, PLZF, was studied in testis tissue and germ stem cells entire the culture. Results: hGSCs clusters from a brain dead patient developed in testicular cell culture and then cultured and propagated up to 6 weeks. During the culture Oct4 were a specific marker for identification of hGSCs in testis tissue. Expression of PLZF was applied on RNA level in germ stem cells. Conclusion: hGSCs indicated by SSCs specific marker can be cultured and propagated for long-term in vitro conditions. This article extracted from Ph.D. Thesis. (Zeinab Piravar) PMID:24639790

Akhondi, Mohammad Mehdi; Mohazzab, Arash; Jeddi-Tehrani, Mahmood; Sadeghi, Mohammad Reza; Eidi, Akram; Khodadadi, Abbas; Piravar, Zeinab

2013-01-01

365

Development of a new microfluidic platform integrating co-cultures of intestinal and liver cell lines.  

PubMed

We developed a new biological model to mimic the organ-organ interactions between the intestine and the liver. We coupled polycarbonate cell culture inserts and microfluidic biochips in an integrated fluidic platform allowing dynamic co-cultures (called IIDMP for Integrated Insert in a Dynamic Microfluidic Platform). The intestinal compartment was simulated using Caco-2 TC7 cells and the liver one by HepG2 C3A. We showed that Caco-2 TC7 viability, barrier integrity and functionality (assessed by paracellular and active transport), were not altered during co-cultures in the bioreactor in comparison with the conventional insert Petri cultures. In parallel, the viability and metabolism of the HepG2 C3A cells were maintained in the microfluidic biochips. Then, as proof of concept, we used the bioreactor to follow the transport of phenacetin through the intestinal barrier and its metabolism into paracetamol by the CYP1A of the HepG2 C3A cells. Our results demonstrated the performance of this bioreactor with cell co-cultures compared to static co-culture controls in which weak biotransformation into paracetamol was detected. Our study illustrated the interest of such a bioreactor combining the advantages of a cell culture barrier and of liver microfluidic cultures in a common framework for in vitro studies. PMID:24662032

Bricks, Thibault; Paullier, Patrick; Legendre, Audrey; Fleury, Marie-José; Zeller, Perrine; Merlier, Franck; Anton, Pauline M; Leclerc, Eric

2014-08-01

366

WHAT IS PLANT TISSUE CULTURE? Plant tissue culture involves the growth of plant cells, tissues or segments for purposes such as  

E-print Network

1 WHAT IS PLANT TISSUE CULTURE? Plant tissue culture involves the growth of plant cells, tissues or segments for purposes such as generating or cloning large amounts of new cells, tissues or plants; to study development; to provide mechanisms of genetic engineering; or to produce valuable chemicals found in plant

Durako, Michael J.

367

Cryopreservation of Specific Pathogen-Free (SPF) Pig Islet Cells: Effect of Culture Time before Cryopreservation and after Thawing  

Microsoft Academic Search

The aim of this study was to determine the optimal conditions (effect of culture time before and after cryopreservation) for cryopreservation of specific pathogen-free pig islet cells.Methods.(1) Glucose-induced insulin secretion by fresh islet cells cultured for 10 days was compared to that by islet cells cryopreserved 7 days after isolation and cultured 3 days after thawing. (2) Islet cells were

Valérie Duvivier; Sylviane Darquy; Gérard Reach

1999-01-01

368

Creation of macropores in three-dimensional bacterial cellulose scaffold for potential cancer cell culture.  

PubMed

There is an increasing need for an effective in vitro model that can resemble the 3-D nature of tumor microenvironments. In this work, a 3-D bacterial cellulose (BC) scaffold with macropores was fabricated by a facile freeze drying method for potential culture of cancer cells. This in vitro study reported, for the first time, the role of macropores in the adjustment of cancer cell behavior when compared with previous results cultured in BC scaffolds without macropores. The scaffold was characterized by SEM and mercury intrusion porosimeter. A human breast cancer cell line (MDA-MB-231) cultured in the macroporous BC scaffold was examined via cell proliferation, histological and SEM analyses. The results demonstrated that the macroporous scaffold provided a good environment for cell viability, adhesion, proliferation, and infiltration. These findings suggested that the macroporous BC scaffold might have great potential for use in the in vitro culture of cancer cells. PMID:25263926

Xiong, Guangyao; Luo, Honglin; Zhu, Yong; Raman, Sudha; Wan, Yizao

2014-12-19

369

Culture and characterization of mammary cancer stem cells in mammospheres.  

PubMed

Mammospheres (MMs) are a model for culturing and maintaining mammary gland stem cells (SCs) or cancer stem cells (CSCs) ex situ. As MMs recapitulate the micro-niche of the mammary gland or a tumor, MMs are a model for studying the properties of SCs or CSCs, and for mapping, isolating, and characterizing the SC/CSC generated lineages. Cancer stem cells share with normal SCs the properties of self-renewal and the capacity to generate all cell types and organ structures of the mammary gland. Analysis of human tumor samples suggests that CSCs are heterogeneous in terms of proliferation and differentiation potential. Mammospheres from CSCs likewise display heterogeneity. This heterogeneity makes analysis of CSC generated MMs challenging. To identify the unique and diverse properties of MM derived CSCs, comparative analysis with MMs obtained from normal SCs is required. Here we present protocols for identifying and enriching cells with SC features from a cancer cell line using the LA7CSCs as a model. A comprehensive and comparative approach for identifying, isolating, and characterizing MMs from SCs and CSCs from human breast is also introduced. In addition, we describe detailed procedures for identifying, isolating, and characterizing mammary gland specific cell types, generated during MM formation. PMID:25388398

Piscitelli, Eleonora; Cocola, Cinzia; Thaden, Frank Rüdiger; Pelucchi, Paride; Gray, Brian; Bertalot, Giovanni; Albertini, Alberto; Reinbold, Rolland; Zucchi, Ileana

2015-01-01

370

Isolation of quiescent and nonquiescent cells from yeast stationary-phase cultures  

PubMed Central

Quiescence is the most common and, arguably, most poorly understood cell cycle state. This is in part because pure populations of quiescent cells are typically difficult to isolate. We report the isolation and characterization of quiescent and nonquiescent cells from stationary-phase (SP) yeast cultures by density-gradient centrifugation. Quiescent cells are dense, unbudded daughter cells formed after glucose exhaustion. They synchronously reenter the mitotic cell cycle, suggesting that they are in a G0 state. Nonquiescent cells are less dense, heterogeneous, and composed of replicatively older, asynchronous cells that rapidly lose the ability to reproduce. Microscopic and flow cytometric analysis revealed that nonquiescent cells accumulate more reactive oxygen species than quiescent cells, and over 21 d, about half exhibit signs of apoptosis and necrosis. The ability to isolate both quiescent and nonquiescent yeast cells from SP cultures provides a novel, tractable experimental system for studies of quiescence, chronological and replicative aging, apoptosis, and the cell cycle. PMID:16818721

Allen, Chris; Büttner, Sabrina; Aragon, Anthony D.; Thomas, Jason A.; Meirelles, Osorio; Jaetao, Jason E.; Benn, Don; Ruby, Stephanie W.; Veenhuis, Marten; Madeo, Frank; Werner-Washburne, Margaret

2006-01-01

371

Development of an Integrated Microfluidic Perfusion Cell Culture System for Real-Time Microscopic Observation of Biological Cells  

PubMed Central

This study reports an integrated microfluidic perfusion cell culture system consisting of a microfluidic cell culture chip, and an indium tin oxide (ITO) glass-based microheater chip for micro-scale perfusion cell culture, and its real-time microscopic observation. The system features in maintaining both uniform, and stable chemical or thermal environments, and providing a backflow-free medium pumping, and a precise thermal control functions. In this work, the performance of the medium pumping scheme, and the ITO glass microheater were experimentally evaluated. Results show that the medium delivery mechanism was able to provide pumping rates ranging from 15.4 to 120.0 ?L·min?1. In addition, numerical simulation and experimental evaluation were conducted to verify that the ITO glass microheater was capable of providing a spatially uniform thermal environment, and precise temperature control with a mild variation of ±0.3 °C. Furthermore, a perfusion cell culture was successfully demonstrated, showing the cultured cells were kept at high cell viability of 95 ± 2%. In the process, the cultured chondrocytes can be clearly visualized microscopically. As a whole, the proposed cell culture system has paved an alternative route to carry out real-time microscopic observation of biological cells in a simple, user-friendly, and low cost manner. PMID:22164082

Lin, Lung; Wang, Shih-Siou; Wu, Min-Hsien; Oh-Yang, Chih-Chin

2011-01-01

372

21 CFR 864.2240 - Cell and tissue culture supplies and equipment.  

Code of Federal Regulations, 2010 CFR

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2010-04-01

373

40 CFR 798.5300 - Detection of gene mutations in somatic cells in culture.  

Code of Federal Regulations, 2012 CFR

...false Detection of gene mutations in somatic cells in culture. 798.5300 Section 798.5300...5300 Detection of gene mutations in somatic cells in culture. (a) Purpose. Mammalian cell culture systems may be used to detect...

2012-07-01

374

40 CFR 798.5300 - Detection of gene mutations in somatic cells in culture.  

Code of Federal Regulations, 2010 CFR

...true Detection of gene mutations in somatic cells in culture. 798.5300 Section 798.5300...5300 Detection of gene mutations in somatic cells in culture. (a) Purpose. Mammalian cell culture systems may be used to detect...

2010-07-01

375

21 CFR 864.2240 - Cell and tissue culture supplies and equipment.  

Code of Federal Regulations, 2011 CFR

... 2011-04-01 2011-04-01 false Cell and tissue culture supplies and equipment...DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2240 Cell and tissue culture supplies and...

2011-04-01

376

21 CFR 864.2220 - Synthetic cell and tissue culture media and components.  

Code of Federal Regulations, 2011 CFR

...2011-04-01 2011-04-01 false Synthetic cell and tissue culture media and components...DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2220 Synthetic cell and tissue culture media and...

2011-04-01

377

21 CFR 864.2220 - Synthetic cell and tissue culture media and components.  

Code of Federal Regulations, 2014 CFR

...2014-04-01 2014-04-01 false Synthetic cell and tissue culture media and components...DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2220 Synthetic cell and tissue culture media and...

2014-04-01

378

21 CFR 864.2220 - Synthetic cell and tissue culture media and components.  

Code of Federal Regulations, 2013 CFR

...2013-04-01 2013-04-01 false Synthetic cell and tissue culture media and components...DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2220 Synthetic cell and tissue culture media and...

2013-04-01

379

21 CFR 864.2240 - Cell and tissue culture supplies and equipment.  

Code of Federal Regulations, 2014 CFR

... 2014-04-01 2014-04-01 false Cell and tissue culture supplies and equipment...DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2240 Cell and tissue culture supplies and...

2014-04-01

380

40 CFR 798.5300 - Detection of gene mutations in somatic cells in culture.  

Code of Federal Regulations, 2014 CFR

...false Detection of gene mutations in somatic cells in culture. 798.5300 Section 798.5300...5300 Detection of gene mutations in somatic cells in culture. (a) Purpose. Mammalian cell culture systems may be used to detect...

2014-07-01

381

21 CFR 864.2220 - Synthetic cell and tissue culture media and components.  

Code of Federal Regulations, 2012 CFR

...2012-04-01 2012-04-01 false Synthetic cell and tissue culture media and components...DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2220 Synthetic cell and tissue culture media and...

2012-04-01

382

40 CFR 798.5300 - Detection of gene mutations in somatic cells in culture.  

Code of Federal Regulations, 2013 CFR

...false Detection of gene mutations in somatic cells in culture. 798.5300 Section 798.5300...5300 Detection of gene mutations in somatic cells in culture. (a) Purpose. Mammalian cell culture systems may be used to detect...

2013-07-01

383

21 CFR 864.2220 - Synthetic cell and tissue culture media and components.  

Code of Federal Regulations, 2010 CFR

...2010-04-01 2010-04-01 false Synthetic cell and tissue culture media and components...DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2220 Synthetic cell and tissue culture media and...

2010-04-01

384

40 CFR 798.5300 - Detection of gene mutations in somatic cells in culture.  

Code of Federal Regulations, 2011 CFR

...false Detection of gene mutations in somatic cells in culture. 798.5300 Section 798.5300...5300 Detection of gene mutations in somatic cells in culture. (a) Purpose. Mammalian cell culture systems may be used to detect...

2011-07-01

385

21 CFR 864.2240 - Cell and tissue culture supplies and equipment.  

Code of Federal Regulations, 2012 CFR

... 2012-04-01 2012-04-01 false Cell and tissue culture supplies and equipment...DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2240 Cell and tissue culture supplies and...

2012-04-01

386

21 CFR 864.2240 - Cell and tissue culture supplies and equipment.  

Code of Federal Regulations, 2013 CFR

... 2013-04-01 2013-04-01 false Cell and tissue culture supplies and equipment...DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2240 Cell and tissue culture supplies and...

2013-04-01

387

TRANSFORMATION OF SUGAR BEET CELL SUSPENSION CULTURES  

Technology Transfer Automated Retrieval System (TEKTRAN)

A sugar beet transformation method was developed using particle bombardment of short-term suspension cultures of a breeding line FC607. Highly embryogenic suspension cultures derived from leaf callus were bombarded with the uidA (GUS) reporter gene under the control of either the osmotin or protein...

388

Alloxan cytotoxicity is highly potentiated by plasma membrane- and lysosomal-associated iron — a study on a model system of cultured J-774 cells  

Microsoft Academic Search

Summary  Pancreatic islet beta cells, and some other cell types, are sensitive to the damaging effects of alloxan. The mechanisms behind the cytotoxicity have not been fully elucidated, although they are considered to be mediated by the formation and effects of reactive oxygen metabolites. In the present study, the cytotoxic effects of alloxan\\/cysteine at high and low concentrations were investigated on

H. Zhang; U. T. Brunk

1993-01-01

389

Human long-term culture initiating cell assay.  

PubMed

The long-term culture initiating cell (LTC-IC) assay, founded on the bone marrow long-term culture (LTC) system, measures primitive hematopoietic stem cells (termed LTC-IC) based on their capacity to produce myeloid progeny for at least 5 weeks. Adaptations of the LTC system including the use of stromal cell lines, application of limiting dilution analysis, and estimation of average hematopoietic progenitor output per LTC-IC under defined conditions have made it possible to accurately determine LTC-IC content in minimally separated and highly purified cell populations from human hematopoietic tissue sources such as bone marrow, peripheral blood, cord blood, fetal liver as well as cord blood and mobilized peripheral blood. Methodologies for measuring human LTC-IC using bulk cultures, limiting dilution analysis, and single cell cultures are described. PMID:23179836

Liu, Min; Miller, Cindy L; Eaves, Connie J

2013-01-01

390

Effect of triazole pesticide formulation on bovine culture cells.  

PubMed

To date, most data about the possible genotoxic effect of triazole pesticides are focused on laboratory animals resulting in limited information on further non-target organisms such as cattle. The objective of the present study was to investigate the effect of triazole (tebuconazole/prothioconazole) fungicide formulation on the induction of chromosomal aberrations (CAs), sister chromatid exchanges (SCEs) and DNA fragmentation in bovine cultured lymphocytes. Our results showed that the fungicide formulation did not induce significant number of CAs in bovine cells after 24 h treatment. Nevertheless, the dose-dependent reduction of mitotic division was observed, with the strongest effect at 30.0 ?g mL(-1) in both donors (P < 0.01 and P < 0.001, respectively). Prolonged 48 h exposure caused the increased level of breaks in treated cultures (3.0-15.0 ?g mL(-1); P < 0.05) and significant decrease in mitotic index (MI). The tested fungicide failed to produce any statistical changes in the SCE frequency neither after 24 h nor 48 h treatment. However, the significant decline of the proliferation index (PI) was observed after 24 h indicating the fungicide influence on cell cycle kinetics. Prolonged 48 h exposure caused cytotoxicity reflecting in lower PI value relative to control mainly at the highest fungicide concentrations (30.0 ?g mL(-1), P < 0.001). Using painting probes for bovine chromosomes 1, 5 and 7 (BTA1, BTA5 and BTA7) only low levels of aneuploidies were detected. Significant increase of polyploidy cells (P < 0.05) was induced by a 3.0 ?g mL(-1) dose of the fungicide after 48 h. DNA fragmentation assay didn't reveal the presence of DNA nucleosome ladder in cell cultures at any time (24 h and 48 h) and fungicide concentration. PMID:24007485

Hole?ková, Beáta; Šiviková, Katarína; Dianovský, Ján; Galdíková, Martina

2013-01-01

391

Production of a recombinant industrial protein using barley cell cultures.  

PubMed

The use of recombinant DNA-based protein production using genetically modified plants could provide a reproducible, consistent quality, safe, animal-component free, origin-traceable, and cost-effective source for industrial proteins required in large amounts (1000s of metric tons) and at low cost (below US$100/Kg). The aim of this work was to demonstrate the feasibility of using barley suspension cell culture to support timely testing of the genetic constructs and early product characterization to detect for example post-translational modifications within the industrial protein caused by the selected recombinant system. For this study the human Collagen I alpha 1 (CIa1) chain gene encoding the complete helical region of CIa1 optimized for monocot expression was fused to its N- and C-terminal telopeptide and to a bacteriophage T4 fibritin foldon peptide encoding sequences. The CIa1 accumulation was targeted to the endoplasmic reticulum (ER) by fusing the CIa1 gene to an ER-directing signal peptide sequence and an ER retention signal HDEL. The construct containing the CIa1 gene was then introduced into immature barley half embryos or barley cells by particle bombardment. Transgenic barley cells resulting from these transformations were grown as suspension cultures in flasks and in a Wave bioreactor producing CIa1 similar to CIa1 purified from the yeast Pichia pastoris based on Western blotting, pepsin resistance, and mass spectroscopy analysis. The barley cell culture derived-CIa1 intracellular accumulation levels ranged from 2 to 9 microg/l illustrating the need for further process improvement in order to use this technology to supply material for product development activities. PMID:18406168

Ritala, A; Wahlström, E H; Holkeri, H; Hafren, A; Mäkeläinen, K; Baez, J; Mäkinen, K; Nuutila, A M

2008-06-01

392

Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing  

PubMed Central

The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses’ vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, analyzed by high resolution mass spectrometry coupled to liquid chromatography (LC MS/MS), and the most interesting candidates were validated by PCR, immunoblotting and immunocytochemistry. A total of 112 proteins were identified, of which 84% were cell surface membrane proteins. Twenty-three of these proteins were concurrently expressed by both cell states, 28 proteins exclusively by native RPE cells. Among the latter were two RPE markers with highly specialized RPE functions: cellular retinaldehyde-binding protein (CRALBP) and retinal pigment epithelium-specific protein 65kDa (RPE65). Furthermore, 61 proteins were only expressed by cultured RPE cells and absent in native cells. As we believe that initiating events, leading to the breakdown of the outer blood-retinal barrier, take place at the cell surface of RPE cells as a particularly exposed barrier structure, this differential characterization of cell surface proteomes of native and cultured equine RPE cells is a prerequisite for future studies. PMID:23203049

Szober, Christoph M.; Hauck, Stefanie M.; Euler, Kerstin N.; Fröhlich, Kristina J. H.; Alge-Priglinger, Claudia; Ueffing, Marius; Deeg, Cornelia A.

2012-01-01

393

Study of heat and radiation response of a malignant, melanin-producing cell line derived from C3H 10T1/2 cells transformed in culture by radiation  

SciTech Connect

The mouse C3H 10T1/2 cell line was transformed to the malignant state using ionizing radiation. One of the transformed lines (R25) that was isolated, displayed some properties similar to malignant melanoma cells. The cells became dark and pigmented after prolonged time in culture and this cell line produced tumors in C3H mice. The radiation survival curve of R25 had a large shoulder which was also observed for human melanoma cell lines. R25 was more resistant to heating at 45.0 degrees C than the normal cell line. Heating at 45.0 degrees C before irradiation resulted in a reduction of the survival curve shoulder. The heat and radiation sensitivity of R25 did not appear to be related to the melanin content of these cells.

Raaphorst, G.P.; Vadasz, J.; Azzam, E.I.

1986-12-01

394

Urokinase production by electrophoretically separated cultured human embryonic kidney cells  

NASA Technical Reports Server (NTRS)

Urokinase is a plasminogen activator found in urine. Relatively pure preparations have been tested in Europe, Japan and the United States for the treatment of deep vein thrombosis and other dangerous blood clots. Human embryonic kidney cell cultures have been found to produce urokinase at much higher concentrations, but less than 5% of the cells in typical cultures are producers. Since human diploid cells become senescent in culture the selection of clones derived from single cells will not provide enough material to be useful, so a bulk purification method is needed for the isolation of urokinase producing cell populations. Preparative cell electrophoresis was chosen as the method, since evidence exists that human embryonic cell cultures are richly heterogeneous with respect to electrophoretic mobility, and preliminary electrophoretic separations on the Apollo-Soyuz space flight produced cell populations that were rich in urokinase production. Similarly, erythropoietin is useful in the treatment of certain anemias and is a kidney cell duct, and electrophoretically enriched cell populations producing this product have been reported. Thus, there is a clear need for diploid human cells that produce these products, and there is evidence that such cells should be separable by free-flow cell electrophoresis.

Kunze, M. E.; Plank, L. D.; Giranda, V.; Sedor, K.; Todd, P. W.

1985-01-01

395

Defining process design space for monoclonal antibody cell culture.  

PubMed

The concept of design space has been taking root as a foundation of in-process control strategies for biopharmaceutical manufacturing processes. During mapping of the process design space, the multidimensional combination of operational variables is studied to quantify the impact on process performance in terms of productivity and product quality. An efficient methodology to map the design space for a monoclonal antibody cell culture process is described. A failure modes and effects analysis (FMEA) was used as the basis for the process characterization exercise. This was followed by an integrated study of the inoculum stage of the process which includes progressive shake flask and seed bioreactor steps. The operating conditions for the seed bioreactor were studied in an integrated fashion with the production bioreactor using a two stage design of experiments (DOE) methodology to enable optimization of operating conditions. A two level Resolution IV design was followed by a central composite design (CCD). These experiments enabled identification of the edge of failure and classification of the operational parameters as non-key, key or critical. In addition, the models generated from the data provide further insight into balancing productivity of the cell culture process with product quality considerations. Finally, process and product-related impurity clearance was evaluated by studies linking the upstream process with downstream purification. Production bioreactor parameters that directly influence antibody charge variants and glycosylation in CHO systems were identified. PMID:20589669

Abu-Absi, Susan Fugett; Yang, LiYing; Thompson, Patrick; Jiang, Canping; Kandula, Sunitha; Schilling, Bernhard; Shukla, Abhinav A

2010-08-15

396

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 with 0.1% gelatin at room temperature for two hours. (150 µl/well of 96 well plate; 12 ml/10 cm; 4 ml/6cm. Plate cells in gelatinized plates (150 µl/well of 96 well plate; 12 ml/10 cm; 4 ml/6cm; 2 ml/well of 6

Oliver, Douglas L.

397

Odontoblast differentiation of human dental pulp cells in explant cultures.  

PubMed

In order to elucidate the mechanisms involved in human dentin formation, we developed a cell culture system to promote differentiation of dental pulp cells into odontoblasts. Explants from human teeth were cultured in Eagle's basal medium supplemented with 10% or 15% fetal calf serum, with or without beta-glycerophosphate (beta GP). Addition of beta GP to the culture medium induced odontoblast features in the cultured pulp cells. Cells polarized and some of them exhibited a typical cellular extension. In some cases, cells aligned with their processes oriented in the same direction and developed junctional complexes similar to the terminal web linking odontoblasts in vivo. Fine structural analyses showed the presence of typical intracellular organelles of the odontoblast body, whereas the process contained only cytoskeleton elements and secretory vesicles. Polarized cells deposited onto the plastic dishes an abundant and organized type I collagen-rich matrix with areas of mineralization appearing thereafter. X-ray microanalysis showed the presence of calcium and phosphorus and the electron diffraction pattern confirmed the apatitic crystal structure of the mineral. High expression of alpha 1 (1) collagen mRNAs was detected in all polarized cells whereas dentin sialoprotein gene was mainly expressed in mineralizing areas. This cell culture system allowed for the differentiation of pulp cells into odontoblasts, at both the morphological and functional level. Moreover, these cells presented a spatial organization similar to the odontoblastic layer. PMID:10652961

Couble, M L; Farges, J C; Bleicher, F; Perrat-Mabillon, B; Boudeulle, M; Magloire, H

2000-02-01

398

Chromatin reorganization and endogenous auxin\\/cytokinin dynamic activity during somatic embryogenesis of cultured cotton cell  

Microsoft Academic Search

We conducted a systematic assessment and comparative study on the biochemical and cellular characteristics of cultured cotton\\u000a cells during the entire process of somatic embryogenesis (SE). All staged cultures were widely investigated in this assay.\\u000a Cell and tissue ectogenesis manipulation combined with flow cytometry (FCM) was employed to cellular study during the whole\\u000a totipotency process of dedifferentiation and redifferentiation. We

Fanchang Zeng; Xianlong Zhang; Shuangxia Jin; Lei Cheng; Shaoguang Liang; Lisong Hu; Xiaoping Guo; Yichun Nie; Jinglin Cao

2007-01-01

399

Amniotic fluid promotes the appearance of neural retinal progenitors and neurons in human RPE cell cultures  

PubMed Central

Purpose Retinal pigment epithelial (RPE) cells are capable of differentiating into retinal neurons when induced by the appropriate growth factors. Amniotic fluid contains a variety of growth factors that are crucial for the development of a fetus. In this study, the effects of human amniotic fluid (HAF) on primary RPE cell cultures were evaluated. Methods RPE cells were isolated from the globes of postnatal human cadavers. The isolated cells were plated and grown in DMEM/F12 with 10% fetal bovine serum. To confirm the RPE identity of the cultured cells, they were immunocytochemically examined for the presence of the RPE cell-specific marker RPE65. RPE cultures obtained from passages 2–7 were treated with HAF and examined morphologically for 1 month. To determine whether retinal neurons or progenitors developed in the treated cultures, specific markers for bipolar (protein kinase C isomer ?, PKC?), amacrine (cellular retinoic acid–binding protein I, CRABPI), and neural progenitor (NESTIN) cells were sought, and the amount of mRNA was quantified using real-time PCR. Results Treating RPE cells with HAF led to a significant decrease in the number of RPE65-positive cells, while PKC?- and CRABPI-positive cells were detected in the cultures. Compared with the fetal bovine serum–treated cultures, the levels of mRNAs quantitatively increased by 2-, 20- and 22-fold for NESTIN, PKC?, and CRABPI, respectively. The RPE cultures treated with HAF established spheres containing both pigmented and nonpigmented cells, which expressed neural progenitor markers such as NESTIN. Conclusions This study showed that HAF can induce RPE cells to transdifferentiate into retinal neurons and progenitor cells, and that it provides a potential source for cell-based therapies to treat retinal diseases. PMID:24265548

Davari, Maliheh; Ahmadieh, Hamid; Sanie-Jahromi, Fateme; Ghaderi, Shima; Kanavi, Mozhgan Rezaei; Samiei, Shahram; Akrami, Hassan; Haghighi, Massoud; Javidi-Azad, Fahimeh

2013-01-01

400

The Influence of Micronutrients in Cell Culture: A Reflection on Viability and Genomic Stability  

PubMed Central

Micronutrients, including minerals and vitamins, are indispensable to DNA metabolic pathways and thus are as important for life as macronutrients. Without the proper nutrients, genomic instability compromises homeostasis, leading to chronic diseases and certain types of cancer. Cell-culture media try to mimic the in vivo environment, providing in vitro models used to infer cells' responses to different stimuli. This review summarizes and discusses studies of cell-culture supplementation with micronutrients that can increase cell viability and genomic stability, with a particular focus on previous in vitro experiments. In these studies, the cell-culture media include certain vitamins and minerals at concentrations not equal to the physiological levels. In many common culture media, the sole source of micronutrients is fetal bovine serum (FBS), which contributes to only 5–10% of the media composition. Minimal attention has been dedicated to FBS composition, micronutrients in cell cultures as a whole, or the influence of micronutrients on the viability and genetics of cultured cells. Further studies better evaluating micronutrients' roles at a molecular level and influence on the genomic stability of cells are still needed. PMID:23781504

Arigony, Ana Lúcia Vargas; de Oliveira, Iuri Marques; Bordin, Diana Lilian; Prá, Daniel; Pêgas Henriques, João Antonio

2013-01-01

401

Investigating the establishment of primary cell culture from different abalone (Haliotis midae) tissues  

PubMed Central

The abalone, Haliotis midae, is the most valuable commodity in South African aquaculture. The increasing demand for marine shellfish has stimulated research on the biology and physiology of target species in order to improve knowledge on growth, nutritional requirements and pathogen identification. The slow growth rate and long generation time of abalone restrict efficient design of in vivo experiments. Therefore, in vitro systems present an attractive alternative for short term experimentation. The use of marine invertebrate cell cultures as a standardised and controlled system to study growth, endocrinology and disease contributes to the understanding of the biology of economically important molluscs. This paper investigates the suitability of two different H. midae tissues, larval and haemocyte, for establishing primary cell cultures. Cell cultures are assessed in terms of culture initiation, cell yield, longevity and susceptibility to contamination. Haliotis midae haemocytes are shown to be a more feasible tissue for primary cell culture as it could be maintained without contamination more readily than larval cell cultures. The usefulness of short term primary haemocyte cultures is demonstrated here with a growth factor trial. Haemocyte cultures can furthermore be used to relate phenotypic changes at the cellular level to changes in gene expression at the molecular level. PMID:20680682

Auzoux-Bordenave, Stéphanie; Niesler, Carola; Roodt-Wilding, Rouvay

2010-01-01

402

Flow perfusion culture of marrow stromal cells seeded on porous biphasic calcium phosphate ceramics.  

PubMed

Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% beta-tricalcium phosphate) scaffolds, cultured for up to 16 days in static or flow perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow perfusion culture whereas they were located only along the scaffold perimeter in static culture. At all culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow perfusion bioreactor for bone tissue engineering applications. PMID:16133930

Holtorf, Heidi L; Sheffield, Tiffany L; Ambrose, Catherine G; Jansen, John A; Mikos, Antonios G

2005-09-01

403

Cytogenetic characterization and cell cycle analysis of three human colon adenocarcinoma cell lines: comparison between two- and three-dimensional cell culture systems.  

PubMed

The aim of the study was to investigate whether changes in the pattern of gene copy number and cell cycle were present passing from the two- to the three-dimensional cell culture system. We used three human colon adenocarcinoma cell lines grown two- and three-dimensionally. We analyzed morphology, karyotype, chromosomal gain and losses, and cell cycle. In three-dimensional cell cultures the growth is delayed and arrested in G1 phase without specific rearrangements in the three-dimensional cultures compared to the two-dimensional cultures. These data suggest that the differences between the two- and three-dimensional cell culture systems do not involve chromosomal rearrangements. PMID:19995228

Roncoroni, Leda; Elli, Luca; Bardella, Maria Teresa; Dogliotti, Elena; Grimoldi, Maria Grazia; Viaggi, Silvia; Erba, Eugenio; Zunino, Annalisa; Doneda, Luisa

2010-01-01

404

Reversed anion selectivity in cultured cystic fibrosis sweat duct cells.  

PubMed

The human genetic disease cystic fibrosis (CF) is characterized by defective epithelial Cl- conductance (GCl). To distinguish the CF-affected GCl from other Cl- channels, we have studied the properties of GCl in normal and CF cells grown from explanted reabsorptive sweat ducts (RD). The cultured cells from normal subjects retained some of the typical duct cell properties. The Na+ conductance inhibitor amiloride hyperpolarized intracellular potentials (Vm) by 10.4 +/- 1.6 mV (n = 12). Substitution of gluconate for Cl- depolarized Vm by 15.5 +/- 1.1 mV (n = 33). The apparent GCl (G'Cl) of normal cells was sensitive to adenosine 3',5'-cyclic monophosphate (forskolin, 10(-6) M), as evidenced by a significant increase (63%, n = 9) in the Cl- gradient induc