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1

Use of aggregating cell cultures for toxicological studies.  

PubMed

Relatively simple techniques are now available which allow the preparation of large quantities of highly reproducible aggregate cultures from fetal rat brain or liver cells, and to grow them in a chemically defined medium. Since these cultures exhibit extensive histotypic cellular reorganization and maturation, they offer unique possibilities for developmental studies. Therefore, the purpose of the present study was to investigate the usefulness of these cultures in developmental toxicology. Aggregating brain cell cultures were exposed at different developmental stages to model drugs (i.e., antimitotic, neurotoxic, and teratogenic agents) and assayed for their responsiveness by measuring a set of biochemical parameters (i.e., total protein and DNA content, cell type-specific enzyme activities) which permit a monitoring of cellular growth and maturation. It was found that each test compound elicited a distinct, dose-dependent response pattern, which may ultimately serve to screen and classify toxic drugs by using mechanistic criteria. In addition, it could be shown that aggregating liver cell cultures are capable of toxic drug activation, and that they can be used in co-culture with brain cell aggregates, providing a potential model for complementary toxicological and metabolic studies. PMID:3141206

Honegger, P; Werffeli, P

1988-10-15

2

Qualitative study of three cell culture methods.  

PubMed

Primary rat hepatocytes were cultured using different in vitro models and the enzyme leakage, albumin secretion, and cytochrome P450 1A (CYP 1A) activity were observed. The results showed that the level of LDH was decreased over time in culture. However, on day 5, LDH showed a significant increase in monolayer culture (MC) while after day 8 no LDH was detectable in sandwich culture (SC). The levels of AST and ALT did not change significantly over the investigated time. The CYP 1A activity was gradually decreased in a time-dependent manner in MC and SC. The decline of CYP 1A was faster in MC than in SC. This effect was partially reversed by using cytochrome P450 (CYP450) inducer such as Omeprazol and 3-methylcholanthrene (3-MC) and the CYP 1A induction was always higher in MC than in SC. In bioreactor basic CYP 1A activity was preserved over 2 weeks and the highest albumin production was observed in bioreactor followed by SC and MC. Taken together, it was indicated each investigated model had its advantages and disadvantages. It was also underlined that various in vitro models may address different questions. PMID:12674760

Wang, Aiguo; Xia, Tao; Ran, Peng; Chen, Xuemin; Nuessler, Andreas K

2002-01-01

3

Mammalian Cell Culture  

Microsoft Academic Search

Mammalian cell culture is used widely in academic, medical and industrial settings. It has provided a means to study the physiology and biochemistry of the cell and developments in the fields of cell and molecular biology have required the use of reproducible model systems that only cultured cell lines can provide. For medical use, cell culture provides test systems to

Simon P. Langdon

4

Characterizing parameters of Jatropha curcas cell cultures for microgravity studies  

NASA Astrophysics Data System (ADS)

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

Vendrame, Wagner A.; Pinares, Ania

2013-06-01

5

Sex stratified neuronal cultures to study ischemic cell death pathways.  

PubMed

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome. PMID:24378980

Fairbanks, Stacy L; Vest, Rebekah; Verma, Saurabh; Traystman, Richard J; Herson, Paco S

2013-01-01

6

A neural cell culture study on thin film electrode materials.  

PubMed

Functional neural stimulation requires good interface between the neural cells and the electrode surfaces. In order to study the effect of electrode materials and surface structure on cell adhesion and biocompatibility, we cultured cortical neurons on thin films of platinum and iridium oxide. We used both flat, as-deposited and laser micro-structured films. The laser micro-structuring consisted of creating regular arrays of micro-bumps or holes with diameters of 4-5 mum and height of about 1.5 mum. The micro-bumps were fabricated onto platinum and iridium film surfaces deposited on borosilicate glass substrates, using mask-projection irradiation with single nano-second pulses from a KrF excimer laser (lambda = 248 nm). Amorphous and crystalline (deposited at 250 degrees C) IrO(2) films were deposited onto the laser micro-structured iridium films by pulsed-DC reactive sputtering to obtain micro-structured IrO(2) films. Cortical neurons isolated from rat embryo brain were cultured onto these film surfaces. Our results indicate that flat and micro-structured film surfaces are biocompatible and non-toxic for neural cell growth. The use of poly-D: -lysine as a mediator for cell adhesion onto the thin film surfaces is also discussed. PMID:17483885

Thanawala, Sachin; Palyvoda, Olena; Georgiev, Daniel G; Khan, Saida P; Al-Homoudi, Ibrahim A; Newaz, Golam; Auner, Gregory

2007-09-01

7

Elicitation studies in cell suspension cultures of Cannabis sativa L  

Microsoft Academic Search

Cannabis sativa L. plants produce a diverse array of secondary metabolites. Cannabis cell cultures were treated with biotic and abiotic elicitors to evaluate their effect on secondary metabolism. Metabolic profiles analysed by 1H NMR spectroscopy and principal component analysis (PCA) showed variations in some of the metabolite pools. However, no cannabinoids were found in either control or elicited cannabis cell

Isvett Josefina Flores-Sanchez; Jaroslav Pe?; Junni Fei; Young Hae Choi; Jaroslav Dušek; Robert Verpoorte

2009-01-01

8

Studies on sphingomyelinase activity in cultured cells and leucocytes  

Microsoft Academic Search

Using both [3H]ceramide-sphingomyelin and [14C]methyl choline-sphingomyelin as substrate, enzyme activity was determined in skin fibroblasts, cultured amnotic fluid cells and leucocytes. Sphingomyelinase activities of fibroblasts from two patients with Niemann-Pick disease were about 1% of the mean control values (22.9 nmol\\/h\\/mg protein and 65.6 nmol\\/h\\/mg protein, with [3H]sphingomyelin and [14C]sphingomyelin respectively). Leucocyte sphingomyelinase activity is much lower than that of

G. T. N. Besley

1978-01-01

9

Study of neurotrophin 3 signaling in primary cultured neurons using multiplex stable isotope labeling with amino acids in cell culture  

PubMed Central

Conventional stable isotope labeling with amino acids in cell culture (SILAC) requires extensive metabolic labeling of proteins, and therefore is difficult to apply to cells that do not divide or are unstable in SILAC culture. Using two different sets of heavy amino acids for labeling allows for straightforward SILAC quantitation using partially labeled cells because the two cell populations are always equally labeled. Here we report the application of this labeling strategy to primary cultured neurons. We demonstrated that protein quantitation was not compromised by incomplete labeling of the neuronal proteins. We used this method to study neurotrophin-3 (NT-3) signaling in primary cultured neurons. Surprisingly our results indicate TrkB signaling is a major component of the signaling network induced by NT-3 in cortical neurons. In addition, involvement of proteins such as VAMP2, Scamp1 and Scamp3 suggest NT-3 may lead to enhanced exocytosis of synaptic vesicles. PMID:21370927

Zhang, Guoan; Deinhardt, Katrin; Chao, Moses V.; Neubert, Thomas A.

2011-01-01

10

Immunological and virological studies of cultured labial biopsy cells from patients with Sjögren's syndrome  

PubMed Central

Labial salivary gland tissues from twenty-five patients were cultured in vitro for virus studies and for use as target cells in cellular and antibody-mediated cytotoxicity assays. Fourteen patients had definite Sjögren's Syndrome (SS), four had possible SS and seven did not have SS. No evidence for the presence of a virus in the cultured cells or after chemical treatment of the cultured cells was obtained. Tubuloreticular structures were present in three of the original biopsies but were not seen in the corresponding cultured cells, although in two of these cell lines rare bundles of intranuclear microfibrils occurred. The significance of these structures is unknown. Autologous serum and autologous lymphocytes were not cytotoxic for the cultured cells. ImagesFig. 1Fig. 2 PMID:4468195

Cremer, Natalie E.; Daniels, T. E.; Oshiro, L. S.; Marcus, F.; Claypool, R.; Sylvester, R. A.; Talal, N.

1974-01-01

11

The use of cultured Drosophila cells for studying the microtubule cytoskeleton.  

PubMed

Cultured Drosophila cell lines have been developed into a powerful tool for studying a wide variety of cellular processes. Their ability to be easily and cheaply cultured as well as their susceptibility to protein knockdown via double-stranded RNA-mediated interference (RNAi) has made them the model system of choice for many researchers in the fields of cell biology and functional genomics. Here we describe basic techniques for gene knockdown, transgene expression, preparation for fluorescence microscopy, and centrosome enrichment using cultured Drosophila cells with an emphasis on studying the microtubule cytoskeleton. PMID:24633795

Nye, Jonathan; Buster, Daniel W; Rogers, Gregory C

2014-01-01

12

Micropatterned superhydrophobic structures for the simultaneous culture of multiple cell types and the study of cell-cell communication.  

PubMed

The ability to control spatial arrangement and geometry of different cell types while keeping them separated and in close proximity for a long time is crucial to mimic and study variety of biological processes in vitro. Although the existing cell patterning technologies allow co-culturing of different cell types, they are usually limited to relatively simple geometry. The methods used for obtaining complex geometries are usually applicable for patterning only one or two cell types. Here we introduce a convenient method for creating patterns of multiple (up to twenty) different cell types on one substrate. The method virtually allows any complexity of cell pattern geometry. Cell positioning on the substrate is realized by a parallel formation of multiple cell-containing microreservoirs confined to the geometry of highly hydrophilic regions surrounded by superhydrophobic borders built-in a fine nanoporous polymer film. As a case study we showed the cross-talk between two cell populations via Wnt signaling molecules propagation during co-culture in a mutual culture medium. PMID:23228425

Efremov, Alexander N; Stanganello, Eliana; Welle, Alexander; Scholpp, Steffen; Levkin, Pavel A

2013-02-01

13

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

14

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

E-print Network

transcription factors, including members of the Snail, Twist, and Zeb families. EMT results in diminishedPRECLINICAL STUDY Prolonged mammosphere culture of MCF-7 cells induces an EMT and repression transition (EMT) also induces stem cell features in normal and transformed mammary cells. We examined whether

Terasaki, Mark

15

Characterization of normal breast epithelial cells in primary cultures: Differentiation and growth factor receptors studies  

Microsoft Academic Search

Summary  The growth and differentiation of normal human mammary epithelial cells (HMEC) were studied after propagation of serial cultures\\u000a from breast tissue biopsies from 42 mammoplasty patients. Cells were grown for up to 7 mo. in low calcium medium. HMEC cultures\\u000a displayed heterogeneous growth patterns, according to the average doubling time of 44 ± 6 h for 32 generations. Proliferation\\u000a peaked

Philippe Berthon; Gianfranco Pancino; Patricia de Cremoux; Alberto Roseto; Christian Gespach; Fabien Calvo

1992-01-01

16

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

17

ASBESTOS AND GASTRO-INTESTINAL CANCER: CELL CULTURE STUDIES  

EPA Science Inventory

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

18

Tissue culture studies on human giant cell tumour and osteosarcoma  

Microsoft Academic Search

Benign giant cell tumours showed a sharp chromosome number mode at 46 with a superficially normal diploid karyotype. Osteosarcomas, however, were characterised by having aneuploid modal numbers. LDH isozyme patterns of giant cell tumours showed a maximum activity in Fractions IV and V, whereas that of osteosarcomas apparently fell in a range of Fraction III.

Seiichi Ishii; Shinya Yamawaki; Tetsuto Sasaki; Masamichi Usui; Yuji Ubayama; Akio Minami; Tomonori Yagi

1978-01-01

19

Stem cell culture engineering  

Microsoft Academic Search

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

Gargi Seth; Catherine M. Verfaillie

2005-01-01

20

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

PubMed Central

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

2013-01-01

21

Optimizing stem cell culture  

PubMed Central

Stem cells always balance between self-renewal and differentiation. Hence, stem cell culture parameters are critical and need to be continuously refined according to progress in our stem cell biology understanding and the latest technological developments. This led to the progressive replacement of ill-defined additives such as serum or feeder cell layers by recombinant cytokines or growth factors. Another example is the control of the oxygen pressure. For many years cell cultures have been done under atmospheric oxygen pressure which is much higher than the one experienced by stem cells in vivo. A consequence of cell metabolism is that cell culture conditions are constantly changing. Therefore, the development of high sensitive monitoring processes and control algorithms is required for ensuring cell culture medium homeostasis. Stem cells also sense the physical constraints of their microenvironment. Rigidity, stiffness and geometry of the culture substrate influence stem cell fate. Hence, nanotopography is probably as important as medium formulation in the optimization of stem cell culture conditions. Recent advances include the development of synthetic bioinformative substrates designed at the micro- and nanoscale level. On going research in many different fields including stem cell biology, nanotechnology, and bioengineering suggest that our current way to culture cells in Petri dish or flasks will soon be outdated as flying across the Atlantic Ocean in the Lindbergh’s plane. PMID:20803548

Van Der Sanden, Boudewijn; Dhobb, Mehdi; Berger, François; Wion, Didier

2010-01-01

22

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

PubMed Central

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

23

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

24

A study of hybridoma cell growth and antibody production kinetics in continuous culture  

Microsoft Academic Search

A detailed study of cell growth and antibody production kinetics in continuous culture found that the specific rate of antibody production reached a maximum saturated profile at a specific growth rate less than the maximum. This observation is novel and of importance in the understanding of the mechanism of antibody production and\\/or antibody transport.

K. S. Low; C. Harbour; J. P. Barford

1987-01-01

25

Histochemical study of brown-fat cells in the golden hamster (Mesocricetus auratus) in cultures  

SciTech Connect

The authors undertake the task of studying the synthesis of certain hormones by brown-fat cells. The authors used brown-fat cells from the golden hamster. The metabolism of brown-fat cells was studied on precultured cells, which made it possible to detect the synthesis of the studied substances rather than their accumulation in the organ. The authors conducted three experiments. First, fragments of brown fat were cultivated in diffusion chambers in vivo. Pieces of brown fat were cultivated in parallel in vitro on agar (organotypic cultures) and on plasma (histotypic cultures). During cultivation in diffusion chambers, the chambers were implanted in the abdominal cavity of young white rats. For in vitro cultivation, TCM 199 plus 15-20% calf serum was used. A total of 36 cultures with 12 cultures in each series of experiments were performed. The auto-radiographic studies of brown-fat cells were conducted on 24-hour cultures and on brown-fat fragments taken from the intact animal. The cultures were incubated with isotopes for 1 h. Either (/sup 3/H)lysine (87.3 Ci/mM specific activity), (/sup 3/H)arginine (16.7 Ci/mM), (/sup 3/H)glycerol (43 Ci/mM), or (/sup 3/H)cholesterol (43 Ci/mM) were added to the medium. After incubation, the cultures were washed three times in pure medium, fixed in Sierra fluid, and embedded in paraffin. The paraffin sections were covered with Ilford K/sub 2/ emulsion, and the preparations were exposed for 20 days at 4/sup 0/C temperature. Radio-immunological methods were used to study the accumulation of estradiol-17-beta in the culture medium by the Dobson method and that of testerone. The culture medium was taken on cultivation days 2,4,6,8, and 10. The medium was changed during cultivation every third day, which made it possible to judge the rates of accumulation of material with increase in the cultivation times.

Sokolov, V.E.; Boyadzhieva-Mikhailova, A.; Koncheva, L.; Angelova, P.; Evgen'eva, T.P.

1985-11-01

26

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

27

Growth, morphology and chemosensitivity studies on postconfluent cells cultured in 'V'-bottomed microtiter plates  

Microsoft Academic Search

This study assessed the growth pattern, cellular organisation and chemosensitivity of established human tumour cell lines growing as postconfluent cultures in 'V'-bottomed, 96-well microtiter plates. Cross-sections of the colon (HT29, SW620, SW1116), ovarian (A2780) and head and neck (UM-SCC-22B) carcinoma microcultures allowed in situ evaluation of the cellular organisation in the wells. After 5 days of growth, every cell line

PE Pizao; DM Lyaruu; GJ Peters; J van Ark-Otte; B Winograd; G Giaccone; HM Pinedo

1992-01-01

28

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

29

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

30

Mouse Pancreas Tissue Slice Culture Facilitates Long-Term Studies of Exocrine and Endocrine Cell Physiology in situ  

PubMed Central

Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ. PMID:24223842

Marciniak, Anja; Selck, Claudia; Friedrich, Betty; Speier, Stephan

2013-01-01

31

Blood, sweat and tears : a case study of the development of cultured red blood cells for transfusion   

E-print Network

This thesis is an in-depth case study of an interdisciplinary, paradigm breaking, research team who are seeking to develop cultured red blood cells (RBCs) for transfusion using stem cells (known as the BloodPharma project). ...

King, Emma Katharine

2013-11-27

32

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

33

Cytotoxicity analysis of alendronate on cultured endothelial cells and subcutaneous tissue. a pilot study.  

PubMed

The use of alendronate, a bisphosphonate which is able to inhibit bone resorption, in order to prevent dental root resorption after tooth replantation would be of clinical relevance. However, this drug must be biocompatible to the periapical tissues. The aim of this study was to analyze the effect of an alendronate paste in polyethyleneglycol (2 g ml(-1)) on endothelial cells in culture (in vitro) and on rat subcutaneous tissue (in vivo). For the in vitro study the paste was applied on round glass coverslips that were immersed into confluent cell cultures (clone Cips). The cell viability percentages of these cultures were obtained 0, 6 and 12 h after contact with the substance. As control, cultures that received plain coverslips were used. This analysis was carried out in triplicate using the Trypan blue dye exclusion assay. For the in vivo study the paste was introduced into polyethylene tubes that were placed into the rat subcutaneous tissue. The rats were killed 7 and 14 days later; then, the tissue sections stained with hematoxylin-eosin were analyzed. In vitro, the alendronate caused a significant decrease in the cell viability in 6 h (P < 0.05) and 12 h (P < 0.01), when compared with the control cultures. In vivo the tissue response was exuberant and similar at the two experimental times. There was a necrosis in a comprehensive area in contact with the open end of the tube. Presence of micro-abscesses and intense inflammatory infiltrate in the hypoderm permeating the muscle fibers and fat lobules were observed. In conclusion, the alendronate paste in polyethylene glycol as used showed to be highly cytotoxic in vitro as well as in vivo. PMID:16262618

Moreira, Maria Stella; Katayama, Emilio; Bombana, Antonio Carlos; Marques, Márcia Martins

2005-12-01

34

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

PubMed Central

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

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

2014-01-01

35

Slice Culture Method for Studying Migration of Neuronal Progenitor Cells Derived from Human Embryonic Stem Cells (hESC).  

PubMed

In this unit we describe an overlay brain slice culture assay for studying migration of transgenic neurospheres derived from human embryonic stem cells (hESC). Neuronal progenitor cells were generated from hESC by derivation of embryoid bodies and rosettes. Rosettes were transfected using the PiggyBac transposon system with either control plasmids (GFP) or plasmid encoding a gene important for migration of neuronal progenitor cells, Doublecortin (DCX). Transfected cells were subsequently grown in low-adhesion plates to generate transgenic human neurospheres (t-hNS). Organotypic slice cultures were prepared from postnatal rat forebrain and maintained using the interface method, before transfected t-hNS were overlaid below the cortex of each hemisphere. After 1 to 5 days, forebrain slices were fixed and processed for immunofluorescence. The distance at which cells migrated from the center of neurospheres to the host forebrain tissue was measured using Image J software. This protocol provides details for using the slice culture method for studying migration and integration of human neuronal cells into the host brain tissue. Curr. Protoc. Stem Cell Biol. 29:1H.7.1-1H.7.14. © 2014 by John Wiley & Sons, Inc. PMID:24838914

Filipovic, Radmila; Kumar, Saranya Santosh; Bahr, Ben A; Loturco, Joseph

2014-01-01

36

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.

37

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

38

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

SciTech Connect

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

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

1984-04-01

39

Continuous dilution culture system for studies on gene-enzyme regulation in synchronous cultures of plant cells  

Microsoft Academic Search

Summary  A thermophilic species ofChlorella was used as a model for higher eucaryotic cells in the development of a continuous dilution system for maintenance of nearly\\u000a constant environmental conditions during synchronous growth of cells cultured at high concentrations. An equilibrium (isopycinic)\\u000a centrifugation procedure for selection of large numbers of highly synchronous daughter cells in Ficoll density gradients was\\u000a also developed. By

Robert R. Schmidt

1974-01-01

40

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

SciTech Connect

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

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

2004-06-01

41

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

42

Cell Culturing of Cytoskeleton  

NASA Technical Reports Server (NTRS)

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

2004-01-01

43

Cell Culturing of Cytoskeleton  

NASA Technical Reports Server (NTRS)

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

2004-01-01

44

Studies of the Transferrin Receptor on both Human Reticulocytes and Nucleated Human Cells in Culture  

PubMed Central

The transferrin receptor, present on reticulocytes and nucleated cells in tissue culture, has been measured with both immunoassay techniques and transferrin binding studies. The total cellular immunoreactive receptor is rapidly lost from erythrocytes during the process of reticulocyte maturation (from as many as 400,000 molecules to <20,000 molecules/reticulocyte). This event parallels the loss of cell surface transferrin binding sites and RNA content, and correlates with previous studies that have measured the decline in hemoglobin synthesis. Nonhemoglobin-producing normal human fibroblasts, which appear to have a much lower iron requirement than reticulocytes, contain similar numbers of immunoreactive receptors per cell (400,000 receptor molecules), when in an active state of proliferation. Although receptor density on fibroblasts is directly related to cell proliferation, our studies demonstrate that nonproliferating fibroblasts still retain significant numbers of immunoreactive receptors (150,000 molecules/cell) and transferrin binding sites. Since additional studies indicate that proliferating cells have increased iron uptake, a simple hypothesis would predict that the parallel increase in transferrin binding sites and total cellular immunoreactive receptor associated with proliferation is related to an increased cellular iron requirement. However, the number of immunoreactive receptor molecules and transferrin binding sites is not changed when cells are grown in iron-deficient media, or in media with added transferrin-iron. This result and the lack of marked differences in receptor number on both hemoglobin-producing and nonhemoglobin-producing cells indicate that other factors besides receptor density play major roles in the regulation of cellular iron uptake, retention, and loss. Images PMID:6804495

Frazier, Janet L.; Caskey, Jennifer H.; Yoffe, Mark; Seligman, Paul A.

1982-01-01

45

Oscillating Cell Culture Bioreactor  

NASA Technical Reports Server (NTRS)

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

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

2010-01-01

46

A Novel Human Cell Culture Model for the Study of Familial Prostate Cancer  

Microsoft Academic Search

Research into molecular and genetic mechanisms underlying familial prostate cancer would be greatly advanced by in vitro models of prostate tumor cells representing primary tumors. We have successfully estab- lished an immortalized human prostate epithelial cell culture derived from primary tumors of familial prostate cancer patients with telomerase. The actively proliferating early-passaged 957E cells were transduced through infection with a

Yutaka Yasunaga; Keiichiro Nakamura; Charles M. Ewing; William B. Isaacs; Bharati Hukku; Johng S. Rhim

47

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

48

Isometric contraction by fibroblasts and endothelial cells in tissue culture: a quantitative study  

PubMed Central

We have used an isometric force transducer to study contraction of two types of nonmuscle cells in tissue culture. This method permits the quantitative measurement of contractile force generated by cells of defined type under the influence of external agents while allowing detailed morphological observation. Chick embryo fibroblasts (CEF), which form a contractile network inside a collagen matrix, and human umbilical vein endothelial cells (HUVE), which are located in a monolayer on the surface of the collagen matrix, were studied. CEF and HUVE in 10% FCS produce a substantial tension of 4.5 +/- 0.2 x 10(4) dynes/cm2 and 6.1 x 10(4) dynes/cm2, respectively. Both cell types contract when stimulated with thrombin, generating a force per cell cross-sectional area of approximately 10(5) dynes/cm2, a value approximately an order of magnitude less than smooth muscle. The integrity of the actin cytoskeleton is essential for force generation, as disruption of actin microfilaments with cytochalasin D results in a rapid disappearance of force. Intact microtubules appear to reduce isometric force exerted by CEF, as microtubule-disrupting drugs result in increased tension. Contraction by HUVE precedes a dramatic rearrangement of actin microfilaments from a circumferential ring to stress fibers. PMID:1556157

1992-01-01

49

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

PubMed Central

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

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

2013-01-01

50

Assisted reproduction with in-vitro-cultured testicular spermatozoa in cases of severe germ cell apoptosis: a pilot study  

Microsoft Academic Search

BACKGROUND: Apoptosis-related cell damage is known to compromise success rates of assisted reproduction with ejaculated spermatozoa. This study was undertaken to determine whether the frequency of apoptosis-related cell damage and reproductive performance of testicular spermatozoa from men with non-obstructive azoospermia can be improved by in-vitro culture. METHODS: Testicular tissue samples were cultured for 2 days in the presence of 50

Jan Tesarik; Ermanno Greco; Carmen Mendoza

2001-01-01

51

Differential Activities of Glycolipid Glycosyltransferases in Tay-Sachs Disease: Studies in Cultured Cells from Cerebrum  

Microsoft Academic Search

Four different glycolipid:glycosyltransferase activities involved in the biosynthesis in vitro of gangliosides and blood group-related glycosphingolipids have been tested in a simian virus 40-transformed glial cell culture derived from the cerebrum of a fetus with Tay-Sachs disease (TSD). The TSD cultured brain cells contained little activity of either UDP-Gal:GM2 (beta 1-3)galactosyltransferase (GalT-3; EC 2.4.1.62), which catalyzes the formation of GM1a

Manju Basu; Kathleen A. Presper; Subhash Basu; Linda M. Hoffman; Steven E. Brooks

1979-01-01

52

Ultrastructural study of long-term canine distemper virus infection in tissue culture cells.  

PubMed Central

The morphogenesis of canine distemper virus was studied in Vero cell cultures for 43 days post-inoculation. Active replication of the virus was observed by electron microscopy and assay from 12 h after inoculation on, and peak production was observed on days 5, 14, and 22. From day 28 on, constant but smaller amounts of infectious virus were detected. Two ultrastructural types of intracytoplasmic nucleoprotein filaments were observed; although they first appeared at different times, their subsequent chronological patterns of development were similar. The cells apparently became free of virus by a mechanism of vacuolation. Intranuclear filaments were seen about day 11 and appeared to increase in number thereafter, whereas the infectious titer declined. Possible mechanisms of persistence are discussed in the light of these findings. Images PMID:7076301

Narang, H K

1982-01-01

53

The Organotypic Longitudinal Spinal Cord Slice Culture for Stem Cell Study  

PubMed Central

The objective of this paper is to describe in detail the method of organotypic longitudinal spinal cord slice culture and the scientific basis for its potential utility. The technique is based on the interface method, which was described previously and thereafter was modified in our laboratory. The most important advantage of the presented model is the preservation of the intrinsic spinal cord fiber tract and the ventrodorsal polarity of the spinal cord. All the processes occurring during axonal growth, regeneration, synapse formation, and myelination could be visualized while being cultured in vitro for up to 4-5 weeks after the slices had been isolated. Both pups and adult animals can undergo the same, equally efficient procedures when going by the protocol in question. The urgent need for an appropriate in vitro model for spinal cord regeneration results from a greater number of clinical trials concerning regenerative medicine in the spinal cord injury and from still insufficient knowledge of the molecular mechanisms involved in the neuroreparative processes. The detailed method of organotypic longitudinal spinal cord slice culture is accompanied by examples of its application to studying biological processes to which both the CNS inhabiting and grafted cells are subjected. PMID:25802530

Sypecka, Joanna; Koniusz, Sylwia; Kawalec, Maria

2015-01-01

54

Calcium oxalate crystals in fetal bovine serum: implications for cell culture, phagocytosis and biomineralization studies in vitro.  

PubMed

Cell culture methods and models are key investigative tools for cell and molecular biology studies. Fetal bovine serum (FBS) is commonly used as an additive during cell culture since its constituents promote cell survival, proliferation and differentiation. Here we report that commercially available FBS from different major suppliers consistently contain precipitated, calcium oxalate crystals-either in the monohydrate (COM) or dihydrate (COD) form. Mineral structure and phase identification of the crystals were determined by X-ray diffraction, chemical composition by energy-dispersive X-ray microanalysis, and imaging and measurement of crystal growth steps by atomic force microscopy-all identified and confirmed crystallographic parameters for COM and COD. Proteins binding to the crystals were identified by immunoblotting, revealing the presence of osteopontin and fetuin-A (alpha(2)HS-glycoprotein)--known regulators of crystal growth found in serum. Macrophage cell cultures exposed to calcium oxalate crystals showed internalization of the crystals by phagocytosis in a process that induced disruption of cell-cell adhesion, release of reactive oxygen species and membrane damage, events that may be linked to the release of inflammatory cytokines by these cells into the culture media. In conclusion, calcium oxalate crystals found in commercially available FBS are toxic to cells, and their presence may confound results from in vitro studies where, amongst others, phagocytosis, biomineralization, renal cell and molecular biology, and drug and biomaterial testing are being examined. PMID:17879965

Pedraza, Claudio E; Chien, Yung-Ching; McKee, Marc D

2008-04-01

55

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

PubMed

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 CD49f(hi) /CD90(lo) cell phenotype produced an aggressive tumor. Multiple lines of evidence demonstrated that inhibiting CD49f decreased the tumor-forming ability. Furthermore, the CD49f(hi) /CD90(lo) 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

2014-08-01

56

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

PubMed

The rostral migratory stream (RMS) is the main pathway by which newly born subventricular zone (SVZ) cells reach the olfactory bulb (OB) in rodents. This migration has been well studied in vivo, but an organotypic in vitro model would facilitate more experimental investigations. Here we introduce a slice culture preparation of the rat forebrain including en suite the rostral part of the lateral ventricle, the RMS and the OB. The preparation was validated with regard to endogenous cell proliferation and migration by tracking bromodeoxyuridine (BrdU)-labelled cells in newly established and 3 and 6 week old cultures. For testing the migratory abilities of exogenous precursor cells, rat SVZ neurospheres and human neural (HNS1 cells) and mesenchymal (hMSC-TERT) stem cell lines were micrografted to the rostral SVZ of 1 and 7 day old cultures. Two weeks later graft derivatives were identified by immunohistochemical staining for human nuclei (HNS1/hMSC-TERT cells) and BrdU (HNS1 cells/neurospheres). Numerous HNS1 cells and BrdU-positive neurosphere cells were found in the RMS. Having reached the OB, subpopulations of the cells expressed the astroglial markers glial fibrillary acidic protein/hAM and the neuronal markers NeuN/tyrosine hydroxylase. Interestingly, the hMSC-TERT cells remained at the implantation site, demonstrating a diversity in migratory capability of different precursor cells. In conclusion, the RMS in rat forebrain slice cultures retains its ability to support migration of endogenous and exogenous neural precursors, making the cultures highly feasible for studies of conditions and factors regulating cell migration. PMID:19646977

Tanvig, Mette; Blaabjerg, Morten; Andersen, Rikke K; Villa, Ana; Rosager, Ann Mari; Poulsen, Frantz R; Martinez-Serrano, Alberto; Zimmer, Jens; Meyer, Morten

2009-10-27

57

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...

58

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

59

Microfluidic Cell Culture Device  

NASA Technical Reports Server (NTRS)

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

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

2014-01-01

60

Organotypic Spinal Cord Slice Culture to Study Neural Stem/Progenitor Cell Microenvironment in the Injured Spinal Cord  

E-print Network

The molecular microenvironment of the injured spinal cord does not support survival and differentiation of either grafted or endogenous NSCs, restricting the effectiveness of the NSC-based cell replacement strategy. Studying the biology of NSCs in in vivo usually requires a considerable amount of time and cost, and the complexity of the in vivo system makes it difficult to identify individual environmental factors. The present study sought to establish the organotypic spinal cord slice culture that closely mimics the in vivo environment. The cultured spinal cord slices preserved the cytoarchitecture consisting of neurons in the gray matter and interspersed glial cells. The majority of focally applied exogenous NSCs survived up to 4 weeks. Pre-exposure of the cultured slices to a hypoxic chamber markedly reduced the survival of seeded NSCs on the slices. Differentiation into mature neurons was severely limited in this co-culture system. Endogenous neural progenitor cells were marked by BrdU incorporation, and applying an inflammatory cytokine IL-1? significantly increased the extent of endogenous neural progenitors with the oligodendrocytic lineage. The present study shows that the organotypic spinal cord slice culture can be properly utilized to study molecular factors from the post-injury microenvironment affecting NSCs in the injured spinal cord. Key words: spinal cord injury, organotypic slice culture, neural stem cells, hypoxia, inflammatory cytokine

61

Fabrication of uniform multi-compartment particles using microfludic electrospray technology for cell co-culture studies  

PubMed Central

In this work, we demonstrate a robust and reliable approach to fabricate multi-compartment particles for cell co-culture studies. By taking advantage of the laminar flow within our microfluidic nozzle, multiple parallel streams of liquids flow towards the nozzle without significant mixing. Afterwards, the multiple parallel streams merge into a single stream, which is sprayed into air, forming monodisperse droplets under an electric field with a high field strength. The resultant multi-compartment droplets are subsequently cross-linked in a calcium chloride solution to form calcium alginate micro-particles with multiple compartments. Each compartment of the particles can be used for encapsulating different types of cells or biological cell factors. These hydrogel particles with cross-linked alginate chains show similarity in the physical and mechanical environment as the extracellular matrix of biological cells. Thus, the multi-compartment particles provide a promising platform for cell studies and co-culture of different cells. In our study, cells are encapsulated in the multi-compartment particles and the viability of cells is quantified using a fluorescence microscope after the cells are stained for a live/dead assay. The high cell viability after encapsulation indicates the cytocompatibility and feasibility of our technique. Our multi-compartment particles have great potential as a platform for studying cell-cell interactions as well as interactions of cells with extracellular factors. PMID:24404050

Liu, Zhou; Shum, Ho Cheung

2013-01-01

62

Fabrication of uniform multi-compartment particles using microfludic electrospray technology for cell co-culture studies.  

PubMed

In this work, we demonstrate a robust and reliable approach to fabricate multi-compartment particles for cell co-culture studies. By taking advantage of the laminar flow within our microfluidic nozzle, multiple parallel streams of liquids flow towards the nozzle without significant mixing. Afterwards, the multiple parallel streams merge into a single stream, which is sprayed into air, forming monodisperse droplets under an electric field with a high field strength. The resultant multi-compartment droplets are subsequently cross-linked in a calcium chloride solution to form calcium alginate micro-particles with multiple compartments. Each compartment of the particles can be used for encapsulating different types of cells or biological cell factors. These hydrogel particles with cross-linked alginate chains show similarity in the physical and mechanical environment as the extracellular matrix of biological cells. Thus, the multi-compartment particles provide a promising platform for cell studies and co-culture of different cells. In our study, cells are encapsulated in the multi-compartment particles and the viability of cells is quantified using a fluorescence microscope after the cells are stained for a live/dead assay. The high cell viability after encapsulation indicates the cytocompatibility and feasibility of our technique. Our multi-compartment particles have great potential as a platform for studying cell-cell interactions as well as interactions of cells with extracellular factors. PMID:24404050

Liu, Zhou; Shum, Ho Cheung

2013-01-01

63

Insect neuronal cultures: an experimental vehicle for studies of physiology, pharmacology and cell interactions  

Microsoft Academic Search

The current status of insect neuronal cultures is discussed and their contribution to our understanding of the insect nervous system is explored. Neuronal cultures have been developed from a wide range of insect species and from all developmental stages. These have been used to study the morphological development of insect neurones and some of the extrinsic factors that affect this

D. J. Beadle

2006-01-01

64

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

65

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

PubMed Central

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

Pal, Anupama; Kleer, Celina G.

2014-01-01

66

Integrated PDMS\\/CMOS Microsystem for Autonomous Incubation and Imaging in Cell Culture Studies  

Microsoft Academic Search

We discuss the design, fabrication and testing of a hybrid microsystem for stand-alone cell culture, incubation and imaging. The micro-incubator is engineered through the integration of two CMOS dies. The first for the heater and temperature sensor, and a second die for imaging. A multilayer PDMS (polydimethylsiloxane) structures is used to create not only the fluidic structures but to encapsulate

Jennifer M. Blain Christen; Andreas G. Andreou

2006-01-01

67

Cell culture's spider silk road.  

PubMed

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

Perkel, Jeffrey

2014-06-01

68

Single-cell growth analysis in a mixed cell culture  

NASA Astrophysics Data System (ADS)

We perform single cell analysis of cell growth in a mixed cell culture. Two species of yeast cells: Saccharomyces cerevisiae and Candida albicans, are optically trapped using focused continuous-wave near infrared laser. Cell growth for both cells is inhibited only when the two species of cells are in contact with each other. This indicates cell-cell interaction mediated cell growth inhibition mechanism. Single cell level analysis of cell growth studied here contributes to the further understanding of yeast growth arrest in a mixed yeast culture.

Ando, Jun; Bato, Mary Grace P.; Daria, Vincent Ricardo

2008-06-01

69

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

70

Haute Culture: Tailoring stem cells  

E-print Network

research projects and its faculty have founded five stem cell-related startup companies and serveHaute Culture: Tailoring stem cells to make us well Tuesday, April 24, 2012 6:00-7:30 p;Haute Culture: Tailoring stem cells to make us well Moderator Brock Reeve, MPhil, MBA Executive Director

Chou, James

71

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

72

An innovative three-dimensional gelatin foam culture system for improved study of glioblastoma stem cell behavior.  

PubMed

Three-dimensional (3-D) tissue engineered constructs provide a platform for examining how the local extracellular matrix contributes to the malignancy of various cancers, including human glioblastoma multiforme. Here, we describe a simple and innovative 3-D culture environment and assess its potential for use with glioblastoma stem cells (GSCs) to examine the diversification inside the cell mass in the 3-D culture system. The dissociated human GSCs were cultured using gelatin foam. These cells were subsequently identified by immunohistochemical staining, reverse transcriptase-polymerase chain reaction, and Western blot assay. We demonstrate that the gelatin foam provides a suitable microenvironment, as a 3-D culture system, for GSCs to maintain their stemness. The gelatin foam culture system contributes a simplified assessment of cell blocks for immunohistochemistry assay. We show that the significant transcription activity of hypoxia and the protein expression of inflammatory responses are detected at the inside of the cell mass in vitro, while robust expression of PROM1/CD133 and hypoxia-induced factor-1 alpha are detected at the xenografted tumor in vivo. We also examine the common clinical trials under this culture platform and characterized a significant difference of drug resistance. The 3-D gelatin foam culture system can provide a more realistic microenvironment through which to study the in vivo behavior of GSCs to evaluate the role that biophysical factors play in the hypoxia, inflammatory responses and subsequent drug resistance. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 103B: 618-628, 2015. PMID:24966152

Yang, Meng-Yin; Chiao, Ming-Tsang; Lee, Hsu-Tung; Chen, Chien-Min; Yang, Yi-Chin; Shen, Chiung-Chyi; Ma, Hsin-I

2015-04-01

73

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

Microsoft Academic Search

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

H. Bielefeldt Ohmann; B. Bloch

1982-01-01

74

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

75

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

76

Fabrication of polymer fiber scaffolds by centrifugal spinning for cell culture studies  

Microsoft Academic Search

We demonstrate a mass production amenable technology for the fabrication of polymer fibers that can be used as 3D scaffolds for cell culture and tissue engineering. As the first attempt, we used centrifugal melt spinning technique to fabricate fiber matrix of poly-lactic-co-glycolic acid (PLGA) which is a well-known biodegradable co-polymer. We then developed a solvent assisted centrifugal spinning technique to

Li Wang; Jian Shi; Li Liu; Emilie Secret; Yong Chen

2011-01-01

77

Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection  

Microsoft Academic Search

BACKGROUND: In order to elucidate how Hepatitis C Virus (HCV) interacts with polarized hepatocytes in vivo and how HCV-induced alterations in cellular function contribute to HCV-associated liver disease, a more physiologically relevant hepatocyte culture model is needed. As such, NASA-engineered three-dimensional (3-D) rotating wall vessel (RWV) bioreactors were used in effort to promote differentiation of HCV-permissive Huh7 hepatoma cells. RESULTS:

Bruno Sainz Jr; Veronica TenCate; Susan L Uprichard

2009-01-01

78

Fermentation with immobilized cell cultures.  

PubMed

For the production of monoclonal antibodies and complex recombinant human proteins or glycoproteins a number of immobilized cell culture systems have been developed. The advantages of such cell culture systems are that cells can be kept in small volumes of cell culture fluid and media can be changed continuously if necessary for induction of product synthesis or removal and harvest of metabolic products. Whereas the hollow fiber and the opticell culture systems can be limited in scaling up the microcarrier system, the fluidized bed bioreactor and the solid bed bioreactor are suitable for scaling up. In contrast to the other systems, the solid bed bioreactor requires no special manipulation for anchoring the cells to the wire springs. In situ cleaning is possible and the beads are reusable. With this cell culture fermentation system, production processes for interferon beta, monoclonal antibodies for interferon alfa and recombinant human tissue plasminogen activator were developed. PMID:3285839

Werner, R G; Merk, W; Walz, F

1988-02-01

79

Stromal cells in long-term murine bone marrow culture: FACS studies and origin of stromal cells in radiation chimeras  

SciTech Connect

Adherent layers from hematopoietically active long-term bone marrow cultures (LTBMC), incubated with fluorescent beads, were analyzed for autofluorescence and phagocytic ability, using a fluorescence-activated cell sorter (FACS). Four groups of cells were separated from the adherent layers, including a group of large polygonal fibroblastoid stromal cells. Long-term chimeras were made by lethal irradiation of CBA/Ca (CBA) and C57Bl6/J (B6) mice and repopulation with phosphoglycerate kinase (PGK-1) alloenzyme-congenic bone marrow cells. Hematopoietically active LTBMC were established from such chimeras, and donor and host contributions of FACS-sorted adherent-layer cells were measured. While macrophages and other hematopoietic cells were of donor origin, the fibroblastoid stromal cells were mainly or entirely host derived.

Lennon, J.E.; Micklem, H.S.

1986-05-01

80

An in vitro system for studying the effects of ozone on mammalian cell cultures and viruses  

SciTech Connect

A unique in vitro system was developed for exposing mammalian cell cultures, viruses, or both to ozone under conditions mimicking those of the respiratory tract. The system used borosilicate glass roller culture bottles equipped with specially designed caps to permit the flow of humidified gas (ozone or filtered air) through the rotating vessels. The system was designed to allow two test cultures and one control culture to be simultaneoulsy exposed to different precisely defined concentrations of ozone. The input and exhaust concentrations of ozone were sequentially monitored with an ultraviolet photometric ozone analyzer. The system was used to determine the reactivity of ozone with several tissue culture media at different flow rates. The reaction rate of ozone with media was shown to be a function of the input concentration and increased as the gas flow rate was increased. Input ozone concentrations measured during 48-hr test exposures remained stable, yielding standard deviations of less than 4%. Exposure of Madin-Darby bovine kidney cells to ozone concentrations of 0.16 and 0.64 ppm for 24 hr resulted in a slight but significant decrease in the synthesis of RNA as measured by the incorporation of (/sup 3/H)uridine into TCA-precipitable material. The synthesis of protein and DNA was not significantly affected by identical treatments. Vesicular stomatitis virus exposed to ozone at concentrations of 0.16 and 0.64 ppm for 12 hr showed marked loss of biological function when compared to identical controls exposed to filtered air. The feasibility of using enveloped viruses as a model for investigation of ozone-induced damage to cellular membranes and membrane proteins is discussed.

Bolton, D.C.; Tarkington, B.K.; Zee, Y.C.; Osebold, J.W.

1982-04-01

81

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

PubMed

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

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

2015-02-01

82

Huanglongbing and psyllid cell cultures  

Technology Transfer Automated Retrieval System (TEKTRAN)

We successfully established cell cultures of the Asian citrus psyllid, Diaphorina citri (Psyllidae: Hemiptera), DcHH-1. The cell culture also supported growth of Candidatus Liberibacter asiaticus. This bacterial pathogen is associated with Huanglongbing, known as citrus greening disease. Research on...

83

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

PubMed

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

2005-12-01

84

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

85

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

PubMed Central

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

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

2014-01-01

86

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

PubMed

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

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

2014-10-01

87

FTIR microscopic studies on normal and H-Ras transfected cultured mouse fibroblast cells  

NASA Astrophysics Data System (ADS)

Infrared (IR) absorption spectra are well known for their selectivity and minutiae fingerprint of molecular structure. The biochemical changes in the sub-cellular levels developing in abnormal cells, including a majority of cancer forms, manifest themselves in different optical signatures, which can be detected in infrared spectroscopy. The molecular vibrational modes which are responsible for IR absorption spectra, are characteristic of the biochemistry of the cells and their sub-cellular components. We measured the infrared absorption spectra of monolayers of cultured normal and ras gene transformed mouse fibroblasts, using microscopic infrared system (micro-FTIR) technique. The absorption for normal cells was higher than the malignant ones in the spectral range 1000 - 1500 and 2800 - 3000 cm-1. The effect on phospholipid metabolism due to ras gene incorporation is also discussed.

Salman, Ahmad; Ramesh, Jagannathan; Grossman, Nili; Hammody, Ziad; Cohen, Beny; Mordechai, Shaul

2000-11-01

88

MATERIALS AND METHODS Cell culture  

E-print Network

cell line hCMEC/D3 which retains the main characteristics of primary brain endothelial cells, has beenMATERIALS AND METHODS Reagents Cell culture The immortalized human brain microvessel endothelial previously described (S1). hCMEC/D3 were grown at a density of 25 000 cells per cm2 in flasks coated with 5

89

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...

90

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

91

Cultural Environmental Studies  

NSDL National Science Digital Library

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

92

Dynamized Preparations in Cell Culture  

PubMed Central

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

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

2009-01-01

93

Effect of plasma needle on cultured cells  

Microsoft Academic Search

To investigate a possible application of plasma in fine surgery, we studied the effects of a small atmospheric glow discharge on living cultured cells. The plasma source used for this purpose was the \\

I. E. Kieft; N. A. Dvinskikh; Jos L. V. Broers; Dick W. Slaaf; Eva Stoffels

2004-01-01

94

Characterization of a microcarrier cell culture system for 23Na MR spectroscopy studies.  

PubMed

A MR spectroscopy method is described for the simultaneous discrimination and observation of sodium from the three compartments created by an intact cell monolayer. Results are reported for Madin Darby Canine Kidney (MDCK) cells, an epithelial-like continuous cell line, cultured on Cytodex 1 microcarrier beads and perfused with medium containing 6 mM dysprosium (III) tripolyphosphate [Dy(TPP)2(7-)] as shift reagent. The sodium spectrum shows three resonances which are assigned to the shifted intrabead (basolateral) and extrabead (apical) pools and the unshifted intracellular pool. Ouabain inhibition of the Na(+)-K(+)-ATPase cellular pump mechanism was used to demonstrate the sensitivity of the method for monitoring intracellular sodium. The supported MDCK cells in this system remained viable after exposure for 5 h to medium containing Dy(TPP)2(7-) at a concentration of 6 mM, as determined by trypan blue dye exclusion and by comparison of the log growth rate and ability to form domes in subsequent generations of exposed cells vs unexposed controls. PMID:1751347

Shedd, S F; Spicer, L D

1991-10-01

95

Type I collagen influences cartilage calcification: an immunoblocking study in differentiating chick limb-bud mesenchymal cell cultures.  

PubMed

Chick limb-bud mesenchymal cells, plated in high-density micro-mass culture, differentiate and form a matrix resembling chick epiphyseal cartilage. In the presence of 4 mM inorganic phosphate or 2.5 mM beta-glycerophosphate mineral deposits upon this matrix forming a mineralized tissue that, based on electron microscopy, x-ray diffraction and Fourier Transform Infrared microspectoscopy, is like that of chick calcified cartilage. In this culture system the initial mineral deposits are found on the periphery of the chondrocyte nodules. During differentiation of the cells in the high-density micro-mass cultures there is a switch from expression of type I collagen to type II, and then to type X collagen. However, type I collagen persists in the matrix. Because there is some debate about whether type I collagen influences cartilage calcification, an immunoblocking technique was used to determine the importance of type I collagen on the mineralization process in this system. Studies using nonspecific goat anti-chick IgG demonstrated that 1-100 ng/ml antibody added with the media after the cartilage nodules had developed (day 7) had no effect on the accumulation of mineral in the cultures. Nonspecific antibody added before day 7 blocked development of the cultures. Parallel solution based cell-free studies showed that IgG did not have a strong affinity for apatite crystals, and had no significant effect on apatite crystal growth. Type I collagen antibodies (1-200 ng/ml) added to cultures one time on day 9 (before mineralization started), or on day 11 (at the start of mineralization), slightly inhibited the accumulation of mineral. There was a statistically significant decrease in mineral accretion with 100 or 200 ng/ml collagen antibody addition continuously after these times. Fab' fragments of nonspecific and type I collagen antibodies had effects parallel to those of the intact antibodies, indicating that the decreased mineralization was not attributable to the presence of the larger, bulkier antibodies. The altered accumulation of mineral was not associated with cell death in the presence of antibody (demonstrated by fluorescent labeling of DNA) or with increased apoptosis (TUNEL-stain). In the immunoblocked cultures, EM analysis demonstrated that mineral continued to deposit on collagen fibrils, but there appeared to be fewer deposits. The data demonstrate that type I collagen is important for the mineralization of these cultures. PMID:10906758

Boskey, A L; Stiner, D; Binderman, I; Doty, S B

2000-07-19

96

HUMAN VASCULAR ENDOTHELIAL CELLS IN CULTURE  

PubMed Central

Human endothelial cells, obtained by collagenase treatment of term umbilical cord veins, were cultured using Medium 199 supplemented with 20% fetal calf serum. Small clusters of cells initially spread on plastic or glass, coalesced and grew to form confluent monolayers of polygonal cells by 7 days. Cells in primary and subcultures were identified as endothelium by the presence of Weibel-Palade bodies by electron microscopy. A morphologically distinct subpopulation of cells contaminating some primary endothelial cultures was selectively subcultured, and identified by ultrastructural criteria as vascular smooth muscle. Autoradiography of endothelial cells after exposure to [3H]thymidine showed progressive increases in labeling in growing cultures beginning at 24 h. In recently confluent cultures, labeling indices were 2.4% in central closely packed regions, and 53.2% in peripheral growing regions. 3 days after confluence, labeling was uniform, being 3.5 and 3.9% in central and peripheral areas, respectively. When small areas of confluent cultures were experimentally "denuded," there were localized increases in [3H]thymidine labeling and eventual reconstitution of the monolayer. Liquid scintillation measurements of [3H]thymidine incorporation in primary and secondary endothelial cultures in microwell trays showed a similar correlation of DNA synthesis with cell density. These data indicate that endothelial cell cultures may provide a useful in vitro model for studying pathophysiologic factors in endothelial regeneration. PMID:4363161

Gimbrone, Michael A.; Cotran, Ramzi S.; Folkman, Judah

1974-01-01

97

A study of protein synthesis in cells cultured from involved psoriatic skin.  

PubMed

Using histochemical techniques an abnormal programme of epidermal differentiation has been well documented in psoriasis. In order to characterise further the biochemistry of this process we have cultured dermal fibroblasts and epidermal keratinocytes from involved psoriatic skin. This has facilitated metabolic radiolabelling of skin cells and analysis of protein synthesis by two-dimensional polyacrylamide gel electrophoresis. The expression of keratin and differentiation markers was identical to that of normal keratinocytes, suggesting that psoriatic epidermal differentiation is not truncated in vitro as has been postulated to be the case in vivo. Low molecular mass components (5-8.5 kDa), previously shown to be upregulated in suprabasal keratinocytes, were detected in epidermal fractions from psoriatic skin enriched for basal cells. Of special interest was a component of 26 kDa, pI 5.9, which was highly upregulated in psoriatic as compared to normal cultured keratinocytes and was not detected in fibroblasts. These findings are in accord with a qualitatively abnormal pattern of differentiation for keratinocytes in the involved psoriatic epidermis. PMID:1915249

Easty, D J; Patel, K; Otto, W R; Dunn, M J; Kiil, J; Evans, D J

1991-01-01

98

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

99

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

PubMed Central

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

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

2011-01-01

100

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

101

Monoclonal antibody to intermediate filaments of cytokeratin type. I. Drug studies and reactivity with cultured cells and tissue sections.  

PubMed

Establishment of a mouse-mouse hybridoma and partial characterization of IgM monoclonal antibody (M-04) identifying cytoplasmic filamentous structures is described. Immunofluorescence performed on a panel of various cultured human cell types as well as on frozen sections of normal and tumour tissues revealed specificity of M-04 antibody for cells of epithelial origin. Using MCF-7 cell line as a model, staining patterns of microtubules, microfilaments and M-04-target filaments in untreated cells were compared with those pretreated with Colcemid and Cytochalasin B. From both differential staining of various cell types and the results of drug studies it is concluded that monoclonal antibody M-04 binds to intermediate filaments of cytokeratin type. Furthermore, restricted expression of M-04 target determinant among epithelial tissues is suggested from the lack of reaction in stratified skin epithelium. PMID:2578996

Bártek, J; Kovarík, J; Lauerová, L; Munzarová, M

1985-01-01

102

[Experimental study in vivo on implantation of autogenous tendon cells after combining culture with carbon fibers].  

PubMed

In order to investigate the possibility of repairing injuried tendon with living artificial tendon, after combining culture, subcultured autogenous tendon cells with carbon fibers were implanted into the calcaneous tendon of rabbits. In different stages, the synthesis of type I collagen and their relevant morphological changes were observed. The results showed as follows: after implantation, tendon cells continued proliferating. Four weeks after implantation, tendon cells were detached from the carbon fibers and proliferated and produced collagen among the carbon fibers. The collagen fibrils were linked with each other to formed a dense structure. In the linkage site, the collagen fibrils originated from the implants joined to that from the ruptured end of the tendon, which meaned that the implant was healed with the recipient tendon. Observed under scanning electronic microscope, the tendon cells were lined among the carbon fibers evenly and in order, the collagen fibrils joined each other and formed an network, the fibrils were lined parallel to the carbon fibers. Under transparent electron microscope, the nucleolus were clear and organelle were abundant. PMID:9867920

Zhang, Q; Yang, Z; Peng, W

1997-05-01

103

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...

104

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

105

Morphological characteristics of cultured olfactory bulb cells  

Microsoft Academic Search

Cultured olfactory bulb cells from embryonic mice had ultrastructural characteristics similar to those of many cell types in the intact adult mouse olfactory bulb. Identified cultured cells included mitral\\/tufted cells, granule cells, short-axon cells, and fibrous and protoplasmic astrocytes. Cultured neurons were found as individual cells, clusters or aggregates. Clusters consisted of a loose array of neurons that appeared to

S. P. Fracek; L. Guo; R. Schafer

1994-01-01

106

Cryopreservation of Dedifferentiated Cell Cultures  

Microsoft Academic Search

When Gottlieb Haberlandt made the first efforts to cultivate single isolated plant cells in salt solutions his goal was to\\u000a prove the totipotency of single cells (Haberlandt 1902). The cultivation of isolated plant cells in a chemically defined culture\\u000a medium became possible only after the discovery and application of auxins (Gautheret 1939). Today plant cells as well as tissues\\u000a can

Elke Heine-Dobbernack; Heiko Kiesecker; Heinz Martin Schumacher

107

Development of a novel perfusion microfluidic cell culture device for cell-based assays  

Microsoft Academic Search

This study reports on the development of a novel perfusion microfluidic cell culture device integrated drug delivering, fluid controlling and cell culturing functions on a two- layer PDMS chip. The device consists of an array of 6 X 6 cell culture chambers, a drug gradient generator and two control valves. Cells are physically trapped and cultured in the center of

Yunhuan Zheng; Jianzhang Wu; Jianbo Shao; Qinghui Jin; Jianlong Zhao

2009-01-01

108

Characterization of olfactory receptor neurons and other cell types in dissociated rat olfactory cell cultures  

Microsoft Academic Search

In dissociated cell cultures, control over the cellular environment facilitates study of the differentiation of mature cellular phenotypes. Central to this approach is a rigorous characterization of the cells that reside in culture. Therefore, we have used a battery of cell type-specific antibody markers to identify the cell types present in dissociated cultures of olfactory mucosal cells (containing cells from

S. K. Pixley

1996-01-01

109

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

PubMed Central

Silicon nitride is a biocompatible material that is currently used as an interfacial surface between cells and large-scale integration devices incorporating ion-sensitive field-effect transistor technology. Here, we investigated whether a poly-L-lysine coated silicon nitride surface is suitable for the culture of PC12 cells, which are widely used as a model for neural differentiation, and we characterized their interaction based on cell behavior when seeded on the tested material. The coated surface was first examined in terms of wettability and topography using contact angle measurements and atomic force microscopy and then, conditioned silicon nitride surface was used as the substrate for the study of PC12 cell culture properties. We found that coating silicon nitride with poly-L-lysine increased surface hydrophilicity and that exposing this coated surface to an extracellular aqueous environment gradually decreased its roughness. When PC12 cells were cultured on a coated silicon nitride surface, adhesion and spreading were facilitated, and the cells showed enhanced morphological differentiation compared to those cultured on a plastic culture dish. A bromodeoxyuridine assay demonstrated that, on the coated silicon nitride surface, higher proportions of cells left the cell cycle, remained in a quiescent state and had longer survival times. Therefore, our study of the interaction of the silicon nitride surface with PC12 cells provides important information for the production of devices that need to have optimal cell culture-supporting properties in order to be used in the study of neuronal functions. PMID:24587271

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

2014-01-01

110

A comparative study of ectonucleotidase and P2 receptor mRNA profiles in C6 cell line cultures and C6 ex vivo glioma model.  

PubMed

Glioblastoma multiforme is the most common type of primary brain tumour and has the worst clinical outcome. Nucleotides represent an important class of extracellular molecules involved in cell proliferation, differentiation and apoptosis. Alterations in purinergic signalling have been implicated in pathological processes, such as cancer, and glioma cell lines are widely employed as a model to study the biology of brain tumours. Increasing evidence, however, suggests that glioma cell lines may not present all the phenotypic and genetic characteristics of the primary tumours. We have compared the biological characteristics of C6 rat glioma cells in culture and the same cells after their implantation in the rat brain and growth in culture (denominated as the C6 ex vivo culture model). Parameters evaluated included cell morphology, differentiation, angiogenic markers, purinergic receptors and ecto-nucleotidase mRNA profile/enzymatic activity. Analysis of the C6 glioma cell line and C6 ex vivo glioma cultures revealed distinct cell morphologies, although cell differentiation and angiogenic marker expressions were similar. Both glioma models co-expressed multiple P2X and P2Y receptor subtypes with some differences. In addition, the C6 glioma cell line and C6 ex vivo glioma cultures exhibited similar extracellular ATP metabolism and cell proliferation behaviour when exposed to cytotoxic ATP concentrations. Thus, the disruption of purinergic signalling is a feature shown not only by glioma cell lineages, but also by primary glioma cultures. Our results therefore suggest the participation of the purinergic system in glioma malignancy. PMID:19023597

Braganhol, Elizandra; Huppes, Daiane; Bernardi, Andressa; Wink, Márcia Rosângela; Lenz, Guido; Battastini, Ana Maria Oliveira

2009-02-01

111

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

112

Olfactory Ensheathing Cell Cultures  

Microsoft Academic Search

\\u000a Over the past several years, neuroscientists have developed a considerable interest in a glial cell found only in the first\\u000a cranial nerve. These glial cells, which are referred to as “olfactory ensheath-ing cells,” provide ensheathment for the unmyelinated\\u000a axons of the olfactory nerve (Doucette, 1984, Doucette, 1986, 11,1; Raisman, 1985). Two major reasons why these cells have become so popular

Ronald Doucette

113

The effect of simulated microgravity on human mesenchymal stem cells cultured in an osteogenic differentiation system: a bioinformatics study.  

PubMed

One proposed strategy for bone regeneration involves ex vivo tissue engineering, accomplished using bone-forming cells, biodegradable scaffolds, and dynamic culture systems, with the goal of three-dimensional tissue formation. Rotating wall vessel bioreactors generate simulated microgravity conditions ex vivo, which lead to cell aggregation. Human mesenchymal stem cells (hMSCs) have been extensively investigated and shown to possess the potential to differentiate into several cell lineages. The goal of the present study was to evaluate the effect of simulated microgravity on all genes expressed in hMSCs, with the underlying hypothesis that many important pathways are affected during culture within a rotating wall vessel system. Gene expression was analyzed using a whole genome microarray and clustering with the aid of the National Institutes of Health's Database for Annotation, Visualization and Integrated Discovery database and gene ontology analysis. Our analysis showed 882 genes that were downregulated and 505 genes that were upregulated after exposure to simulated microgravity. Gene ontology clustering revealed a wide variety of affected genes with respect to cell compartment, biological process, and signaling pathway clusters. The data sets showed significant decreases in osteogenic and chondrogenic gene expression and an increase in adipogenic gene expression, indicating that ex vivo adipose tissue engineering may benefit from simulated microgravity. This finding was supported by an adipogenic differentiation assay. These data are essential for further understanding of ex vivo tissue engineering using hMSCs. PMID:20807102

Sheyn, Dima; Pelled, Gadi; Netanely, Dvir; Domany, Eytan; Gazit, Dan

2010-11-01

114

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

115

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

116

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

117

Rotating cell culture systems for human cell culture: human trophoblast cells as a model.  

PubMed

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

Zwezdaryk, Kevin J; Warner, Jessica A; Machado, Heather L; Morris, Cindy A; Höner zu Bentrup, Kerstin

2012-01-01

118

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

PubMed Central

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

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

2014-01-01

119

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

PubMed

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 models that recapitulate the 3D architecture of tissues observed in vivo. The application of these models to the study of infectious diseases is discussed. PMID:20948552

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

2010-11-01

120

31P Nuclear Magnetic Resonance Study of the Metabolic Pools of Adenosine Triphosphate in Cultured Bovine Adrenal Medullary Chromaffin Cells  

Microsoft Academic Search

31P NMR was used to resolve and determine the relative quantity and mobility of ATP in the cytosolic and vesicular compartments of isolated adrenomedullary chromaffin cells. The cells were cultured on microcarrier beads and superfused with an oxygenated medium--thereby permitting dense suspensions of viable cells to be maintained in the NMR probe for extended time periods. Under these conditions, distinct

George R. Painter; Emanuel J. Diliberto; Jane Knoth

1989-01-01

121

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

122

CD40 ligand is necessary and sufficient to support primary diffuse large B-cell lymphoma cells in culture: a tool for in vitro preclinical studies with primary B-cell malignancies  

PubMed Central

Established cell lines are utilized extensively to study tumor biology and preclinical therapeutic development; however, they may not accurately recapitulate the heterogeneity of their corresponding primary disease. B-cell tumor cells are especially difficult to maintain under conventional culture conditions, limiting access to samples that faithfully represent this disease for preclinical studies. Here, we used primary canine diffuse large B-cell lymphoma to establish a culture system that reliably supports the growth of these cells. CD40 ligand, either expressed by feeder cells or provided as a soluble two-trimeric form, was sufficient to support primary lymphoma cells in vitro. The tumor cells retained their original phenotype, clonality and known karyotypic abnormalities after extended expansion in culture. Finally, we illustrate the utility of the feeder cell-free culture system for comparable assessment of cytotoxicity using dog and human B-cell malignancies. We conclude this system has broad applications for in vitro preclinical development for B-cell malignancies. PMID:22229753

Ito, Daisuke; Frantz, Aric M.; Williams, Christina; Thomas, Rachael; Burnett, Robert C.; Avery, Anne C.; Breen, Matthew; Mason, Nicola J.; O’Brien, Timothy D.; Modiano, Jaime F.

2013-01-01

123

Studies on the glucocorticoid-receptor blocking action of RU 38486 in cultured ACTH-secreting human pituitary tumour cells and normal rat pituitary cells.  

PubMed

The glucocorticoid-receptor blocking actions of RU 38486, a new compound with anti-progesterone activity, have been investigated in cultured human ACTH-secreting pituitary tumour cells and normal rat pituitary cells. Pre-incubation of human pituitary tumour cells for 24 h with RU 38486 (1 microM) did not influence basal or CRF-stimulated ACTH release. RU 38486 (100 nM-1 microM) significantly overcame or prevented the dexamethasone (100 nM-1 microM)-induced inhibition of CRF-stimulated ACTH release by the cultured tumour cells prepared from 2 patients with Cushing's disease. The tumour cells of a third patient were insensitive to CRF. Pre-incubation for 24 h with 1 microM RU 38486 facilitated CRF-stimulated ACTH release significantly. Studies with cultured normal rat pituitary cells showed that the inhibiting effect of 24 h pre-incubation with 10 and 50 nM dexamethasone on CRF-stimulated ACTH release could be acutely (measured over 4 h) overruled in a dose-dependent way by RU 38486 (100 nM, 1 and 10 microM), while pre-incubation for 24 h of these cells with RU 38486 (100 nM and 1 microM) significantly attenuated the acute inhibiting effect of 1 microM dexamethasone on CRF-stimulated ACTH-release. The results of these in vitro experiments are discussed against the background of the possible therapeutic use RU 38486 in patients with Cushing's syndrome in order to block the deleterious effects of high circulating cortisol concentrations. PMID:2988258

Lamberts, S W; Bons, E G; Uitterlinden, P

1985-05-01

124

Isolation and culture of protoplasts from cotton cell cultures  

E-print Network

ISOLATION AND CULTURE OF PROTOPLASTS FROM COTTON CELL CULTURES A Thesis by JOHN JAMES FINER Submitted to the Graduate College of Texas ASM University in partial fulfillment of the requirement for the degree of MASTER OF SCIENCE May 1981... Major Subject: Plant Physiology ISOLATION AND CULTURE OF PROTOPLASTS FROM COTTON CELL CULTURES A Thesis by John James Finer Approved as tc style and content by: (Chairman ot Committee) (Member) (Member) (Member) (Head oF Department) May 1981...

Finer, John James

1981-01-01

125

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

PubMed Central

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

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

2013-01-01

126

Studies on the porphobilinogen deaminase–uroporphyrinogen cosynthetase system of cultured soya-bean cells  

PubMed Central

1. Porphobilinogenase was isolated and purified from soya-bean callus tissue; its components, porphobilinogen deaminase and uroporphyrinogen isomerase, were separated and purified. 2. The purified porphobilinogenase was resolved into two bands on starch-gel electrophoresis. The molecular weights of porphobilinogenase, deaminase and isomerase fractions were determined by the gel-filtration method. Porphobilinogenase activity was affected by the presence of air; uroporphyrinogens were only formed under anaerobic conditions, although substrate consumption was the same in the absence of oxygen as in its presence. 3. pH-dependence of both porphobilinogenase and deaminase was the same and a sharp optimum at pH 7.2 was obtained. Isomerase was heat-labile, but the presence of ammonium ions or porphobilinogen afforded some protection against inactivation. The action of several compounds added to the system was studied. Cysteine, thioglycollate, ammonium ions and hydroxylamine inhibited porphobilinogenase; certain concentrations of sodium and magnesium salts enhanced activity; some dicarboxylic acids and 2-methoxy-5-nitrotropone inhibited the deaminase. 4. ?-Aminolaevulate and ethionine in the culture media stimulated porphyrin synthesis and increased porphobilinogenase activity, whereas iron deficiency resulted in porphyrin accumulation. 5. The development of chlorophyll and porphobilinogenase on illumination of dark-grown callus was followed. 6. A hypothetical scheme is suggested for the enzymic synthesis of uroporphyrinogens from porphobilinogen. PMID:5165654

Llambías, Elena B. C.; Batlle, Alcira M. Del C.

1971-01-01

127

Studies on the porphobilinogen deaminase-uroporphyrinogen cosynthetase system of cultured soya-bean cells.  

PubMed

1. Porphobilinogenase was isolated and purified from soya-bean callus tissue; its components, porphobilinogen deaminase and uroporphyrinogen isomerase, were separated and purified. 2. The purified porphobilinogenase was resolved into two bands on starch-gel electrophoresis. The molecular weights of porphobilinogenase, deaminase and isomerase fractions were determined by the gel-filtration method. Porphobilinogenase activity was affected by the presence of air; uroporphyrinogens were only formed under anaerobic conditions, although substrate consumption was the same in the absence of oxygen as in its presence. 3. pH-dependence of both porphobilinogenase and deaminase was the same and a sharp optimum at pH 7.2 was obtained. Isomerase was heat-labile, but the presence of ammonium ions or porphobilinogen afforded some protection against inactivation. The action of several compounds added to the system was studied. Cysteine, thioglycollate, ammonium ions and hydroxylamine inhibited porphobilinogenase; certain concentrations of sodium and magnesium salts enhanced activity; some dicarboxylic acids and 2-methoxy-5-nitrotropone inhibited the deaminase. 4. delta-Aminolaevulate and ethionine in the culture media stimulated porphyrin synthesis and increased porphobilinogenase activity, whereas iron deficiency resulted in porphyrin accumulation. 5. The development of chlorophyll and porphobilinogenase on illumination of dark-grown callus was followed. 6. A hypothetical scheme is suggested for the enzymic synthesis of uroporphyrinogens from porphobilinogen. PMID:5165654

Llambías, E B; Battle, A M

1971-01-01

128

Studies on the regulation of the lipid-linked oligosaccharide pathway in cultured cells  

SciTech Connect

Although the reactions involved in the formation of Glc3Man9(GlcNAc)2-PP-dolichol are reasonably well understood, little is known about the regulation of these reactions. Previous studies in the authors lab and the labs of Lennarz and Robbins showed that when protein synthesis was inhibited with puromycin (P) or cycloheximide (C), mannose (man) incorporation into lipid-linked oligosaccharides (LLO) was also inhibited. This inhibition of LLO could be due to limitations in the amount of dol-P as a result of absence of oligosaccharide acceptor, or to accumulation of a metabolic inhibitor. They have examined the effect of P and C on incorporation of TH-man and TH-GlcN into LLO and protein in MDCK cells. Both P and C inhibited man incorporation into LLO and protein, but formation of dol-P-man was not affected. On the other hand, GlcN incorporation into dol-PP-GlcNAc and dol-PP-(GlcNAc)2 was inhibited by P and C, as were incorporation into LLO and protein. Man incorporation into LLO was also inhibited by hydroxynorvaline, a thr analog that inhibits glycosylation. Inhibition of man incorporation into LLO and protein by P and C could not be overcome by adding dol-P or dol to the cells, although both lipids greatly stimulated man incorporation into lipids and protein in normal cells. Enzyme measurements of control or inhibited cells did not show any difference. These results suggest that inhibition of LLO is due to inhibition of dol-PP-GlcNAc formation, perhaps by accumulation of a metabolic inhibitor.

Pan, Y.T.; Elbein, A.D.

1987-05-01

129

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

NASA Astrophysics Data System (ADS)

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

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

130

Morphological characteristics of cultured olfactory bulb cells  

Microsoft Academic Search

Cultured olfactory bulb cells from embryonic mice had ultrastructural characteristics similar to those of many cell types\\u000a in the intact adult mouse olfactory bulb. Identified cultured cells included mitral\\/tufted cells, granule cells, short-axon\\u000a cells, and fibrous and protoplasmic astrocytes. Cultured neurons were found as individual cells, clusters or aggregates. Clusters\\u000a consisted of a loose array of neurons that appeared to

S. P. Fracek; L. Guo; R. Schafer

1994-01-01

131

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

132

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

133

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

E-print Network

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

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

134

TISSUE CULTURE STUDIES  

PubMed Central

Some of the compounds in the active fraction of ultrafiltrates of chick embryo extract have been identified as taurine, serine, glutamic acid, xanthine, uracil, glucose-6-phosphate, glucose, ferrous iron, and inorganic phosphate. Based on the identity of these compounds a synthetic replacement for the ultrafilterable portion of chick embryo extract has been devised. There is an additional nutritional requirement that can be met by vitamin B12. Folic acid appears to be beneficial to the system though the requirements of this or any of the above compounds except vitamin B12 remain for future research. The low nitrogen content of the isolated fraction and the synthetic mixture suggests that the main nutrition of chick cells in roller tube cultures is derived from the non-dialyzable portion of the medium. PMID:13109159

Rosenberg, Sheldon; Kirk, Paul L.

1953-01-01

135

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

136

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

137

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

138

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

139

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

140

Establishment of a primary culture of Echinococcus multilocularis germinal cells.  

PubMed

This study was designed to establish an in vitro primary culture of germinal cells of Echinococcus multilocularis, a parasite that causes alveolar echinococcosis of the liver (AEL). We also investigated the temperature-dependency of the cultured cells. The germinal cells, which originated from a human lesion, were cultured by an original fluid-suspension method at 25 degrees C or 37 degrees C for 4 weeks. Anchorage-dependent and -independent cells were observed by light microscopy, transmission electron microscopy, and immunocytochemistry to confirm their origin. Cell number and viability were examined by immunocytochemistry and mitochondrial exclusion test. The cultured cells were also inoculated into jirds (Meriones unguiculatus) to evaluate metacestode formation. Morphology and immunocytochemistry showed that the cultured cells were typically germinal cells. The cell number declined gradually over the 4-week culture period, but viability remained at 50% at 3 weeks. These findings were not associated with either of the two culture temperatures; moreover, host-associated cells were not noted in the cultured cells at 25 degrees C. The implanted cells formed metacestodes in the jird peritoneal cavity, and their histology demonstrated mature and typical alveolar-type echinococcal cysts. We successfully established an in vitro primary culture of germinal cells. This should contribute to future studies, and, hence, a better outcome for patients with AEL. PMID:9213248

Yamashita, K; Uchino, J; Sato, N; Furuya, K; Namieno, T

1997-06-01

141

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

142

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

PubMed

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

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

2015-04-01

143

Differentiated cultures of primary hamster tracheal airway epithelial cells.  

PubMed

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

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

2004-01-01

144

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

E-print Network

sex differentiation and the onset or arrest of meiosis in female or male germ cells. Wolff and his (Wolffand Haffen, 1965), thus indicating that testicular sex differentiation might be independentSome relationships among germ, satellite and interstitial cells during chick gonad differentiation

Boyer, Edmond

145

Cocaine-induced kidney toxicity: an in vitro study using primary cultured human proximal tubular epithelial cells.  

PubMed

Renal failure resulting from cocaine abuse has been well documented, although the underlying mechanisms remain to be investigated. In the present study, primary cultured human proximal tubular epithelial cells (HPTECs) of the kidney were used to investigate its ability to metabolize cocaine, as well as the cytotoxicity induced by cocaine and its metabolites benzoylecgonine (BE), ecgonine methyl ester (EME) and norcocaine (NCOC). Gas chromatography/ion trap-mass spectrometry (GC/IT-MS) analysis of HPTECs exposed to cocaine (1 mM) for 72 h confirmed its metabolism into EME and NCOC, but not BE. EME levels increased along the exposure time to cocaine, while NCOC concentration diminished after reaching a maximum at 6 h, indicating a possible secondary metabolism for this metabolite. Cocaine promoted a concentration-dependent loss of cell viability, whereas BE and EME were found to be non-toxic to HPTECs at the tested conditions. In contrast, NCOC revealed to have higher intrinsic nephrotoxicity than the parent compound. Moreover, cocaine-induced cell death was partially reversed in the presence of ketoconazole (KTZ), a potent CYP3A inhibitor, supporting the hypothesis that NCOC may play a role in cocaine-induced nephrotoxicity. Cocaine-induced cytotoxicity was found to involve intracellular glutathione depletion at low concentrations and to induce mitochondrial damage at higher concentrations. Under the present experimental conditions, HPTECs death pathway followed an apoptotic pattern, which was evident for concentrations as low as 0.1 mM. PMID:21983858

Valente, Maria João; Henrique, Rui; Vilas-Boas, Vânia; Silva, Renata; Bastos, Maria de Lourdes; Carvalho, Félix; Guedes de Pinho, Paula; Carvalho, Márcia

2012-02-01

146

Calcium uptake studies of 1,4-dihydropyridine agonists into rabbit aortic smooth muscle cells in culture  

SciTech Connect

The effects of the three dihydropyridine calcium channel agonists (/plus minus/)BAY K 8644, (+)202-791 and (/plus minus/)CGP 28392 on /sup 45/Ca/sup + +/ uptake were studied in cultures of rabbit aortic smooth muscle cells. At 10/sup /minus/7/M each agonist enhanced /sup 45/Ca/sup + +/ uptake in 15-50 mM K/sup +/ but had no effect on the basal /sup 45/Ca/sup + +/ uptake at 5 mM K/sup +/. At the uptake threshold of 15 mM K/sup +/ each agonist potentiated /sup 45/Ca/sup + +/ uptake in a dose-dependent manner with half maximal effects at 2.4 nM for (/plus minus/)BAY K 8644, 22 nM for (+)202-791 and 18 nM for (/plus minus/)CGP 28392. The agonists showed no significant antagonistic activity. Responses were antagonized competitively by nifedipine and non-competitively by (/plus minus/)D-600. The /sup 45/Ca/sup + +/ uptake-dose response curves and the half maximal effects of the three agonists were over the same range of concentrations as their inhibition of (/sup 3/H)nitrendipine binding to rat ventricular receptor membrane preparations. The data suggest that these cells mimic the calcium uptake by the intact aorta better than commercial vascular smooth muscle lines or cardiac cells.

Papaioannou, S.; Panzer-Knodle, S.; Yang, P.C.

1989-01-01

147

Interpretation of the Multinucleated Giant Cell in Human Trophoblast Cultures  

Microsoft Academic Search

Morphology of human trophoblast culture has been studied in relation to certain conditions such as duration of the pregnancy, age of the culture, and strength of the trypsinization. ‘Multinucleated giant cells’ appear to be actually fortuitous transversal sections of chorionic buds. Their presence and aspects depend on the degree of trypsinization. Origin of epithelioid polygonal cells and of fibroblast cells

J. Foldes; Tehila Kehaty; Jeanna Schwartz

1973-01-01

148

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

149

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

PubMed Central

The microsporidia have recently been recognized as a group of pathogens that have potential for waterborne transmission; however, little is known about the effects of routine disinfection on microsporidian spore viability. In this study, in vitro growth of Encephalitozoon syn. Septata intestinalis, a microsporidium found in the human gut, was used as a model to assess the effect of chlorine on the infectivity and viability of microsporidian spores. Spore inoculum concentrations were determined by using spectrophotometric measurements (percent transmittance at 625 nm) and by traditional hemacytometer counting. To determine quantitative dose-response data for spore infectivity, we optimized a rabbit kidney cell culture system in 24-well plates, which facilitated calculation of a 50% tissue culture infective dose (TCID50) and a minimal infective dose (MID) for E. intestinalis. The TCID50 is a quantitative measure of infectivity and growth and is the number of organisms that must be present to infect 50% of the cell culture wells tested. The MID is as a measure of a system's permissiveness to infection and a measure of spore infectivity. A standardized MID and a standardized TCID50 have not been reported previously for any microsporidian species. Both types of doses are reported in this paper, and the values were used to evaluate the effects of chlorine disinfection on the in vitro growth of microsporidia. Spores were treated with chlorine at concentrations of 0, 1, 2, 5, and 10 mg/liter. The exposure times ranged from 0 to 80 min at 25°C and pH 7. MID data for E. intestinalis were compared before and after chlorine disinfection. A 3-log reduction (99.9% inhibition) in the E. intestinalis MID was observed at a chlorine concentration of 2 mg/liter after a minimum exposure time of 16 min. The log10 reduction results based on percent transmittance-derived spore counts were equivalent to the results based on hemacytometer-derived spore counts. Our data suggest that chlorine treatment may be an effective water treatment for E. intestinalis and that spectrophotometric methods may be substituted for labor-intensive hemacytometer methods when spores are counted in laboratory-based chlorine disinfection studies. PMID:10742198

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

2000-01-01

150

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

151

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

152

Plant cell suspension cultures: some engineering considerations.  

PubMed

Higher plants are the source of a vast array of biochemicals which are used as drugs, pesticides, flavourings and fragrances. For some of these compounds, plant cell culture can provide a potential production alternative to traditional cultivation methods or chemical synthesis routes. Many systems have been patented and the last 20 years have seen considerable industrial and academic interest in the development of large scale cultures to produce pharmaceutically active, high value substances. However, the industrial application of plant cell suspension cultures has, to date, been limited. Commercialisation has essentially been impeded by economic feasibility, arising from both biological and engineering considerations. This paper reviews the commercial development of the technology to date and focuses on the impact of specific engineering-related factors, in particular, the shear sensitivity of plant cell suspension cultures. Evidence of sensitivity to hydrodynamic shear in bioreactors has generally been attributed to the physical characteristics of the suspended cells. Recent studies indicate that shear sensitivity may not be as important, in some cases, as initially anticipated. PMID:9487717

Kieran, P M; MacLoughlin, P F; Malone, D M

1997-12-17

153

Biological properties of different type carbon particles in vitro study on primary culture of endothelial cells.  

PubMed

Carbon powders have extended surface of carbon layers, which is of significant biomedical importance since the powders are employed to cover implants material. Carbon Powder Particles are produced by different methods: by a detonation method, by RF PACVD (Radio Frequency Plasma Activated Chemical Vapour Deposition) or MW/RF PCVD (Microwave/Radio Frequency Plasma Activated Chemical Vapour Deposition) and others. Our previous data showed that Carbon Powder Particles may act as antioxidant and/or anti-inflammatory factor. However the mechanism of such behavior has been not fully understood. The aim of the work was tested influence carbon powders manufactured by Radio Frequency Plasma Activated Chemical Vapour Deposition RFPACVD method and detonation method on selected parameters of human endothelial cells, which play a crucial role in the regulation of the circulation and vascular wall homeostasis. Graphite powder was used as a control substance. Endothelial cells are actively involved in a wide variety of processes e.g., inflammatory responses to a different type of stimuli (ILs, TNF-alpha) or regulating vasomotor tone via production of vasorelaxants and vasocontrictors. Biological activation is dependent on the type and quantity of chemical bonds on the surface of the powders. The effect of powders on the proliferation of HUVECs (Human Umbilical Vein Endothelial Cells) was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay. We found decreased cell proliferation after 72 h treatment with graphite as well as Carbon Powder Particles. PMID:20352757

Czerniak-Reczulska, M; Niedzielski, P; Balcerczyk, A; Bartosz, G; Karowicz-Bili?ska, A; Mitura, K

2010-02-01

154

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

Microsoft Academic Search

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

Zuzanna Bukowy; Ewa Zi?tkiewicz; Micha? Witt

2011-01-01

155

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

E-print Network

, vascular and respiratory epithelium, and arterial smooth muscle cells. For years, the Limulus amoebocyte with high recovery. Additionally, the S1 peptide is highly resistant to a wide range of pH's and ionic- duce endotoxin-free samples with high protein recovery. Endotoxin Removal from Proteins using Endo

Lebendiker, Mario

156

Bioreactor studies on the effect of dissolved oxygen concentrations on growth and differentiation of carrot ( Daucus carota L.) cell cultures  

Microsoft Academic Search

A bioreactor control system was used to investigate the effects of two dissolved oxygen concentrations (10% and 100%) on the growth and differentiation of Daucus carota L. cell cultures. The strategy used allowed the dissolved oxygen concentration to be controlled without the need for changing either the agitator speed or the total gas flow rate. During the proliferation phase, reducing

Véronique Jay; Simone Genestier; Jean-Claude Courduroux

1992-01-01

157

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

PubMed Central

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

Stephens, D S

1989-01-01

158

Probing nanoparticle interactions in cell culture media.  

PubMed

Nanoparticle research is often performed in vitro with little emphasis on the potential role of cell culture medium. In this study, gold nanoparticle interactions with cell culture medium and two cancer cell lines (human T-cell leukemia Jurkat and human pancreatic carcinoma PANC1) were investigated. Gold nanoparticles of 10, 25, 50, and 100 nm in diameter at fixed mass concentration were tested. Size distributions and zeta potentials of gold nanoparticles suspended in deionized (DI) water and Dulbecco's Modified Eagle's Media (DMEM) supplemented with fetal calf serum (FCS) were measured using dynamic light scattering (DLS) technique. In DI water, particle size distributions exhibited peaks around their nominal diameters. However, the gold nanoparticles suspended in DMEM supplemented with FCS formed complexes around 100 nm, regardless of their nominal sizes. The DLS and UV-vis spectroscopy results indicate gold nanoparticle agglomeration in DMEM that is not supplemented by FCS. The zeta potential results indicate that protein rich FCS increases the dispersion quality of gold nanoparticle suspensions through steric effects. Cellular uptake of 25 and 50 nm gold nanoparticles by Jurkat and PANC1 cell lines were investigated using inductively coupled plasma-mass spectroscopy. The intracellular gold level of PANC1 cells was higher than that of Jurkat cells, where 50 nm particles enter cells at faster rates than the 25 nm particles. PMID:22421416

Sabuncu, Ahmet C; Grubbs, Janna; Qian, Shizhi; Abdel-Fattah, Tarek M; Stacey, Michael W; Beskok, Ali

2012-06-15

159

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

160

[Changes in the number of sister chromatid exchanges in cultured Chinese hamster cells with limited proliferation. Additional studies].  

PubMed

During "stationary phase ageing" of cultured Chinese hamster cells (B11dii-FAF28 line, 2372a clone), i. e. while decreasing the proliferation rate and in the stationary growth phase the frequency of spontaneous sister chromatid exchanges (SCE) progressively increases (from 2- to 23-day "age"); the frequency of thiophosphamide-induced (1h) SCE increases from 2- to 23-day "age" by the same value as the frequency of spontaneous SCE; the cells deepen into the R-phase of the cell cycle. PMID:3113020

Khokhlov, A N; Chirkova, E Iu; Chebotarev, A N

1987-01-01

161

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

PubMed

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

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

2013-11-01

162

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

163

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...

164

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

165

Differential proliferative responses of cultured Schwann cells to axolemma- and myelin-enriched fractions. I. Biochemical studies  

PubMed Central

Cultured rat Schwann cells were treated for 72 h with axolemma- and myelin-enriched fractions prepared from rat brainstem. [3H]Thymidine was added to the cultures 48 h before the termination of the experiment. Although, both fractions produced a dose-dependent uptake of label into Schwann cells, the shape of the dose response curves and rates at which [3H]thymidine was incorporated were different. The axolemma-enriched fraction produced a sigmoid dose response curve with a Hill coefficient of 2.05. The dose response curve for myelin rose sharply and saturated at a level that was approximately 50% of the maximal response observed with axolemma. Schwann cells that had been treated with axolemma exhibited little change in the rate of [3H]thymidine incorporation from 36-72 h after the addition of the membranes. In contrast, Schwann cells accumulated label three times faster during the 48-72-h period following the addition of myelin to the cultures when compared with the rate during the preceding 12-h interval. Furthermore, the mitogenic activity of the myelin-enriched fraction was decreased by the addition of ammonium chloride, a lysosomal inhibitor, whereas the activity of the axolemmal fraction was not impaired. PMID:6501427

1984-01-01

166

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

167

Methods for decreasing hydrophobicity of polydimethylsiloxane (PDMS) using surface treatments and gel mixtures for use in parallel fluid flow cell culture studies  

Microsoft Academic Search

Polydimethylsiloxane (PDMS) is a silica-based elastomer used in many biological applications in microfluidics and the growing of cell cultures. A PDMS biochip has been designed for cell culture testing, but due to PDMS's natural hydrophobicity, water pressures in the chip's microchannels make it difficult to accurately distribute fluids to the cell cultures. It is hypothesized that this effect can be

Derek Caetano-Anollés; Yuan Wen; Shang-Tian Yang; L. James Lee

168

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

169

[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

170

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

171

Liver cells culture on three-dimensional micropatterned polydimethylsiloxane surfaces  

Microsoft Academic Search

Current in vitro cell culture technologies present some limitations as they can't simulate or mimic in vivo situations. Indeed, in vivo cells function in a three-dimensional (3D) structure where they have a close contact with adjacent cells. In this study, human hepatocarcinoma Hep G2 cells were cultured on 3D micropatterned polydimethylsiloxane (PDMS) substrates. Using soft-lithography techniques, arrays of octagonal macropillars

Fanny Evenou; Teruo Fujii; Yasuyuki Sakai

172

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

Microsoft Academic Search

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

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

2011-01-01

173

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

174

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

175

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

176

Acetaldehyde and hexanaldehyde from cultured white cells  

PubMed Central

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

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

2009-01-01

177

Patterning of polymeric cell culture substrates.  

PubMed

The purpose of this chapter is to provide a summary of polymer patterning technologies for biological applications and detailed instructions for resist-free deep ultraviolet (UV) patterning of poly(styrene). Photochemical modifications of this polymer yield unstable peroxides together with stable oxidized chemical groups. The altered physicochemical properties of the polymer surface influence protein adsorption and cell adhesion. HepG2 (human hepatoma cell line), fibroblasts (L929, murine fibroblast line), and other cell lines exhibit strong adhesion on areas of UV-irradiated polymer. Masked irradiations open a simple, fast (cell patterns are obtained within a few hours), and economical route to obtain chemically patterned cell culture substrates. The described protocol is advantageous compared to silane-based patterning techniques on glass or thiol-based patterning on gold because of the elimination of any chemical treatment and the small size of achieved structures. The protocol is compatible with common clean room technologies; however, even without access to a clean room, structured substrates can be produced. The described technique can be a useful tool for a variety of cell cultures used to study biological processes like intercellular communication and organogenesis and for applications like biosensing or tissue engineering. PMID:24439278

Welle, Alexander; Weigel, Simone; Bulut, Özgül Demir

2014-01-01

178

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

179

Culturing adult stem cells from mouse small intestinal crypts.  

PubMed

In recent years, the study of primary cells in culture has evolved from an extraphysiological, two-dimensional platform to novel, three-dimensional platforms in which the addition of matrix components and/or supporting cells provide an ex vivo niche. Such studies have provided the basis on which to study more advanced physiological processes in detail, including multilayered, long-term cultures, epithelial-stromal interactions, and stem cell behaviors that more closely recapitulate normal morphology than two-dimensional culture. Various techniques for three-dimensional organotypic culture and crypt culture of primary cells from mouse and human small intestine and colon have been described. These methods have allowed for the study of specific stem cell characteristics, including survival, self-renewal, and long-term growth in culture, as well as the ability to propagate all the appropriate progenitor and postmitotic lineages. These assays have become a widely accepted functional measure of "stemness" and, in combination with lineage-tracing experiments in various genetically engineered mouse models, have been critical in the identification of specific markers of intestinal stem cells. In this protocol we draw upon recently described methods for the isolation and culture of mouse small intestinal enterospheres/enteroids from isolated crypts and/or single cells. Cultures of murine colon epithelium, as well as human small intestine and colon, require additional growth factors not discussed here. The description provided here represents current knowledge, and it is possible, if not likely, that modifications in the future will emerge. PMID:25834260

Hamilton, Kathryn E; Crissey, Mary Ann S; Lynch, John P; Rustgi, Anil K

2015-01-01

180

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

181

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration.  

PubMed

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair. PMID:25742570

de Luca, Alba C; Faroni, Alessandro; Reid, Adam J

2015-01-01

182

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration  

PubMed Central

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair. PMID:25742570

de Luca, Alba C.; Faroni, Alessandro; Reid, Adam J.

2015-01-01

183

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

184

J Cell Biochem . Author manuscript Optimizing stem cell culture  

E-print Network

J 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 balance between self-renewal and differentiation. Hence, stem cell culture parameters are critical

Paris-Sud XI, Université de

185

Ascorbic acid transport into cultured pituitary cells  

SciTech Connect

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

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

1986-05-01

186

Correlated mass spectrometry imaging and confocal Raman microscopy for studies of three-dimensional cell culture sections.  

PubMed

A novel method of correlated imaging, combining confocal Raman microscopy (CRM) and matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) was developed in order to investigate the structural and chemical diversity inherent in three-dimensional (3D) cell cultures. These 3D spheroidal cell cultures are high throughput in vitro model systems that recapitulate some of the chemical and physiological gradients characteristic of tissues. As a result, they are ideal for testing new imaging approaches due to the native diversity of cellular phenotypes found within a single culture. Individually, confocal Raman microscopy (CRM) and mass spectrometry imaging (MSI) produce different kinds of chemical information. CRM imaging reveals differences in cellular integrity and protein secretion across a typical near-equatorial transverse slice, while MSI shows localization of small molecules to discrete regions of the spheroid section. Correlating information obtained from these disparate imaging methods begins with an external fiducial mask, added to the spheroidal samples to orient image acquisition on the two orthogonal platforms. Rather than combine the images directly, principal component analysis is used to reveal the most chemically-informative elements, which are then combined using digital image correlation. Using this approach, relationships between the principal components of each method are visualized so that they may be compared on commensurate spatial length scales. PMID:25030970

Ahlf, Dorothy R; Masyuko, Rachel N; Hummon, Amanda B; Bohn, Paul W

2014-09-21

187

C6 ceramide dramatically enhances docetaxel-induced growth inhibition and apoptosis in cultured breast cancer cells: A mechanism study.  

PubMed

Here we reported that co-administration of docetaxel and a cell-permeable short-chain ceramide (C6) resulted in a striking increase in growth inhibition and apoptosis in primary and transformed breast cells (MCF-7 and MDA-231), which were associated with mitochondrial permeability transition pore (mPTP) opening, a significant reactive oxygen species (ROS) production and the pro-apoptotic AMP-Protein Kinase (AMPK) as well as c-Jun N-terminal kinases (JNK) activations. Contrarily, the mPTP blocker sanglifehrin A (SfA) or the ROS scavenger N-acetyl-l-cysteine (NAC) largely inhibited co-administration-induced cytotoxicity. Further, cyclosporin A (CsA), the inhibitor of cyclophilin-D (Cyp-D, the key mPTP component), as well as Cyp-D RNA silencing also suppressed breast cancer cell death by the co-treatment, while cells overexpressing Cyp-D showed hypersensitivity to docetaxel. Meanwhile, JNK and AMPK inhibition alleviated cell death induced by the co-administration in cultured breast cancer cells. Significantly, C6 ceramide plus docetaxel caused dramatic human epidermal growth factor receptor (HER)-1/-2 degradation and downstream Akt/Erk inhibition in HER-2 expressing MDA-231 cells. These in vitro findings provide confidence in support of further development of C6 ceramide as an adjunct of docetaxel for the treatment of the metastatic breast cancer. PMID:25576381

Yang, Lan; Zheng, Li-Yun; Tian, Ye; Zhang, Zhi-Qing; Dong, Wan-Li; Wang, Xu-Fen; Zhang, Xiao-Ying; Cao, Cong

2015-03-01

188

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...

189

Gametogony of Sarcocystis sp. in Cell Culture  

Microsoft Academic Search

Sexual stages and cystlike bodies of Sarcocystis sp., a protozoan parasite found in muscles of reptiles, birds, and mammals, including man, developed in cell culture. Motile organisms, obtained from leg muscles of wild grackles, were inoculated into cell line cultures of embryonic bovine kidney. Mature micro- and macrogametes and the cystlike forms were found 30 and 42 hours after inoculation,

Ronald Fayer

1972-01-01

190

Measles virus: study of induced transfer of biological properties. II. Fluorescence studies on rescue of neurotropic strain in cell culture.  

PubMed

HeLa cells are non-permissive for the neurotropic suckling mouse strain of measles virus (MMV), but are permissive for cell-adapted Edmonston strain of measles virus (EDm). Fluorescence and electron microscopy demonstrated no membrane fluorescence and no membrane associated viral components, as well as characteristic lack of nuclear antigen after MMV infection of HeLa cells. This appearance differs markedly from the membrane, cytoplasmic and nuclear fluorescence after Edm infection of HeLa cells. Fluorescence microscopy demonstrates fusion of the two dissimilar syncytia after mixed infection. This suggests Edm envelopment of MMV nucleocapsids may be the means of MMV rescue in this system. PMID:788682

Weil, M L; Imagawa, D T

1976-01-01

191

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

192

Horizontally rotated cell culture system with a coaxial tubular oxygenator  

NASA Technical Reports Server (NTRS)

The present invention relates to a horizontally rotating bioreactor useful for carrying out cell and tissue culture. For processing of mammalian cells, the system is sterilized and fresh fluid medium, microcarrier beads, and cells are admitted to completely fill the cell culture vessel. An oxygen containing gas is admitted to the interior of the permeable membrane which prevents air bubbles from being introduced into the medium. The cylinder is rotated at a low speed within an incubator so that the circular motion of the fluid medium uniformly suspends the microbeads throughout the cylinder during the cell growth period. The unique design of this cell and tissue culture device was initially driven by two requirements imposed by its intended use for feasibility studies for three dimensional culture of living cells and tissues in space by JSC. They were compatible with microgravity and simulation of microgravity in one G. The vessels are designed to approximate the extremely quiescent low shear environment obtainable in space.

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

1991-01-01

193

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

194

Uptake and washout of borocaptate sodium and borono-phenylalanine in cultured melanoma cells: a multi-nuclear NMR study.  

PubMed

The cellular uptake and washout of the two principal boron neutron capture therapy (BNCT) agents, borocaptate sodium (BSH) and borono-phenylalanine (BPA), were monitored on-line, noninvasively, using nuclear magnetic resonance (NMR) spectroscopy. The uptake and washout of inorganic borate (B(i)) was also followed for comparison. M2R mouse melanoma cells grown on polystyrene microspheres were perfused inside the NMR sample tube. (11)B NMR was used to detect the presence of B(i), BSH and BPA, and (19)F NMR was applied to detect fluorinated BPA ((19)F-BPA). The results revealed chemical modifications of BSH due to spontaneous formation of the borocaptate dimer, BSSB, in the culture medium. BPA readily formed a complex with glucose contained in the culture medium but was also converted in the cells to a yet unidentified compound in a reaction that probably involves the hydrolysis of BPA and the release of B(i). The cellular accumulation ratio for BPA was significantly higher than 1 and was also significantly higher than that for BSH. On the other hand, the cellular retention time observed for BSH was much longer than for BPA, indicating a strong trapping of BSH in cells. PMID:10856971

Panov, V; Salomon, Y; Kabalka, G W; Bendel, P

2000-07-01

195

Mammalian cell cultures for biologics manufacturing.  

PubMed

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

Kantardjieff, Anne; Zhou, Weichang

2014-01-01

196

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

197

Longterm cultures of sheep thyroid cells.  

PubMed

A thyroid tissue culture system has been established in which follicular morphology can be preserved for at least 30 days. The system is highly dependent on whether or not the medium supporting the cells is changed during the culture period. The high levels of TSH (40 mU/ml) normally used in thyroid culture systems enhance follicular morphology but are not a prerequisite for differentiation. In the absence of medium changes follicular morphology improves for up to 20 days after initiation of the culture. Thereafter, the cells die unless the medium is changed. If differentiation is to be preserved after 20 days the medium into which the cells are transferred must be "conditioned" by preincubation with thryoid cultures. Regular medium changes into fresh medium causes the cultures to lose their differentiated characteristics and revert to a conventional monolayer. The capacity of these cultures to trap iodide has been quantified using a new method. The method is based on comparison of the 125I--iodide retained by thyroid cells with that retained in a undifferentiated established cell line (CHO--K1). The results demonstrated that trapping can be preserved for at least 20 days in cells cultured in the presence of TSH, provided the medium is not changed; and that under appropriate conditions the cells can trap iodide even in the absence of TSH stimulation. The extent to which the above cultures proliferate is also investigated. At the relatively high innoculation of cells used in primary cultures little proliferation takes place even when the cells are stimulated by TSH. However, regular medium changed induce some growth. In the absence of medium changes the cells die after 15-20 days. Those grown in the presence of TSH live slightly longer than those grown in its absence. Subculturing thyroid cells and innoculating them at low densities in Petri dishes leads to substantial cell proliferation whether or not TSH is present. The doubling time of cells in these cultures is the order of 1 to 2 days. The population resulting from this growth exhibit both epithelial and fibroblast like morphology although the former predominates. When cells from primary thryoid cultures are reseeded at very low concentrations (approximately 10(3) cells/Petri dish) about 3-10 per cent of the population give rise to viable macroscopic clones. PMID:6996408

O'Connor, M K; Malone, J F; Cullen, M J

1980-01-01

198

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

199

High-power acoustic insult to living cultured cells as studied by high-frequency scanning acoustic microscopy  

NASA Astrophysics Data System (ADS)

A plurality of articles discussing combined effects of acoustic high-pressure (mechanical factor) and heat (thermal factor) caused by acoustic vibration on biological tissues and cells has been published. Herein, we contribute the preliminary results describing the behavior of living human skin cells when separately applying shock waves and thermal insult to them. First, we gradually increased temperature of a culturing medium from 37.5 to 52 degree(s)C using the heat plate with temperature controller, and carried out in-situ observation of the cells grown on a substrate via the medium using a scanning acoustic microscope. Second, we provided the pressure using high power ultrasonic pulses generated by a laser induced ultrasonic shock wave system to the cells, wherein the pressure caused by the pulses was measured by a hydrophone, and wherein temperature was monitored by thermocouples. The cells were observed just after giving the impact. The difference between phenomena indicating cellular insult and injury (e.g., shrinkage or lift-off) were clearly visualized by the scanning acoustic microscope with frequency at 1.0 GHz.

Miyasaka, Chiaki; Tittmann, Bernhard R.

2002-06-01

200

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

201

Microassay of decarboxylation reactions in cultured cells.  

PubMed

The currently described methods for determination of decarboxylation reaction rates in cultured cells require large quantities of cells and often involve cell manipulation prior to assay. We describe a simple microassay for the rapid measurement of various decarboxylation reaction rates in intact cultured cells. The assay is based on the traditional measurement of 14CO2 generated from 14C-labeled substrates. Key to the method is a novel modification of the standard petri dish. Pyruvate dehydrogenase, branched chain alpha-ketoacid dehydrogenase, alpha-ketoglutarate dehydrogenase, and ornithine decarboxylase activities were determined in adult cardiomyocyte cultures containing only 0.1-0.5 mg of protein per culture dish. Efficiency of 14CO2 collection ranged between 94 and 100%. Pharmacological enhancement or inhibition of pyruvate dehydrogenase activity was easily detected in the culture system. This new method simplifies the measurement of various decarboxylation reaction rates in cultured cells and allows rapid, reproducible measurements to be made on small numbers of cells without perturbation of the culture conditions or the cells themselves. PMID:8238897

Bartos, D; Vlessis, A A; Muller, P; Mela-Riker, L; Trunkey, D D

1993-09-01

202

Skeletal muscle satellite cells cultured in simulated microgravity.  

PubMed

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

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

1997-05-01

203

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

204

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

205

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

206

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

207

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

208

Development of a disposable three-compartment micro-cell culture device for toxicokinetic study in humans and its preliminary evaluation  

Microsoft Academic Search

For animal-free in vitro prediction of systemic toxicity in humans, one possible approach is multi- compartmental micro-cell culture device (on-chip human) where important organs-derived cells are simultaneously cultured in a physiologically-relevant perfusion circuit using microfluidic technologies. We newly developed a disposable-type three-compartment micro-cell culture based on polydimethylsiloxane (PDMS) and small magnetic stirrer-based pumps. By changing several valves on the device,

Hidenari Nakayama; Hiroshi Kimura; Kikuo Komori; Teruo Fujii; Yasuyuki Sakai

209

Stromal cells from human long-term marrow cultures, but not cultured marrow fibroblasts, phagocytose horse serum constituents: studies with a monoclonal antibody that reacts with a species-specific epitope common to multiple horse serum proteins.  

PubMed

This report describes an IgG1 mouse monoclonal antibody derived after immunization of mice with washed stromal cells from human, long-term bone marrow cultures. The antigen recognized by the antibody (BMS-1) is a carbohydrate-containing prosthetic group that is common to and specific for multiple horse serum proteins. These proteins are avidly ingested by stromal cells and concentrated in endocytic vesicles. Cultured smooth muscle cells took up the horse proteins in a similar manner to marrow stromal cells while cultured marrow fibroblasts, endothelial cells, and hepatoma cells did not. These data indicate that marrow stromal cells specifically accumulate horse serum proteins which might partially explain the horse serum requirement for long-term marrow culture maintenance. The data also suggest further similarities between marrow stromal and smooth muscle cells and additional differences between marrow fibroblasts and marrow stromal cells. PMID:3780891

Charbord, P; Tippens, D; Wight, T S; Gown, A M; Singer, J W

1987-01-01

210

Functional activity of mitochondria in cultured neural precursor cells.  

PubMed

We studied mitochondrial transmembrane potential of neural precursor cells forming neurospheres in culture. Uneven energization of mitochondria in neurosphere cells was detected. Heterogeneity of cells by the mitochondrial potential increased with neurosphere enlargement during culturing. Decrease in the mitochondrial potential in the central cells in large spheres, presumably caused by insufficient diffusion of oxygen and nutrients, can provoke their damage and death. Population of cells with high mitochondrial potential responded to addition of the nuclear dye by a decrease in mitochondrial potential, which can indicate functioning of ABCG2 complex in these cells, characteristic of undifferentiated stem cells. These data will help to create optimum conditions for culturing of neural stem cells for the maintenance of their maximum functional and proliferative activity. PMID:16929986

Plotnikov, E Yu; Marei, M V; Podgornyi, O V; Aleksandrova, M A; Zorov, D B; Sukhikh, G T

2006-01-01

211

Vitamin A Modifies the Glycopeptide Composition of Cultured Sertoli Cells  

Microsoft Academic Search

Sertoli cells obtained from prepubertal rat testes were cultured in the presence or absence of retinol. Incorpora- tion of monosaccharides and glycopeptide composition of the cells were studied under two experimental condi- tions. The results indicate that retinol increases the amount of mannose and glucosamine incorporated into cellular glycoconjugates. The labeled glycopeptides obtained from control and retinol-treated cells were separated

M. GALDIERI; L. NISTICO

1986-01-01

212

Endothelial cell culture on dacron fabrics of different configurations.  

PubMed

The growth of tissue-cultured aortic endothelial cells from the calf using 12 different configurations of Dacron polyester (U.S. Catheter and Instrument Co.) as substrates was studied. Scanning electron microscopy showed maximum cell coverage on tightly knit configurations, whereas loose knits and velours did not support cell growth. PMID:150420

Eskin, S G; Trevino, L; Chimoskey, J E

1978-07-01

213

Extraction parameters for metabolomics from cultured cells.  

PubMed

The successful extraction of metabolites is a critical step in metabolite profiling. By optimizing metabolite extraction, the range and quantitative capacity of metabolomics studies can be improved. We considered eight separate extraction protocols for the preparation of a metabolite extract from cultured mammalian cells. Parameters considered included temperature, pH, and cell washing before extraction. The effects on metabolite recovery were studied using a liquid chromatography high-resolution mass spectrometry (LC-HRMS) platform that measures metabolites of diverse chemical classes, including amino acids, lipids, and sugar derivatives. The temperature considered during the extraction or the presence of formic acid, a commonly used additive, was shown to have minimal effects on the measured ion intensities of metabolites. However, washing of samples before metabolite extraction, whether with water or phosphate-buffered saline, exhibited dramatic effects on measured intensities of both intracellular and extracellular metabolites. Together, these findings present a systematic assessment of extraction conditions for metabolite profiling. PMID:25613493

Ser, Zheng; Liu, Xiaojing; Tang, Ngoc Nu; Locasale, Jason W

2015-04-15

214

Development of a micro-scale perfusion 3D cell culture biochip with an incorporated electrical impedance measurement scheme for the quantification of cell number in a 3D cell culture construct  

Microsoft Academic Search

This article reports a perfusion 3D cell culture biochip with an incorporated electrical impedance measurement-based scheme\\u000a for the quantification of cell number in the 3D cell culture construct. The biochip consists of culture chamber and fluidic\\u000a channel for perfusion 3D cell culture followed by assembling the electrodes for impedance measurement. In this study, breast\\u000a cancer cell line culture was performed

Kin Fong Lei; Min-Hsien Wu; Pei-You Liao; Yan-Ming Chen; Tung-Ming Pan

215

Bioprocessing technology for plant cell suspension cultures  

Microsoft Academic Search

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

Wei wen Su

1995-01-01

216

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

217

Co-Culture of ? TC-6 Cells and ? TC-1 Cells: Morphology and Function  

PubMed Central

Background In vitro experiments using only ?-cell lines instead of islets are limited because pancreatic islets are composed of four different types of endocrine cells. Several recent studies have focused on cellular interactions among these cell types, especially ?- and ?-cells. Because islet isolation needs time and experience, we tested a simple co-culture system with ?- and ?-cells. Their morphology and function were assessed by comparison to each single cell culture and pancreatic islets. Methods ? TC-6 cells and ? TC-1 cells were maintained in Dulbecco's Minimal Essential Medium containing 5 mM glucose and 10% fetal bovine serum. Cells were mixed at a 1:1 ratio (5×105) in 6-well plates and cultured for 24, 48, and 72 hours. After culture, cells were used for insulin and glucagon immunoassays and tested for glucose-stimulated insulin secretion (GSIS). Results ? TC-6 and ? TC-1 cells became condensed by 24 hours and were more strongly compacted after 48 hours. ? TC-1 cells showed both ?-? and ?-? cell contacts. GSIS increased with increasing glucose concentration in co-cultured cells, which showed lower secreted insulin levels than ? TC-1 cells alone. The increase in the secreted insulin/insulin content ratio was significantly lower for co-cultured cells than for ?-cells alone (P=0.04). Compared to islets, the ?-/?-cell co-culture showed a higher ratio of GSIS to insulin content, but the difference was not statistically significant (P=0.09). Conclusion ? TC-6 and ? TC-1 cells in the co-culture system showed cell-to-cell contacts and a similar stimulated insulin secretion pattern to islets. The co-culture system may be used to better mimic pancreatic islets in in vitro assessments. PMID:25325280

Kim, Sung Man; Lee, Eun Ju; Jung, Hye Sook; Han, Na; Kim, You Jeong; Kim, Tae Kyoon; Kim, Tae Nyun; Kwon, Min Jeong; Lee, Soon Hee; Park, Jeong Hyun; Rhee, Byoung Doo

2015-01-01

218

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

PubMed

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

Vijaya, K; Ananthan, S; Nalini, R

1995-12-01

219

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

220

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

221

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

PubMed

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

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

2015-03-01

222

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

223

Growth of melanocytes in human epidermal cell cultures  

SciTech Connect

Epidermal cell cultures were grown in keratinocyte-conditioned medium for use as burn wound grafts; the melanocyte composition of the grafts was studied under a variety of conditions. Melanocytes were identified by immunohistochemistry based on a monoclonal antibody (MEL-5) that has previously been shown to react specifically with melanocytes. During the first 7 days of growth in primary culture, the total number of melanocytes in the epidermal cultures decreased to 10% of the number present in normal skin. Beginning on day 2 of culture, bipolar melanocytes were present at a mean cell density of 116 +/- 2/mm2; the keratinocyte to melanocyte ratio was preserved during further primary culture and through three subpassages. Moreover, exposure of cultures to mild UVB irradiation stimulated the melanocytes to proliferate, suggesting that the melanocytes growing in culture maintained their responsiveness to external stimuli. When the sheets of cultured cells were enzymatically detached from the plastic culture flasks before grafting, melanocytes remained in the basal layer of cells as part of the graft applied to the patient.

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

1990-08-01

224

Characterization of human fungiform papillae cells in culture.  

PubMed

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

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

2011-09-01

225

Spheroid cultures promote the stemness of corneal stromal cells.  

PubMed

Several culture methods generated spheroids of rabbit and mouse corneal stromal cells (CSCs) in vitro. In this study, rabbit CSC spheroids were positively expressed the mesenchymal and stem cell phenotypes, which contained immunopositive for vimentin (a mesenchymal cell marker) and CD34 (a stem cell marker), as well as mRNA expression of nestin (a neural stem cell marker) and Nanog (a stem cell marker), in suspension or adherent cultures that were induced by methylcellulose, a rotary cell culture system (RCCS) or reprogramming proteins and VPA. Mouse CSCs showed poor growth and hardly formed spheroids after treatment with methylcellulose or reprogramming proteins and VPA. Our work has laid a promising foundation to elucidate CSCs and the further use of CSC spheroids for reprogramming, bioprinting and tissue engineering. PMID:25497331

Li, Hongyang; Dai, Ying; Shu, Jianchang; Yu, Rongjie; Guo, Yonglong; Chen, Jiansu

2015-02-01

226

Hypergravity signal transduction and gene expression in cultured mammalian cells  

NASA Technical Reports Server (NTRS)

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

Kumei, Y.; Whitson, P. A.

1994-01-01

227

Microfabricated polymeric vessel mimetics for 3-D cancer cell culture  

PubMed Central

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

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

2013-01-01

228

Isolation and culture of primary equine tracheal epithelial cells.  

PubMed

Culture of airway epithelial cells is a useful model to investigate physiology of airway epithelia and airway disease mechanisms. In vitro models of airway epithelial cells are established for various species. However, earlier published method for isolation and culture of equine tracheal epithelial cells requires significant improvements. In this report, the development of a procedure for efficient isolation, characterization, culture, and passage of primary equine tracheal epithelial cells are described. Epithelial cells were isolated from adult equine trachea by exposing and stripping the mucosal epithelium from the adjacent connective tissue and smooth muscle. The tissue was minced and dissociated enzymatically using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution for 2 h at 37 degrees C. Cells were collected by sieving and centrifugation, and contaminating fibroblasts were removed by differential adhesion. This procedure resulted in a typical yield of 1 x 10(7) cytokeratin-positive epithelial cells per gram tracheal lining tissue. Viability was 95% by trypan blue exclusion and isolates contained approximately 94% cytokeratin-positive cells of epithelial origin. Cells seeded at a density of 6.9 x 10(4) cells/cm2 in serum-free airway epithelial cell growth medium formed monolayers near confluency within a week. Confluent cells were dissociated using dispase II and first passages (P1) and second passages (P2) were successfully established in serum-free medium. Collagen coating of tissue culture flask was not required for cell adhesion, and cultures could be maintained at the level of P2 over 30 d. In the present study, we could establish a high-yield protocol for isolation and culture of equine tracheal epithelial cells that can serve for in vitro/ex vivo studies on the (patho-)physiology of equine airway disease as well as pharmacological and toxicological targets relevant to airway diseases. PMID:18594938

Shibeshi, Workineh; Abraham, Getu; Kneuer, Carsten; Ellenberger, Christin; Seeger, Johannes; Schoon, Heinz-Adolf; Ungemach, Fritz R

2008-01-01

229

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

PubMed

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

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

2013-12-01

230

A new bioreactor system for animal cell culture  

E-print Network

A NEW BIOREACTOR SYSTEM FOR ANIMAL CELL CULTURE A Thesis by PATRICK BERNARD MONAHAN Submitted to the Office of Graduate Studies of Texas ASM University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE August... 1989 Major Subject: Chemical Engineering A NEW BIOREACTOR SYSTEM FOR ANIMAL CELL CULTURE A Thesis by PATRICK BERNARD MONAHAN Approved as to style and content by: le C. Engler (Member) D. Hanson (Member) R. Flumer elt (Head of Department...

Monahan, Patrick Bernard

1989-01-01

231

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

232

Lipoprotein binding to cultured human hepatoma cells.  

PubMed Central

Binding of various 125I-lipoproteins to hepatic receptors was studied on cultured human hepatoma cells (Hep G2). Chylomicrons, isolated from a chylothorax, chylomicron remnants, hypertriglyceridemic very low-density lipoproteins, normotriglyceridemic very low-density lipoproteins (NTG-VLDL), their remnants, low-density lipoproteins (LDL), and HDL-E (an Apo E-rich high-density lipoprotein isolated from the plasma of a patient with primary biliary cirrhosis) were bound by high-affinity receptors. Chylomicron remnants and HDL-E were bound with the highest affinity. The results, obtained from competitive binding experiments, are consistent with the existence of two distinct receptors on Hep G2 cells: (a) a remnant receptor capable of high-affinity binding of triglyceride-rich lipoproteins and HDL-E, but not of Apo E free LDL, and (b) a LDL receptor capable of high-affinity binding of LDL, NTG-VLDL, and HDL-E. Specific binding of Apo E-free LDL was completely abolished in the presence of 3 mM EDTA, indicating that binding to the LDL receptor is calcium dependent. Specific binding of chylomicron remnants was not inhibited by the presence of even 10 mM EDTA. Preincubation of the Hep G2 cells in lipoprotein-containing medium resulted in complete suppression of LDL receptors but did not affect the remnant receptors. Hep G2 cells seem to be a suitable model for the study of hepatic receptors for lipoprotein in man. Images PMID:3038957

Krempler, F; Kostner, G M; Friedl, W; Paulweber, B; Bauer, H; Sandhofer, F

1987-01-01

233

Culture, Immortalization, and Characterization of Human Meibomian Gland Epithelial Cells  

PubMed Central

Purpose. Meibomian gland epithelial cells are essential in maintaining the health and integrity of the ocular surface. However, very little is known about their physiological regulation. In this study, the cellular control mechanisms were explored, first to establish a defined culture system for the maintenance of primary epithelial cells from human meibomian glands and, second, to immortalize these cells, thereby developing a preclinical model that could be used to identify factors that regulate cell activity. Methods. Human meibomian glands were removed from lid segments after surgery, enzymatically digested, and dissociated. Isolated epithelial cells were cultured in media with or without serum and/or 3T3 feeder layers. To attempt immortalization, the cells were exposed to retroviral human telomerase reverse transcriptase (hTERT) and/or SV40 large T antigen cDNA vectors, and antibiotic-resistant cells were selected, expanded, and subcultured. Analyses for possible biomarkers, cell proliferation and differentiation, lipid-related enzyme gene expression, and the cellular response to androgen were performed with biochemical, histologic, and molecular biological techniques. Results. It was possible to isolate viable human meibomian gland epithelial cells and to culture them in serum-free medium. These cells proliferated, survived through at least the fifth passage, and contained neutral lipids. Infection with hTERT immortalized these cells, which accumulated neutral lipids during differentiation, expressed multiple genes for lipogenic enzymes, responded to androgen, and continued to proliferate. Conclusions. The results show that human meibomian gland epithelial cells may be isolated, cultured, and immortalized. PMID:20335607

Liu, Shaohui; Hatton, Mark P.; Khandelwal, Payal

2010-01-01

234

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

235

A comparative study of different in vitro lung cell culture systems to assess the most beneficial tool for screening the potential adverse effects of carbon nanotubes.  

PubMed

To determine the potential inhalatory risk posed by carbon nanotubes (CNTs), a tier-based approach beginning with an in vitro assessment must be adopted. The purpose of this study therefore was to compare 4 commonly used in vitro systems of the human lung (human blood monocyte-derived macrophages [MDM] and monocyte-derived dendritic cells [MDDC], 16HBE14o- epithelial cells, and a sophisticated triple cell co-culture model [TCC-C]) via assessment of the biological impact of different CNTs (single-walled CNTs [SWCNTs] and multiwalled CNTs [MWCNTs]) over 24h. No significant cytotoxicity was observed with any of the cell types tested, although a significant (p < .05), dose-dependent increase in tumor necrosis factor (TNF)-? following SWCNT and MWCNT exposure at concentrations up to 0.02mg/ml to MDM, MDDC, and the TCC-C was found. The concentration of TNF-? released by the MDM and MDDC was significantly higher (p < .05) than the TCC-C. Significant increases (p < .05) in interleukin (IL)-8 were also found for both 16HBE14o- epithelial cells and the TCC-C after SWCNTs and MWCNTs exposure up to 0.02mg/ml. The TCC-C, however, elicited a significantly (p < .05) higher IL-8 release than the epithelial cells. The oxidative potential of both SWCNTs and MWCNTs (0.005-0.02mg/ml) measured by reduced glutathione (GSH) content showed a significant difference (p < .05) between each monoculture and the TCC-C. It was concluded that because only the co-culture system could assess each endpoint adequately, that, in comparison with monoculture systems, multicellular systems that take into consideration important cell type-to-cell type interactions could be used as predictive in vitro screening tools for determining the potential deleterious effects associated with CNTs. PMID:24284789

Clift, Martin J D; Endes, Carola; Vanhecke, Dimitri; Wick, Peter; Gehr, Peter; Schins, Roel P F; Petri-Fink, Alke; Rothen-Rutishauser, Barbara

2014-01-01

236

Infrared Spectroscopy of Plant Cell Cultures 1  

PubMed Central

Infrared spectroscopy was used to examine suspension-cultured pear (Pyrus communis L.) and Spartina pectinata cells. Noninvasive measurements were made using internal reflectance sampling. Spectra of actively growing cells exhibited a pronounced absorbance at 2343 reciprocal centimeters. The absorbance peak was identified and verified as CO2 dissolved in water. This peak was absent in nonviable cells. Peak height was directly proportional to percent viability in artificial mixtures of viable and nonviable cells, indicating that the level of intracellular CO2 production could be used as a viability determinant for plant cells. Suspension-cultured cells were slowly cooled to subzero temperatures and analyzed for viability using infrared spectroscopy and tetrazolium staining. Both methods showed similar trends in viability assessment. Infrared spectroscopy could provide a more detailed understanding of cell viability and allow measurement on a noninvasive basis. PMID:16668026

Sowa, Sharon; Towill, Leigh E.

1991-01-01

237

Ultrastructural Studies of Spontaneous in Vitro Transformation of Cultured Marrow Monocyte-Macrophage Cells from a Patient with Congenital Hypoplastic Anemia  

Microsoft Academic Search

The CM-S cell line was established from the bone marrow of a patient suffering from congenital hypoplastic anemia (syndrome of Diamond-Blackfan). The cells grew in suspension in liquid culture and were dependent for their continuous replication in vitro on growth factors produced by the same cells seeded at high density. Initially, undifferentiated blasts, immature myeloid, megakaryocytic and, rarely, erythroid cells

Yula Sambuy; Luigi G. Spagnoli; Eliana Vigneti; Roberto P. Revoltella

1985-01-01

238

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

239

Toxicity of some dental cements in a cell culture system.  

PubMed

A cell culture method has been used to study the effect of zinc phosphate cement (De Trey's Zinc Zement Improved), zinc silicophosphate cement (Fluoro-Thin) and polycarboxylate cement (Durelon) on animal cells. Disks (20 x 1 mm) of the materials were placed in the center of plastic Petri dishes and subsequently incubated with human epithelial cells. Cell multiplication, medium pH and the release of cement constituents were measured. All three cements exhibited a cytotoxic effect, which was most pronounced in the cultures with zinc silicophosphate cement and polycarboxylate cement. The results also indicated that cell growth on the surface of the disks is a more sensitive indicator of cytotoxicity than cell growth around the disks. pH of the medium was only slightly affected in cultures with polycarboxylate cement, whereas a decrease was found in cultures with zinc phosphate cement and especially with zinc silicophosphate cement. A rapid release of phosphate was found in cultures with zinc silicophosphate cement. Zinc was released into the medium from disks of zinc phosphate cement, zinc silicophosphate cement and polycarboxylate cement--exceeding the toxicity level for the present cell line after 24 h. In cultures with zinc silicophosphate cement and polycarboxylate cement the release of fluoride reached toxic levels within the same time interval. PMID:22926

Leirskar, J; Helgeland, K

1977-09-01

240

Pitfalls in cell culture work with xanthohumol.  

PubMed

Xanthohumol, the most abundant prenylated chalcone in hop (Humulus lupulus L.) cones, is well known to exert several promising pharmacological activities in vitro and in vivo. Among these, the chemopreventive, anti-inflammatory and anti-cancer effects are probably the most interesting. As xanthohumol is hardly soluble in water and able to undergo conversion to isoxanthohumol we determined several handling characteristics for cell culture work with this compound. Recovery experiments revealed that working with xanthohumol under cell culture conditions requires a minimal amount of 10% FCS to increase its solubility to reasonable concentrations (-50-75 micromol/l) for pharmacological in vitro tests. Additionally, more than 50% of xanthohumol can be absorbed to various plastic materials routinely used in the cell culture using FCS concentrations below 10%. In contrast, experiments using fluorescence microscopy in living cells revealed that detection of cellular intake of xanthohumol is hampered by concentrations above 1% FCS. PMID:22393838

Motyl, M; Kraus, B; Heilmann, J

2012-01-01

241

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

242

High density cell culture by membrane-based cell recycle.  

PubMed

Enhancement of productivity of a bioprocess necessitates continuous operation of bioreactors with high biomass concentrations than are possible in conventional batch, fedbatch or continuous modes of culture. Membrane-based cell recycle has been effectively used to maintain high cell concentrations in bioreactors. This review compares membranebased cell recycle operation with other such high density cell culture systems as immobilized cell reactors and reactors with cell recycle by centrifugation or gravity sedimentation. A theoretical of production of primary and secondary metabolites in membrane-based recycle systems is presented. Operation of this type of system is discussed with examples from aerobic and anaerobic fermentations. PMID:14548467

Chang, H N; Yoo, I K; Kim, B S

1994-01-01

243

The consensus mechanics of cultured mammalian cells  

PubMed Central

Although understanding cells' responses to mechanical stimuli is seen as increasingly important for understanding cell biology, how to best measure, interpret, and model cells' mechanical properties remains unclear. We determine the frequency-dependent shear modulus of cultured mammalian cells by using four different methods, both unique and well established. This approach clarifies the effects of cytoskeletal heterogeneity, ATP-dependent processes, and cell regional variations on the interpretation of such measurements. Our results clearly indicate two qualitatively similar, but distinct, mechanical responses, corresponding to the cortical and intracellular networks, each having an unusual, weak power-law form at low frequency. The two frequency-dependent responses we observe are remarkably similar to those reported for a variety of cultured mammalian cells measured with different techniques, suggesting it is a useful consensus description. Finally, we discuss possible physical explanations for the observed mechanical response. PMID:16793927

Hoffman, Brenton D.; Massiera, Gladys; Van Citters, Kathleen M.; Crocker, John C.

2006-01-01

244

Human Primary Lung Endothelial Cells in Culture  

PubMed Central

Pulmonary endothelial functions are critical to maintain the low pressure of the pulmonary circulation and effective diffusion capacity of the lung. To investigate pulmonary endothelial cell biology in healthy or diseased lungs, we developed methods to harvest and culture pure populations of primary pulmonary arterial endothelial cells and microvascular endothelial cells from human lung explanted at time of transplantation or from donor lungs not used in transplantation. The purity and characteristics of cultured endothelial cells is ascertained by morphologic criteria using phase contrast and electron microscopy; phenotypic expression profile for endothelial specific proteins such as endothelial nitric oxide synthase, platelet/endothelial cell adhesion molecule, and von Willbrand factor; and endothelial function assays such as Dil-acetylated low-density lipoprotein uptake and tube formation. This detailed method provides researchers with the ability to establish cells for molecular, genetic, and biochemical investigation of human pulmonary vascular diseases. PMID:22427538

Xu, Weiling; Mavrakis, Lori; Aldred, Micheala A.; Asosingh, Kewal; Erzurum, Serpil C.

2012-01-01

245

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

246

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

PubMed Central

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

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

2013-01-01

247

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

248

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

249

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

250

Buffer Combinations for Mammalian Cell Culture  

Microsoft Academic Search

The growth and metabolism of cultured mammalian cells are markedly affected by the pH variation in ordinary bicarbonate-buffered media(pH 8.0 to 6.9). Those pH swings can be reduced and the pH of the culture can be stabilized as desired in the range pH 6.4 to 8.3 by appropriate combinations of two or three organic buffers, each at 10 to 15

Harry Eagle

1971-01-01

251

Formation of Odontoblast-Like Cells from Cultured Human Dental Pulp Cells on Dentin In Vitro  

Microsoft Academic Search

Recent characterization of human dental pulp stem cells has shed new light on the understanding of the odontoblastic lineage. The purpose of the study was to characterize human adult dental pulp cells isolated and cultured in vitro and to examine the cell differentiation potential grown on dentin. We observed that some pulp cells isolated with an enzyme-digestion approach proliferated at

George T.-J. Huang; Kristina Shagramanova; Selina W. Chan

2006-01-01

252

Foreign language education and cultural studies  

Microsoft Academic Search

Some basic definitional and theoretical issues concerning the role of cultural studies in foreign language teaching are discussed. The paper is critical of the tourist?consumer model of intercultural contact and of the agnostic or minimalist approaches to cultural studies often associated with allegedly culture?free classifications of language functions. Assumptions of some existing approaches to cultural studies are examined. Four key

Michael Byram

1988-01-01

253

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

254

Micro3D cell culture devices for single cell analysis  

Microsoft Academic Search

In this paper we present two novel devices for the culturing of single cells or small clusters of cells in an array format. The focus of these devices is to create local 3-dimensional microenvironments for the cells which mimic more closely the adhesion state in an in vivo situation. The devices were manufactured using fast processes and cost-effective materials like

M. R. Dusseillerl; D. Schlaepfer; A. Ferrari; R. Kroschewski; M. Textorl

2005-01-01

255

Simulation of ischemic reperfusion in endothelial cell culture increases apoptosis.  

PubMed

The endothelial layer of the myocardial vasculature serves as an important protective barrier between blood and myocardium. Ischemic reperfusion (I/R) of the endothelium has been shown to initiate a series of events that leads to ischemic reperfusion injury in the heart. At the onset of ischemic reperfusion, endothelial cells initiate apoptosis, a process whereby the cells self-destruct. Ischemic reperfusion was simulated to study its effects on the induction of apoptosis in cultured human endothelial cells (ECV 304). In addition, the cells were treated with nitric oxide (NO) to test its effect on induction of apoptosis. To mimic hypoxia, four ECV 304 cultures were placed in a medium that had been bubbled with pure nitrogen gas for 24 hours. A continuous flow of nitrogen gas was applied to the culture flasks during the course of the 2-hour ischemic period. After 2 hours, the nitrogen was removed from the hypoxic cultures to simulate reperfusion. Exposure to NO was achieved through the NO-donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP) at 100 microM, Cell cultures were exposed to hypoxia only, hypoxia and SNAP, and SNAP only. One positive control was established by exposure to staurosporine. A second positive control was established by exposure to a 30-min heat treatment at 43 degrees C. Two cultures were left untreated to serve as negative controls. All cell cultures were incubated for 4 hours. Apoptosis was detected by the binding of annexin V-fluorescein isothiocyanate (annexin V-FITC). In addition, morphologic changes detected by electron microscopy were used. Apoptosis increased in all treated cultures, excluding SNAP only treated cells. It was concluded that I/R may lead to induction of apoptosis. PMID:11680731

Holleyman, C; Larson, D; Hunter, K

2001-09-01

256

Cell wall proteomics of sugarcane cell suspension cultures.  

PubMed

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

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

2014-03-01

257

Apolipoprotein E Regulates Primary Cultured Human Mesangial Cell Proliferation  

Microsoft Academic Search

Background: The role of apolipoprotein (apo) E in kidney disease is still unclear. Animal studies have been performed, but it is doubtful if the conclusions are applicable to human beings. The objective of this study was to determine how apo E acts on human kidneys using primary cultured normal human mesangial cells (NHMCs) rather than animals used in previous studies.

Yixiang Zhang; Yuichiro Yasumoto; Toru Ikeda; Syozo Takenouchi; Atsushi Sogabe; Tsuyoshi Nosaki; Xiaofang Che; Chunlei Zheng; Misako Haraguchi; Shin-ichi Akiyama; Hirohito Tsubouchi

2006-01-01

258

Social Studies: Selected Cultures. Grade 6.  

ERIC Educational Resources Information Center

This revised teachers guide attempts to facilitate the study of selected cultures through a conceptual approach and multimedia instruction in a spiral curriculum. There are six units: 1) Cultures and Archaeology --cultural factors, cultural study, artifacts, fossils, archaeological sites and evidence; 2) Food Gathering Complex --life styles,…

Taylor, Marshall R.

259

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

260

Phenotypic changes in satellite glial cells in cultured trigeminal ganglia.  

PubMed

Satellite glial cells (SGCs) are specialized cells that form a tight sheath around neurons in sensory ganglia. In recent years, there is increasing interest in SGCs and they have been studied in both intact ganglia and in tissue culture. Here we studied phenotypic changes in SGCs in cultured trigeminal ganglia from adult mice, containing both neurons and SGCs, using phase optics, immunohistochemistry and time-lapse photography. Cultures were followed for up to 14 days. After isolation virtually every sensory neuron is ensheathed by SGCs, as in the intact ganglia. After one day in culture, SGCs begin to migrate away from their parent neurons, but in most cases the neurons still retain an intact glial cover. At later times in culture, there is a massive migration of SGCs away from the neurons and they undergo clear morphological changes, and at 7 days they become spindle-shaped. At one day in culture SGCs express the glial marker glutamine synthetase, and also the purinergic receptor P2X7. From day 2 in culture the glutamine synthetase expression is greatly diminished, whereas that of P2X7 is largely unchanged. We conclude that SGCs retain most of their characteristics for about 24 h after culturing, but undergo major phenotypic changes at later times. PMID:22032231

Belzer, Vitali; Shraer, Nathanael; Hanani, Menachem

2010-11-01

261

Prevention and Detection of Mycoplasma Contamination in Cell Culture  

PubMed Central

One of the main problems in cell culture is mycoplasma infection. It can extensively affect cell physiology and metabolism. As the applications of cell culture increase in research, industrial production and cell therapy, more concerns about mycoplasma contamination and detection will arise. This review will provide valuable information about: 1. the ways in which cells are contaminated and the frequency and source of mycoplasma species in cell culture; 2. the ways to prevent mycoplasma contamination in cell culture; 3. the importance of mycoplasma tests in cell culture; 4. different methods to identify mycoplasma contamination; 5. the consequences of mycoplasma contamination in cell culture and 6. available methods to eliminate mycoplasma contamination. Awareness about the sources of mycoplasma and pursuing aseptic techniques in cell culture along with reliable detection methods of mycoplasma contamination can provide an appropriate situation to prevent mycoplasma contamination in cell culture. PMID:23508237

Nikfarjam, Laleh; Farzaneh, Parvaneh

2012-01-01

262

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

263

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

PubMed

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

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

2013-06-01

264

Dynamics of limiting cell wall porosity in plant suspension cultures  

Microsoft Academic Search

.   Changes in the limiting porosity of cell walls, i.e. the size limit for permeation of neutral molecules through the wall,\\u000a were studied in several higher-plant cell-suspension cultures. For this purpose, samples of biomass fixed at different cultivation\\u000a times were investigated using a method based on size-exclusion chromatography of polydisperse dextrans before and after equilibration\\u000a with the extracted cell clusters.

Christine Titel; Holger Woehlecke; Izzidin Afifi; Rudolf Ehwald

1997-01-01

265

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.

266

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

267

Transcriptome data analysis for cell culture processes.  

PubMed

In the past decade, DNA microarrays have fundamentally changed the way we study complex biological systems. By measuring the expression levels of thousands of transcripts, the paradigm of studying organisms has shifted from focusing on the local phenomena of a few genes to surveying the whole genome. DNA microarrays are used in a variety of ways, from simple comparisons between two samples to more intricate time-series studies. With the large number of genes being studied, the dimensionality of the problem is inevitably high. The analysis of microarray data thus requires specific approaches. In the case of time-series microarray studies, data analysis is further complicated by the correlation between successive time points in a series.In this review, we survey the methodologies used in the analysis of static and time-series microarray data, covering data pre-processing, identification of differentially expressed genes, profile pattern recognition, pathway analysis, and network reconstruction. When available, examples of their use in mammalian cell cultures are presented. PMID:22194060

Castro-Melchor, Marlene; Le, Huong; Hu, Wei-Shou

2012-01-01

268

Date Palm Cell and Protoplast Culture  

Microsoft Academic Search

\\u000a This chapter describes the current status of cell and protoplast cultures in date palm (Phoenix dactylifera L.). Critically important steps toward plant regeneration from recalcitrant date palm protoplasts have been achieved in the\\u000a recent past. Callus regeneration was achieved in commercial cvs. Deglet Noor, Takerboucht, Barhee and Zaghloul. The use of\\u000a feeder layer was the main factor for inducing cell

A. Assani; D. Chabane; H. Shittu; N. Bouguedoura

269

Cytotoxicity effects of amiodarone on cultured cells  

Microsoft Academic Search

Amiodarone is a potent anti-arrhythmic drug used for the treatment of cardiac arrhythmias. Although, the effects of amiodarone are well characterized on post-ischemic heart and cardiomyocytes, its toxicity on extra-cardiac tissues is still poorly understood. To this aim, we have monitored the cytotoxicity effects of this drug on three cultured cell lines including hepatocytes (HepG2), epithelial cells (EAhy 926) and

Emna El Golli-Bennour; Amel Bouslimi; Olfa Zouaoui; Safa Nouira; Abdellatif Achour; Hassen Bacha

270

Metabolic measurements in cell culture and tissue constructs  

NASA Astrophysics Data System (ADS)

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

Rolfe, P.

2008-10-01

271

ANTHOCYANIN (ACN) STABILITY IN CELL CULTURE MEDIA  

Technology Transfer Automated Retrieval System (TEKTRAN)

Anthocyanins (ACNs) are potential oxygen radical scavengers that have coronary vasoactive and vasoprotective properties. Cell or tissue culture systems have been used to examine the bioactivity and mechanisms of action of ACNs on the vascular system. However, due to their unique chemical structure, ...

272

Novel Micropatterned Cardiac Cell Cultures with Realistic Ventricular Microstructure  

PubMed Central

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

Badie, Nima; Bursac, Nenad

2009-01-01

273

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

274

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

275

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

276

HISTOCHEMICAL AND ULTRASTRUCTURAL STUDIES ON HELA CELL CULTURES EXPOSED TO SPINDLE INHIBITORS WiTH SPECIAL REFERENCE TO THE INTERPHASE CELL  

Microsoft Academic Search

SUMMARY Manimahian cells of the HeLa (S3) strain, when exposed to sliindle inhibitors, have been found to undergo several morphologucal transformations during interpha.se as well as during mitosis. Some of these have been studied both histochenuically and ul(rastructurahly. '(he lysosomes, rel)resented by the nuiultivesicular bodies in HeLa cells, form clusters and hieconue circunuferentially disposed instead of occupying thse polarized juxtanuclear

ELLIOTT ROBBINS; NICHOLAS K. GONATAS

277

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

278

Preparation of neuronal co-cultures with single cell precision.  

PubMed

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

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

2014-01-01

279

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

NASA Technical Reports Server (NTRS)

The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European 'Biorack' provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the 'Biorack' facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatant in-flight), injection port, and supernatant collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatant, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground-based investigations simulating the conditions expected in the flight experiment. Several parameters including cell concentration, time between cell loading and activation, and storage temperature on cell survival were examined to characterise cell response and optimise the experiments to be flown aboard the Space Shuttle. Results indicate that the objectives of the experiments could be met with delays up to 5 days between cell loading into the hardware and initial in flight experiment activation, without the need for medium exchange. Experiment hardware of this kind, which is adaptable to a wide range of cell types and can be easily interfaced to different spacecraft facilities, offers the possibility for a wide range of experimenters successfully and easily to utilise future flight opportunities.

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

1998-01-01

280

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

2015-04-01

281

Organellar DNA replication in Nicotiana tabacum cultured cells.  

PubMed

In the diploid vegetative plant cell, the nuclear DNA is present in two copies, whereas the chloroplast and mitochondria genomes are present in a higher and variable copy number. We have studied the replication of the nuclear, chloroplast and mitochondrial DNA in cultured Nicotiana tabacum cells using density and radioactive markers. Essentially all the 10,000 chloroplast genomes in a given cell replicate in one cell cycle as do all the mitochondrial DNA molecules. No measurable level of unreplicated organellar DNA molecules can be detected in these cells. PMID:2102874

Infante, D; Weissbach, A

1990-06-01

282

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

283

Effects of carbon monoxide on cardiac muscle cells in culture  

SciTech Connect

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

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

1988-09-01

284

Antibody inhibition of human cytomegalovirus spread in epithelial cell cultures  

PubMed Central

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

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

2013-01-01

285

Biotransformation of salicylaldehyde to salicin using Varthemia persica cell suspension cultures  

Microsoft Academic Search

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

Gholamreza Asghari; Maryam Mosaeybi

286

Growth and Maintenance of Human Giant-cell Bone Tumors (Osteoclastomas) in Continuous Culture  

Microsoft Academic Search

Tissue culture of six human giant cell tumors of bone was undertaken (one on two occasions). All of them were cultivated successfully. Five cultures have shown outgrowth in about 25 days. In the remaining two cases continuous culture (nine generations) was successfully carried out. From this study the following results were obtained: the giant cells usually persist for about 48

H. Gallardo; Eugenia S. de Lustig; F. Schajowicz

1970-01-01

287

Characterization of cultured multipotent zebrafish neural crest cells.  

PubMed

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

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

2014-02-01

288

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

PubMed Central

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

2014-01-01

289

Gene Delivery Through Cell Culture Substrate Adsorbed DNA Complexes  

PubMed Central

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

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

2008-01-01

290

Culture of Human Endothelial Cells Derived from Umbilical Veins. IDENTIFICATION BY MORPHOLOGIC AND IMMUNOLOGIC CRITERIA  

PubMed Central

Endothelial cells were isolated from freshly obtained human umbilical cords by collagenase digestion of the interior of the umbilical vein. The cells were grown in tissue culture as a homogeneous population for periods up to 5 mo and some lines were subcultured for 10 serial passages. During the logarithmic phase of cell growth, cell-doubling time was 92 h. Light, phase contrast, and scanning electron microscopy demonstrated that cultured human endothelial cells grew as monolayers of closely opposed, polygonal large cells whereas both cultured human fibroblasts and human smooth muscle cells grew as overlapping layers of parallel arrays of slender, spindle-shaped cells. By transmission electron microscopy, cultured endothelial cells were seen to contain cytoplasmic inclusions (Weibel-Palade bodies) characteristic of in situ endothelial cells. These inclusions were also found in endothelial cells lining umbilical veins but were not seen in smooth muscle cells or fibroblasts in culture or in situ. Cultured endothelial cells contained abundant quantities of smooth muscle actomyosin. Cultured endothelial cells also contained ABH antigens appropriate to the tissue donor's blood type; these antigens were not detectable on cultured smooth muscle cells or fibroblasts. These studies demonstrate that it is possible to culture morphologically and immunologically identifiable human endothelial cells for periods up to 5 mo. Images PMID:4355998

Jaffe, Eric A.; Nachman, Ralph L.; Becker, Carl G.; Minick, C. Richard

1973-01-01

291

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

292

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

293

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

294

FEMINISM AND CULTURAL STUDIES IN ASIA  

Microsoft Academic Search

This paper marks significant moments in the trajectory of cultural studies in Asia in relation to feminism. It begins by contrasting the relationship of feminism and cultural studies in Western contexts such as Britain, where feminism disrupted a prior field, to feminism's foundational relationship to cultural studies in many Asian contexts, including India. The paper goes on to speculate on

Tejaswini Niranjana

2007-01-01

295

Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices.  

PubMed

Culture of cells using various microfluidic devices is becoming more common within experimental cell biology. At the same time, a technological radiation of microfluidic cell culture device designs is currently in progress. Ultimately, the utility of microfluidic cell culture will be determined by its capacity to permit new insights into cellular function. Especially insights that would otherwise be difficult or impossible to obtain with macroscopic cell culture in traditional polystyrene dishes, flasks or well-plates. Many decades of heuristic optimization have gone into perfecting conventional cell culture devices and protocols. In comparison, even for the most commonly used microfluidic cell culture devices, such as those fabricated from polydimethylsiloxane (PDMS), collective understanding of the differences in cellular behavior between microfluidic and macroscopic culture is still developing. Moving in vitro culture from macroscopic culture to PDMS based devices can come with unforeseen challenges. Changes in device material, surface coating, cell number per unit surface area or per unit media volume may all affect the outcome of otherwise standard protocols. In this review, we outline some of the advantages and challenges that may accompany a transition from macroscopic to microfluidic cell culture. We focus on decisive factors that distinguish macroscopic from microfluidic cell culture to encourage a reconsideration of how macroscopic cell culture principles might apply to microfluidic cell culture. PMID:25105943

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

2015-01-15

296

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

297

sup 31 P nuclear magnetic resonance study of the metabolic pools of adenosine triphosphate in cultured bovine adrenal medullary chromaffin cells  

Microsoft Academic Search

³¹P NMR was used to resolve and determine the relative quantity and mobility of ATP in the cytosolic and vesicular compartments of isolated adrenomedullary chromaffin cells. The cells were cultured on microcarrier beads and superfused with an oxygenated medium-thereby permitting dense suspensions of viable cells to be maintained in the NMR probe for extended time periods. Under these conditions, distinct

G. R. Painter; E. J. Jr. Diliberto; J. Knoth

1989-01-01

298

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...

299

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

300

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

PubMed

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. PMID:11094818

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

2000-09-01

301

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

302

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

303

A comparative study of the proliferation and osteogenic differentiation of human periodontal ligament cells cultured on ?-TCP ceramics and demineralized bone matrix with or without osteogenic inducers in vitro.  

PubMed

The repair of bone defects that result from periodontal diseases remains a clinical challenge for periodontal therapy. ?-tricalcium phosphate (?-TCP) ceramics are biodegradable inorganic bone substitutes with inorganic components that are similar to those of bone. Demineralized bone matrix (DBM) is an acid-extracted organic matrix derived from bone sources that consists of the collagen and matrix proteins of bone. A few studies have documented the effects of DBM on the proliferation and osteogenic differentiation of human periodontal ligament cells (hPDLCs). The aim of the present study was to investigate the effects of inorganic and organic elements of bone on the proliferation and osteogenic differentiation of hPDLCs using three-dimensional porous ?-TCP ceramics and DBM with or without osteogenic inducers. Primary hPDLCs were isolated from human periodontal ligaments. The proliferation of the hPDLCs on the scaffolds in the growth culture medium was examined using a Cell?Counting kit?8 (CCK-8) and scanning electron microscopy (SEM). Alkaline phosphatase (ALP) activity and the osteogenic differentiation of the hPDLCs cultured on the ?-TCP ceramics and DBM were examined in both the growth culture medium and osteogenic culture medium. Specific osteogenic differentiation markers were examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). SEM images revealed that the cells on the ?-TCP were spindle-shaped and much more spread out compared with the cells on the DBM surfaces. There were no significant differences observed in cell proliferation between the ?-TCP ceramics and the DBM scaffolds. Compared with the cells that were cultured on ?-TCP ceramics, the ALP activity, as well as the Runx2 and osteocalcin (OCN) mRNA levels in the hPDLCs cultured on DBM were significantly enhanced both in the growth culture medium and the osteogenic culture medium. The organic elements of bone may exhibit greater osteogenic differentiation effects on hPDLCs than the inorganic elements. PMID:25738431

An, Shaofeng; Gao, Yan; Huang, Xiangya; Ling, Junqi; Liu, Zhaohui; Xiao, Yin

2015-05-01

304

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

305

Culture Models of Human Mammary Epithelial Cell Transformation  

Microsoft Academic Search

Human pre-malignant breast diseases, particularly ductal carcinoma in situ (DCIS)3 already display several of the aberrant phenotypes found in primary breast cancers, including chromosomal abnormalities, telomerase activity, inactivation of the p53 gene, and overexpression of some oncogenes. Efforts to model early breast carcinogenesis in human cell cultures have largely involved studies of in vitro transformation of normal finite lifespan human

Martha R. Stampfer; Paul Yaswen

2000-01-01

306

Gymnemic Acid Production in Suspension Cell Cultures of Gymnema sylvestre  

Microsoft Academic Search

Use of Gymnema sylvestre, commonly known as Periploca of woods an Indian medicinal woody climber has increased recently due to the pharmaceutical potential of gymnemic acids, found in its leaves. Gymnemic acids has been reported to effect a natural treatment for diabetes. This study developed a novel cell culture system for in vitro growth and production of this species, suggesting

C. Subathra Devi; S. Murugesh; V. Mohana Srinivasan

2006-01-01

307

Bacteriorhodopsin production by cell recycle culture of Halobacterium  

E-print Network

Bacteriorhodopsin production by cell recycle culture of Halobacterium halobium Sang Yup Lee*, Ho halobium R1 was cultured with cell recycle in a bioreactor equipped with an external hollow fiber membrane- rhodopsin production. The results obtained from batch and cell recycle culture of H. halobium R1

308

An Introductory Undergraduate Course Covering Animal Cell Culture Techniques  

ERIC Educational Resources Information Center

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

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

2004-01-01

309

Apoptosis in Batch Cultures of Chinese Hamster Ovary Cells  

E-print Network

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

Sinskey, Anthony J.

310

Cultural Studies in the English Classroom.  

ERIC Educational Resources Information Center

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

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

311

Cell Culture-Derived Influenza Vaccines  

Microsoft Academic Search

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

Philip R. Dormitzer

312

Arabinogalactan proteins are involved in cell aggregation of cell suspension cultures of Beta vulgaris L  

Microsoft Academic Search

Arabinogalactan proteins (AGPs) are glycoproteins present at cell surfaces. Although exact functions of AGPs remain elusive,\\u000a they are implicated in plant growth and development. The aim of this study was to evaluate the role of AGPs in the process\\u000a of cell aggregation of Beta vulgaris L. suspension cultures. It was observed that B. vulgaris suspension cultures accumulated AGPs in parallel

Jacqueline Capataz-Tafur; Gabriela Trejo-Tapia; Mario Rodríguez-Monroy; Gabriela Sepúlveda-Jiménez

2011-01-01

313

Heat shock induces protein tyrosine phosphorylation in cultured cells  

Microsoft Academic Search

We examined the effect of heat shock on protein tyrosine phosphorylation in cultured animal cells using antiphosphotyrosine antibodies in immuno- blotting and immunofluorescence microscopy experi- ments. Heat shock significantly elevated the level of phosphotyrosine in proteins in most of the cultured cells examined, including fibroblasts, epithelial cells, nerve cells, and muscle cells, but not in Rous sarcoma virus-transformed fibroblasts. The

P. A. Maher; Elena B. Pasquale

1989-01-01

314

A specific Fc gamma receptor on cultured rat mesangial cells  

Microsoft Academic Search

Mesangial cells represent specialized pericytes in the renal glomerulus that contribute to the regulation of a variety of glomerular functions. Recently we and others have shown that cultured mesangial cells bind and take up immune complexes in an Fc-dependent manner leading in turn to generation of PGE2, reactive oxygen, and platelet-activating factor. The present studies were designed to further characterize

A. Santiago; J. Satriano; S. DeCandido; H. Holthofer; R. Schreiber; J. Unkeless; D. Schlondorff

1989-01-01

315

Cell tri-culture for cardiac vascularization.  

PubMed

Poor graft survival is a critical obstacle toward production of clinically relevant engineered tissues. Here we utilize a multicellular culturing approach for induction of vascular networks embedded within cardiac tissue constructs. The construct is composed of human cardiomyocytes, endothelial cells (ECs), and embryonic fibroblast cells co-seeded onto highly porous three-dimensional (3D) scaffolds. The resulting vascularized cardiac constructs showed microstructural details characteristic of cardiomyocytes and nascent vessels and exhibited synchronous beating activity in vitro. Upon implantation, stable grafts were formed presenting intense vascularization, with evidence of anastomosis between the pre-formed endothelial capillaries and host neovessels. PMID:25070333

Lesman, Ayelet; Gepstein, Lior; Levenberg, Shulamit

2014-01-01

316

Isolation, culture, and transplantation of muscle satellite cells.  

PubMed

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

317

Impact of static magnetic fields on human myoblast cell cultures.  

PubMed

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

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

2011-12-01

318

Morphological Differences between Circulating Tumor Cells from Prostate Cancer Patients and Cultured Prostate Cancer Cells  

PubMed Central

Circulating tumor cell (CTC) enumeration promises to be an important predictor of clinical outcome for a range of cancers. Established CTC enumeration methods primarily rely on affinity capture of cell surface antigens, and have been criticized for underestimation of CTC numbers due to antigenic bias. Emerging CTC capture strategies typically distinguish these cells based on their assumed biomechanical characteristics, which are often validated using cultured cancer cells. In this study, we developed a software tool to investigate the morphological properties of CTCs from patients with castrate resistant prostate cancer and cultured prostate cancer cells in order to establish whether the latter is an appropriate model for the former. We isolated both CTCs and cultured cancer cells from whole blood using the CellSearch® system and examined various cytomorphological characteristics. In contrast with cultured cancer cells, CTCs enriched by CellSearch® system were found to have significantly smaller size, larger nuclear-cytoplasmic ratio, and more elongated shape. These CTCs were also found to exhibit significantly more variability than cultured cancer cells in nuclear-cytoplasmic ratio and shape profile. PMID:24416373

Park, Sunyoung; Ang, Richard R.; Duffy, Simon P.; Bazov, Jenny; Chi, Kim N.; Black, Peter C.; Ma, Hongshen

2014-01-01

319

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

320

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

321

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

322

Establishment of three-dimensional cultures of human pancreatic duct epithelial cells  

Microsoft Academic Search

Three-dimensional (3D) cultures of epithelial cells offer singular advantages for studies of morphogenesis or the role of cancer genes in oncogenesis. In this study, as part of establishing a 3D culture system of pancreatic duct epithelial cells, we compared human pancreatic duct epithelial cells (HPDE-E6E7) with pancreatic cancer cell lines. Our results show, that in contrast to cancer cells, HPDE-E6E7

Angelica M. Gutierrez-Barrera; David G. Menter; James L. Abbruzzese; Shrikanth A. G.. Reddy

2007-01-01

323

Differentiation of mouse iPS cells into ameloblast-like cells in cultures using medium conditioned by epithelial cell rests of Malassez and gelatin-coated dishes.  

PubMed

Induced pluripotent stem (iPS) cells are generated from adult cells and are potentially of great value in regenerative medicine. Recently, it was shown that iPS cells can differentiate into ameloblast-like cells in cultures using feeder cells. In the present study, we sought to induce differentiation of ameloblast-like cells from iPS cells under feeder-free conditions using medium conditioned by cultured epithelial cell rests of Malassez (ERM) cells and gelatin-coated dishes. Two culture conditions were compared: co-cultures of iPS cells and ERM cells; and, culture of iPS cells in ERM cell-conditioned medium. Differentiation of ameloblast-like cells in the cultures was assessed using real-time RT-PCR assays of expression of the marker genes keratin 14, amelogenin, and ameloblastin and by immunocytochemical staining for amelogenin. We found greater evidence of ameloblast-like cell differentiation in the cultures using the conditioned medium. In the latter, the level of amelogenin expression increased daily and was significantly higher than controls on the 7th, 10th, and 14th days. Expression of ameloblastin also increased daily and was significantly higher than controls on the 14th day. The present study demonstrates that mouse iPS cells can be induced to differentiate into ameloblast-like cells in feeder-free cell cultures using ERM cell-conditioned medium and gelatin-coated dishes. PMID:25319805

Yoshida, Koki; Sato, Jun; Takai, Rie; Uehara, Osamu; Kurashige, Yoshihito; Nishimura, Michiko; Chiba, Itsuo; Saitoh, Masato; Abiko, Yoshihiro

2014-10-16

324

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

325

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

326

A Novel Serum-Free Method for Culturing Human Prenatal Retinal Pigment Epithelial Cells  

Microsoft Academic Search

PURPOSE. Established techniques for culturing primary human retinal pigment epithelial (RPE) cells have facilitated the labo- ratory investigation of this multipurpose retinal cell layer. How- ever, most culture methods involve the use of animal serum to establish and maintain RPE monolayers, which can complicate efforts to define and study factors involved in the maturation and function of these cells. Therefore,

David M. Gamm; J. Nicholas Melvan; Rebecca L. Shearer; Isabel Pinilla; Grzegorz Sabat; Clive N. Svendsen; Lynda S. Wright

327

Microfabricated polyester conical microwells for cell culture applications†  

PubMed Central

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

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

2012-01-01

328

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

329

Culture and characterization of rat hair follicle stem cells.  

PubMed

The purpose of this study was to establish methods for isolation, culture, expansion, and characterization of rat hair follicle stem cells (rHFSCs). Hair follicles were harvested from 1-week-old Sprague-Dawley rats and digested with dispase and collagenase IV. The bulge of the hair follicle was dissected under a microscope and cultured in Dulbecco's modified Eagle's medium/F12 supplemented with KnockOut™ Serum Replacement serum substitute, penicillin-streptomycin, L-glutamine, non-essential amino acids, epidermal growth factor, basic fibroblast growth factor, polyhydric alcohol, and hydrocortisone. The rHFSCs were purified using adhesion to collagen IV. Cells were characterized by detecting marker genes with immunofluorescent staining and real-time polymerase chain reaction (PCR). The proliferation and vitality of rHFSCs at different passages were evaluated. The cultured rHFSCs showed typical cobblestone morphology with good adhesion and colony-forming ability. Expression of keratin 15, integrin ?6, and integrin ?1 were shown by immunocytochemistry staining. On day 1-2, the cells were in the latent phase. On day 5-6, the cells were in the logarithmic phase. Cell vitality gradually decreased from the 7th passage. Real-time PCR showed that the purified rHFSCs had good vitality and proliferative capacity and contained no keratinocytes. Highly purified rHFSCs can be obtained using tissue culture and adhesion to collagen IV. The cultured cells had good proliferative capacity and could therefore be a useful cell source for tissue-engineered hair follicles, vessels, and skin. PMID:25407732

Quan, Renfu; Zheng, Xuan; Ni, Yueming; Xie, Shangju; Li, Changming

2014-11-19

330

Developing cell culture-derived pandemic vaccines.  

PubMed

The growing prospect of avian influenza viruses achieving sustained interhuman transmission, combined with the recent emergence of a novel swine-origin A/H1N1 influenza strain, has brought the issue of influenza vaccine production capacity into sharp focus. It is becoming increasingly clear that traditional egg-based manufacturing processes may be insufficient to meet global vaccine demands in a pandemic situation that is caused by a highly pathogenic influenza virus. This review introduces the concepts of modern, cell culture-derived influenza vaccines and their manufacture, and explains the advantages of these vaccines in terms of both speed and efficiency of production as well as immunogenic efficacy. Vaccine production technologies using the mammalian cell lines Vero, MDCK and PER.C6, as well as the baculovirus/insect cell platform, are described in detail. Clinical data are provided from cell culture-derived vaccines that are at an advanced stage of development, and insights are provided into recent developments in the preclinical evaluation of more experimental technologies. PMID:20140813

Barrett, P Noel; Portsmouth, Daniel; Ehrlich, Hartmut J

2010-02-01

331

Adhesive forces in embryonic stem cell cultures  

PubMed Central

Most cell culture systems grow and spread as contact-inhibited monolayers on flat culture dishes, but the embryonic stem cell (ESC) is one of the cell phenotypes that prefer to self-organize as tightly packed three-dimensional (3D) colonies. ESC also readily form 3D cell aggregates, called embryoid bodies (EB) that partially mimic the spatial and temporal processes of the developing embryo. Here, the rationale for ESC aggregation, rather than “spreading” on gelatin-coated or mouse embryonic fibroblast (MEF)-coated dishes, is examined through the quantification of the expression levels of adhesion molecules on ESC and the calculation of the adhesive forces on ESC. Modeling each ESC as a dodecahedron, the adhesive force for each ESC-ESC binding was found to be 9.1 × 105 pN, whereas, the adhesive force for ESC-MEF binding was found to be an order of magnitude smaller at 7.9 × 104 pN. We also show that E-cadherin is the dominating molecule in the ESC-ESC adhesion and blocking E-cadherin leads to a significant reduction in colony formation. Here, we mathematically describe the preference for ESC to self-assemble into ESC-ESC aggregates and 3D colonies, rather than to bind and spread on gelatin or MEF-coated dishes, and have shown that these interactions are predominantly due to E-cadherin expression on ESC. PMID:22274712

Blancas, Alicia A; Chen, Chi-Shuo; Stolberg, Sarah

2011-01-01

332

Primary culture of human adipocyte precursor cells: expansion and differentiation.  

PubMed

Culture of adipose tissue precursor cells allows gaining insight into the sequential processes involved in adipocyte development. Furthermore, the secretory properties associated with these cellular changes can be studied. Although clonal cell lines are valuable tools for the identification of mechanisms associated with proliferation or differentiation such models do not necessarily represent the complexity of adipose tissue physiology. Primary cell culture systems may be closer to physiology and circumvent some of these restrictions. One advantage is that phenotypic properties of the tissue donor such as gender, age, or body weight are still, at least partially retained in vitro. In primary culture, also differences between various adipose depots can be studied either as a condition per se or in the cellular context of the stromal-vascular (SV) fraction. Furthermore, artificial stressors such as hypoxia and other relevant conditions can be applied to elucidate their functional role. Finally, cultures of human adipose precursor cells may also be used as a screening tool for potential novel drug targets to modulate adipocyte differentiation and biology. PMID:22057455

Skurk, Thomas; Hauner, Hans

2012-01-01

333

Human keratinocyte culture. Identification and staging of epidermal cell subpopulations.  

PubMed Central

Stratification of human epidermal cells into multilayered sheets composed of basal and suprabasal layers (resembling the stratum germinativum and stratum spinosum of the epidermis) was studied in a dermal component-free culture system. Although no stratum corneum developed in vitro, this culture system provided a method to study early events in human keratinocyte differentiation. Multiparameter flow cytometric analysis of acridine orange-stained epidermal cells from these cultures revealed three distinct subpopulations differing in cell size, RNA content, and cell cycle kinetics. The first subpopulation was composed of small basal keratinocytes with low RNA content and a long generation time. The second subpopulation consisted of larger keratinocytes, having higher RNA content and a significantly shorter generation time. Finally, the third subpopulation contained the largest cells, which did not divide, and represent the more terminally differentiated keratinocytes. This in vitro approach provides discriminating cytochemical parameters by which the maturity of the epidermal cell sheets can be assessed prior to grafting onto human burn patients. Images PMID:2418062

Staiano-Coico, L; Higgins, P J; Darzynkiewicz, Z; Kimmel, M; Gottlieb, A B; Pagan-Charry, I; Madden, M R; Finkelstein, J L; Hefton, J M

1986-01-01

334

Cell-cycle arrest of plant suspension cultures by tunicamycin.  

PubMed

The effect of tunicamycin, an inhibitor of N-glycosylation of proteins, on growth and on synthesis of DNA and protein was studied in suspension cultures from Nicotiana tabacum and Catharanthus rosea. In the presence of 0.1-1 ?g · ml(-1) tunicamycin, cell division and DNA synthesis stopped in cells which had been proliferating logarithmically, but protein formation continued. Cytophotometric determination of the nuclear DNA content in Catharanthus cells showed that a cell-cycle arrest had occurred in G1 phase. Metabolic labelling of cells with the glycoprotein precursors glucosamine or mannose was inhibited, too. The results indicate that one or more glycoproteins are needed for the cell to pass through the G1 phase, as was recently postulated for animal and yeast cells. PMID:24233741

Ettlinger, C; Schindler, J; Lehle, L

1986-05-01

335

Application of cultured endothelial cells of the brain microvasculature in the study of the blood-brain barrier  

Microsoft Academic Search

Summary The endothelial cells of the brain microvasculature provide both physical and enzymatic barriers to the exchange of molecules between the extracellular fluid environment of the brain and the systemic circulation. To better understand these barrier properties and the factors that influence them at the cellular level, an in vitro model of the blood-brain barrier has been developed using primary

Donald W. Miller; Kenneth L. Audus; Ronald T. Borchardt

1992-01-01

336

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

337

Selective and organotypic culture of intrahepatic bile duct cells from adult pig liver.  

PubMed

Secondary culture of nontransformed bile duct epithelium has been difficult to achieve. STO feeder cell-dependent secondary cultures of adult pig bile duct cells were established from primary cultures of adult pig liver cells. Adult pig hepatocytes exhibited limited or no replication and were lost from the secondary culture at Passage 3 or 4. In contrast, adult pig bile duct cells replicated and were carried for 4-8 passages in secondary culture. A simple method to produce nearly pure pig intrahepatic bile duct cultures was first to freeze a relatively crude liver cell preparation. Upon subsequent thawing, all hepatocytes and most macrophages were lysed. Bile duct cells composed 95% of the surviving cells after the freeze/thaw, and they grew out rapidly. The bile duct cells grew on top of the STO feeder cells as closely knit epithelial, colonial outgrowths. Histocytochemical and biochemical analyses demonstrated high levels of gamma-glutamyltranspeptidase activity and low levels of P450 activity in the bile duct cultures. The bile duct cells spontaneously adopted a multicellular ductal morphology after 7-10 d in static culture which was similar to that found in in vivo pig liver. Transmission electron microscopic examination revealed complex junctions and desmosomes typical of epithelium, and lumenally projecting cilia typical of in vivo intrahepatic bile ductules. This simple method for the coculture of pig intrahepatic bile duct cells which adopt in vivo-like structure may facilitate biological studies of this important, but difficult to culture, cell type. PMID:9870528

Talbot, N C; Caperna, T J

1998-01-01

338

An in vitro culturing model for rabbit dural cells.  

PubMed

The objectives of this study were (a) to construct an in vitro model of rabbit dural healing, (b) to test the influence of collagen, laminin, and poly-L-lysine on the migration and proliferation of dural cells, and (c) to study the healing mechanism of duraplasty. Rabbit dural pieces (1.5 cm x 1.5 cm) were perforated in their central part with a 2 mm punch to mimic a dural defect. The dural pieces were cultured in 24-well plates that had been coated with collagen, laminin, or poly-L-lysine, and the influence of different extracellular matrices on migration and proliferation of dural cells was observed. Cells were subcultured on slides for immunocytochemistry to study their characteristics; dural healing was observed by scanning electron microscopy. The results demonstrated that only the dural pieces that were cultured on collagen-coated wells showed migration of cells into the central defect after a period of 8 to 10 days and that healing of the dural defect occurred by 13 to 15 days. The cultured dural cells stained strongly positive with an antibody to vimentin, but negative with an antibody to factor VIII. New collagen fibers were observed in the dural defects. This report demonstrates that an in vitro model for dural healing was successfully constructed in collagen-coated wells; the results implicate cellular migration of fibroblasts from the dural defect margin as an important mechanism of wound healing following duraplasty. PMID:16951277

Zhou, Feng; Chen, Gao; Zhang, Jian-Min; Huang, Zheng-Song

2006-01-01

339

Rotating bio-reactor cell culture apparatus  

NASA Technical Reports Server (NTRS)

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

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

1991-01-01

340

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

341

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

PubMed

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

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

2015-03-01

342

Cell types in rat liver cultures: their identification and isolation  

Microsoft Academic Search

This paper reviews the various types of cells in the liver in vivo and in hepatic cellular suspensions produced by perfusion of the liver with collagenase solutions. Methods to identify and isolate different types of hepatic cells are discussed. In vitro culture of various types of liver cells is reviewed and the identification of cultured cells is considered.

J. W. Grisham

1983-01-01

343

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

344

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

345

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

346

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

PubMed Central

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

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

1994-01-01

347

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

348

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

349

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

350

Neonatal rat heart cells cultured in simulated microgravity  

NASA Technical Reports Server (NTRS)

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

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

1997-01-01

351

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

352

Epigenomic Consequences of Immortalized Plant Cell Suspension Culture  

Microsoft Academic Search

Plant cells grown in culture exhibit genetic and epigenetic instability. Using a combination of chromatin immunoprecipitation and DNA methylation profiling on tiling microarrays, we have mapped the location and abundance of histone and DNA modifications in a continuously proliferating, dedifferentiated cell suspension culture of Arabidopsis. We have found that euchromatin becomes hypermethylated in culture and that a small percentage of

Milos Tanurdzic; Matthew W Vaughn; Hongmei Jiang; Tae-Jin Lee; R. Keith Slotkin; Bryon Sosinski; William F Thompson; R. W Doerge; Robert A Martienssen

2008-01-01

353

Optimization of a MRC5 Cell Culture Process for the Production of a Smallpox Vaccine  

Microsoft Academic Search

A cell culture process adaptable to produce smallpox vaccine at large scale has been developed. To achieve this, Design of\\u000a Experiments (DOE) was applied to identify and optimize critical cell culture process parameters for MRC-5 cell growth and\\u000a recovery during cell expansion. For cell growth, a 25?1 partial factorial (two level, five factor, 16 conditions) study was designed to evaluate

Florence Wu; Kesav Reddy; Isabelle Nadeau; John Gilly; Sara Terpening; David J. Clanton

2005-01-01

354

Metabolomic profiling of cultured cancer cells.  

PubMed

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

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

2014-01-01

355

Salt tolerance in cultured cells of Spartina pectinata  

Microsoft Academic Search

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

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

1985-01-01

356

Recombinant Protein Production and Insect Cell Culture and Process  

NASA Technical Reports Server (NTRS)

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

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

1997-01-01

357

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

358

Organotypic slice cultures to study oligodendrocyte dynamics and myelination.  

PubMed

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo. PMID:25177825

Hill, Robert A; Medved, Jelena; Patel, Kiran D; Nishiyama, Akiko

2014-01-01

359

Analysis of cell identity, morphology, apoptosis and mitotic activity in a primary neural cell culture system in Drosophila  

PubMed Central

In Drosophila, most neurogenetic research is carried out in vivo. Mammalian research demonstrates that primary cell culture techniques provide a powerful model to address cell autonomous and non-autonomous processes outside their endogenous environment. We developed a cell culture system in Drosophila using wildtype and genetically manipulated primary neural tissue for long-term observations. We assessed the molecular identity of distinct neural cell types by immunolabeling and genetically expressed fluorescent cell markers. We monitored mitotic activity of cell cultures derived from wildtype and tumorous larval brains. Our system provides a powerful approach to unveil developmental processes in the nervous system and to complement studies in vivo. PMID:22554060

2012-01-01

360

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

361

Isolation and culture of primary glioblastoma cells from human tumor specimens.  

PubMed

Cultured tumor cells are a central tool in cancer research and have provided fundamental insights in tumor biology. Recent evidence, however, indicates that classically established cell lines from different tumors, including glioblastoma, do not fully reflect the genotypes and phenotypes of the respective primary tumors. By contrast, primary cells, isolated from human tumor samples and maintained in serum-free spheroid cultures at low passage under defined growth factor conditions, reproduce key aspects of tumor cell physiology much more faithfully. Among the tumor cell characteristics that are better represented in primary glioblastoma cell cultures is the self-renewal and differentiation potential of the tumor cells. Indeed, a large body of evidence from the past decade indicates that glioblastomas and other tumors are composed of a hierarchy of heterogeneous types of cells, which are generated and maintained by cells that share characteristics of stem cells. This cancer stem cell/tumor initiating cell population is optimally preserved and maintained in primary glioblastoma cultures. Here, we describe a method for the isolation and culture of primary tumor cells from human glioblastomas in serum-free conditions, which allows the routine generation and proper maintenance of tumor cells as spheroid cultures. Such primary tumor cultures can serve as a model of choice for the study of the mechanisms behind key aspects of glioblastoma biology, including tumorigenicity, stem cell hierarchy, invasion, and therapeutic resistance. PMID:25388399

Seidel, Sascha; Garvalov, Boyan K; Acker, Till

2015-01-01

362

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

363

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

364

Milk stimulates growth of prostate cancer cells in culture.  

PubMed

Concern has been expressed about the fact that cows' milk contains estrogens and could stimulate the growth of hormone-sensitive tumors. In this study, organic cows' milk and two commercial substitutes were digested in vitro and tested for their effects on the growth of cultures of prostate and breast cancer cells. Cows' milk stimulated the growth of LNCaP prostate cancer cells in each of 14 separate experiments, producing an average increase in growth rate of over 30%. In contrast, almond milk suppressed the growth of these cells by over 30%. Neither cows' milk nor almond milk affected the growth of MCF-7 breast cancer cells or AsPC-1 pancreatic cancer cells significantly. Soy milk increased the growth rate of the breast cancer cells. These data indicate that prostate and breast cancer patients should be cautioned about the possible promotional effects of commercial dairy products and their substitutes. PMID:22043817

Tate, Patricia L; Bibb, Robert; Larcom, Lyndon L

2011-11-01

365

Monitoring pH and dissolved oxygen in mammalian cell culture using optical sensors  

Microsoft Academic Search

Here, we have studied two parameters critical to process control in mammalian cell culture; dissolved oxygen (dO2) and pH, measured with fluorescent sensors thus allowing the study of the metabolic state of cells in culture without removing\\u000a or damaging cells during cultivation. Two cell lines, namely, NS0 and CHO were batch-grown in 24-well plates at different\\u000a serum concentrations with the

Mariam Naciri; Darrin Kuystermans; Mohamed Al-Rubeai

2008-01-01

366

An immortalized cell culture from a malignant mixed tumor of the lacrimal gland.  

PubMed

Tumor cells from a malignant mixed tumor of the lacrimal gland were maintained in tissue culture for more than 55 generations. Comparative immunohistochemical analysis was performed on whole tumor sections and on the tumor cell culture to define the origin of the cells in culture. The cultured cells expressed cytokeratin, smooth-muscle actin, S-100 protein, and vimentin and were negative for glial fibrillary acidic protein. Tumor sections expressed cytokeratin but were negative for muscle-specific actin, vimentin, and glial fibrillary acidic protein. Through tissue culture studies of salivary gland epithelial neoplasias, which are very similar to lacrimal gland epithelial neoplasias, pluripotential stem cells have been identified. Similar tissue culture analysis of lacrimal gland epithelial neoplasms can be a valuable tool for studying the origin of these uncommon tumors. PMID:9306436

Lauer, S A; Levin, R J; Bradley, M K; Rosenbaum, P S; Rameau, R

1997-09-01

367

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

368

[Effect of chemical carcinogens on the ultrastructure of cultured differentiating myogenic cells].  

PubMed

The electron microscope studies have been carried out on primary monolayer tissue culture obtained from body tissues of rat and C3H mouse embryos. The cells of tissue culture were mainly myoblasts and fibroblast-like cells. The cultures were treated with two different carcinogenic substances--benz(a)-pyrene (BP) and methylnitrosonitroguanidine (MNNG). The changes were uniform and showed some alterations in formation of cell complexes, the inhibition of development and maturing of muscle elements, and distrophy of cytoplasmic organelles. The revealed distinction in morphological reaction of myogenic cells to the effect of BP and MNNG were wave-like myofibrillar structures in MNNG-treated cultures. PMID:571154

Pinchuk, V G; Livshits, V L; Shuklinov, V A; Tregubova, N A

1979-01-01

369

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

370

Organelle transport in cultured Drosophila cells: S2 cell line and primary neurons.  

PubMed

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport. PMID:24300413

Lu, Wen; Del Castillo, Urko; Gelfand, Vladimir I

2013-01-01

371

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

372

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

373

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

NASA Technical Reports Server (NTRS)

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

Krikorian, Abraham D.

1993-01-01

374

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

Code of Federal Regulations, 2010 CFR

...2010-04-01 false Tissue culture media for human ex vivo tissue and cell culture...Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture...a) Identification. Tissue culture media for human ex vivo tissue and cell...

2010-04-01

375

Myocytes and fibroblasts exhibit functional synergism in mixed cultures of neonatal rat heart cells.  

PubMed

Cardiac cells obtained from neonatal rat heart contain a mixed population of cell types that can be enriched in culture in either myocytes or fibroblast-like cells. A metabolic comparison of mixed heart cell cultures with enriched cultures of the same age-in-culture and initial cell density showed that mixed cultures used glucose more rapidly than either enriched myocytes or fibroblasts. Mixed cultures were shown to respond to deprivation of insulin and of serum with decreases in the rate of glucose usage and decreases in the protein content of cells, whereas enriched cultures did not respond in the expected manner to insulin deprivation. Mixed, 11-day-old cells also exhibited greater increases in cellular protein and greater resistance to the stress of starvation than enriched cultures. Palmitate usage, however, was similar in all cultures examined. We conclude that mixed cultures may serve as a better model system to study cardiac metabolism and to monitor the effects of drugs and hormones on the neonatal myocardium. In addition, it is clear from our results that myocytes and fibroblastic-like cells coexist in a metabolically functional synergism. PMID:6361043

Schroedl, N A; Hartzell, C R

1983-12-01

376

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

377

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...

378

Media, media practice and cultural studies  

E-print Network

MA in Digital Media MA in Filmmaking MA in Gender and Media MA in Media and Cultural Studies MA in Digital Documentary, MA in Digital Media, MA in Filmmaking A first- or upper second-class undergraduateMedia, media practice and cultural studies MPhil, PhD Normally a Masters degree with a Merit

Sussex, University of

379

Cultural Studies in Turkey: Education and Practice  

ERIC Educational Resources Information Center

In this essay, the authors aim at contributing to the debate on "International Perspectives on Cultural Studies in/and Education" by presenting a perspective from Turkey, and problematizing the issues that are encountered in the country in the instruction and practice of cultural studies. They start with a brief survey of the Ege University…

Pultar, Gonul; Kirtunc, Ayse Lahur

2004-01-01

380

Anthropology and Popular Culture: A Case Study.  

ERIC Educational Resources Information Center

The study of popular culture in the United States is an appropriate anthropological endeavor, as evidenced in a case study of the volcanic eruption of Mt. St. Helens in Oregon. By examining its popular arts, anthropologists gain understanding of the culture and its people. For example, an analysis of reactions to the Mt. St. Helens eruption…

Estes, Jack

381

Glycosylation-mediated phenylpropanoid partitioning in Populus tremuloides cell cultures  

Microsoft Academic Search

BACKGROUND: Phenylpropanoid-derived phenolic glycosides (PGs) and condensed tannins (CTs) comprise large, multi-purpose non-structural carbon sinks in Populus. A negative correlation between PG and CT concentrations has been observed in several studies. However, the molecular mechanism underlying the relationship is not known. RESULTS: Populus cell cultures produce CTs but not PGs under normal conditions. Feeding salicyl alcohol resulted in accumulation of

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

2009-01-01

382

Imaging the division process in living tissue culture cells  

PubMed Central

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

Khodjakov, Alexey; Rieder, Conly L.

2008-01-01

383

A cell culture model of the blood-brain barrier  

PubMed Central

Endothelial cells that make up brain capillaries and constitute the blood-brain barrier become different from peripheral endothelial cells in response to inductive factors found in the nervous system. We have established a cell culture model of the blood-brain barrier by treating brain endothelial cells with a combination of astrocyte-conditioned medium and agents that elevate intracellular cAMP. These cells form high resistance tight junctions and exhibit low rates of paracellular leakage and fluid-phase endocytosis. They also undergo a dramatic structural reorganization as they form tight junctions. Results from these studies suggest modes of manipulating the permeability of the blood-brain barrier, potentially providing the basis for increasing the penetration of drugs into the central nervous system. PMID:1661734

1991-01-01

384

First-Year English and Cultural Studies Handbook First-Year English and Cultural Studies  

E-print Network

First-Year English and Cultural Studies Handbook First-Year English and Cultural Studies Handbook Department of English & Cultural Studies Contents Checklist for Essay Writing 1 Essay Format 3 Essay Documentation 3 Plagiarism 6 Essay Policies 7 Essay Grading Criteria 7 Who to Contact with Problems 8 English

Haykin, Simon

385

First-Year English and Cultural Studies Handbook First-Year English and Cultural Studies  

E-print Network

First-Year English and Cultural Studies Handbook First-Year English and Cultural Studies Handbook Department of English & Cultural Studies 2010-11 Contents Checklist for Essay Writing 1 Essay Format 3 Essay Documentation 3 Plagiarism 6 Essay Policies 7 Essay Grading Criteria 7 Who to Contact with Problems 8 English

Thompson, Michael

386

Culture of Rodent Spermatogonial Stem Cells, Male Germline Stem Cells of the Postnatal Animal  

PubMed Central

Spermatogonial stem cells (SSCs), postnatal male germline stem cells, are the foundation of spermatogenesis, during which an enormous number of spermatozoa is produced daily by the testis throughout life of the male. SSCs are unique among stem cells in the adult body because they are the only cells that undergo self-renewal and transmit genes to subsequent generations. In addition, SSCs provide an excellent and powerful model to study stem cell biology because of the availability of a functional assay that unequivocally identifies the stem cell. Development of an in vitro culture system that allows an unlimited supply of SSCs is a crucial technique to manipulate genes of the SSC to generate valuable transgenic animals, to study the self-renewal mechanism, and to develop new therapeutic strategies for infertility. In this chapter, we describe a detailed protocol for the culture of mouse and rat SSCs. A key factor for successful development of the SSC culture system was identification of in vitro growth factor requirements for the stem cell using a defined serum-free medium. Because transplantation assays using immunodeficient mice demonstrated that extrinsic factors for self-renewal of SSCs appear to be conserved among many mammalian species, culture techniques for SSCs of other species, including farm animals and humans, are likely to be developed in the coming 5–10 years. PMID:18442644

Kubota, Hiroshi; Brinster, Ralph L.

2014-01-01

387

Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells.  

PubMed Central

The movements of E-cadherin, epidermal growth factor receptor, and transferrin receptor in the plasma membrane of a cultured mouse keratinocyte cell line were studied using both single particle tracking (SPT; nanovid microscopy) and fluorescence photobleaching recovery (FPR). In the SPT technique, the receptor molecules are labeled with 40 nm-phi colloidal gold particles, and their movements are followed by video-enhanced differential interference contrast microscopy at a temporal resolution of 33 ms and at a nanometer-level spatial precision. The trajectories of the receptor molecules obtained by SPT were analyzed by developing a method that is based on the plot of the mean-square displacement against time. Four characteristic types of motion were observed: (a) stationary mode, in which the microscopic diffusion coefficient is less than 4.6 x 10(-12) cm2/s; (b) simple Brownian diffusion mode; (c) directed diffusion mode, in which unidirectional movements are superimposed on random motion; and (d) confined diffusion mode, in which particles undergoing Brownian diffusion (microscopic diffusion coefficient between 4.6 x 10(-12) and 1 x 10(-9) cm2/s) are confined within a limited area, probably by the membrane-associated cytoskeleton network. Comparison of these data obtained by SPT with those obtained by FPR suggests that the plasma membrane is compartmentalized into many small domains 300-600 nm in diameter (0.04-0.24 microns2 in area), in which receptor molecules are confined in the time scale of 3-30 s, and that the long-range diffusion observed by FPR can occur by successive movements of the receptors to adjacent compartments. Calcium-induced differentiation decreases the sum of the percentages of molecules in the directed diffusion and the stationary modes outside of the cell-cell contact regions on the cell surface (which is proposed to be the percentage of E-cadherin bound to the cytoskeleton/membrane-skeleton), from approximately 60% to 8% (low- and high-calcium mediums, respectively). Images FIGURE 2 FIGURE 5 FIGURE 6 FIGURE 8 FIGURE 9 FIGURE 10 FIGURE 11 FIGURE 12 FIGURE 14 PMID:8298032

Kusumi, A; Sako, Y; Yamamoto, M

1993-01-01

388

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 ...

389

Abnormal Deposition of Extracellular Matrix Proteins by Cultured Smooth Muscle Cells from Human Varicose Veins  

Microsoft Academic Search

The aim of the present study was to verify whether the modifications of the extracellular matrix, described in varicose veins, are also present in cultures of smooth muscle cells from human varicose veins. The accumulation of collagen type III and fibronectin was determined by immunofluorescence in cultures of smooth muscle cells at passage 2–3 during the proliferation phase. After 5

Patricia Sansilvestri-Morel; Isabelle Nonotte; Marie-Pierre Fournet-Bourguignon; Alain Rupin; Jean-Noël Fabiani; Tony J. Verbeuren; Paul M. Vanhoutte

1998-01-01

390

Mitosis in Tissue Cultures of Human Giant Cell Tumors of Bone  

Microsoft Academic Search

In in vitro tissue cultures of human bone tumors (osteoclastomas, chondroblastomas) containing numerous multinucleated cells, the finding of tri-, quadri- and other types of multipolar mitosis has been studied. The presence of multipolar mitosis in the cultures and its role in the formation of multinucleated cells suggest the existence of a combined mechanism of mitosis and fusion for the formation

Graciela Duran Troise; Eugenia S. de Lustig; F. Schajowicz; H. Gallardo

1973-01-01

391

An introductory undergraduate course covering animal cell culture techniques.  

PubMed

Animal cell culture is a core laboratory technique in many molecular biology, developmental biology, and biotechnology laboratories. Cell culture is a relatively old technique that has been sparingly taught at the undergraduate level. The traditional methodology for acquiring cell culture training has been through trial and error, instruction when undertaking the first graduate student position, or instruction when hired for a specific industrial cell culture position. However, cell culture is an important candidate course for any biotechnology-related training program because it is a technique that must be performed by investigators before they perform many molecular procedures, and vertebrate cell culture is becoming increasingly important for biomanufacturing of therapeutic proteins. Therefore, a cell culture techniques course is an important offering for undergraduate students who aspire to graduate training, and also undergraduate students who will seek employment with biotechnology companies immediately after graduation. Recently, a cell culture techniques course was developed and delivered to students at North Carolina State University as a component of an undergraduate Biotechnology minor curricula. Currently, the instructors at North Carolina State University are seeking to provide students with the necessary technical and critical reasoning skills to successfully perform animal cell culture. PMID:21706746

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

2004-09-01

392

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 plant growth regulators, the cells in the callus are induced to organize and form new plant organs or segments for purposes such as generating or cloning large amounts of new cells, tissues or plants; to study

Durako, Michael J.

393

In vitro cultured Spodoptera frugiperda insect cells: Model for oxidative stress-induced apoptosis  

Microsoft Academic Search

Cellular imbalance in the levels of antioxidants and reactive oxygen species (ROS) is directly associated with a number of\\u000a pathological states and results in programmed cell death or apoptosis. We demonstrate the use ofin vitro culturedSpodoptera frugiperda (sf9) insect cells as a model to study oxidative stress induced programmed cell death. Apoptosis ofin vitro cultured sf9 cells was induced by

Seyed E. Hasnain; Tarvinder K. Taneja; Nand K. Sah; Manjari Mohan; Niteen Pathak; Sudhir Sahdev; Mohammad Athar; Satish M. Totey; Rasheedunnisa Begum

1999-01-01

394

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

PubMed

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

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

2014-08-01

395

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

396

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

397

Biological characterization of woven fabric using two- and three-dimensional cell cultures.  

PubMed

The integration and long-term functional retention of tissue implants are both strongly linked to the implant material characteristics. As a first approach, the cytocompatibility and bioactivity of such materials are evaluated using in vitro-based cell culture models. Typically, in vitro bioactivity is assessed by seeding single cells onto the test material to evaluate certain parameters such as cell adhesion, survival, proliferation, and functional differentiation. Probably, due to the reduction from three dimensional (3D) toward the two dimensional (2D) situation the data obtained from 2D culture models falls short of predicting the in vivo behavior of the biomaterial in question. In this study, a three dimensional (3D) in vitro cell culture model was applied to evaluate the bioactivity of well characterized fiber-based scaffolds using scaffold colonization as a bioactivity indicator. Cell behavior in this culture model was evaluated against a classical comparable, 2D cell culture system using polyethylene terephthalat and polyamide 6.6 fabrics. By using the 3D culture model, however, differences in cell population performance as a function of fiber diameter and mesh angle were evident. The use of 3D cell culture model clearly outperformed typical cell culture setup as means to evaluate cell population-scaffold interaction. PMID:22275338

Moczulska, M; Bitar, M; Swi?szkowski, W; Bruinink, A

2012-04-01

398

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

PubMed Central

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

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

2014-01-01

399

Effects of Cell Type and Culture Media on Interleukin-6 Secretion in Response to Environmental Particles  

PubMed Central

Cultured lung cells provide an alternative to animal exposures for comparing the effects of different types of air pollution particles. Studies of particulate matter in vitro have reported proinflammatory cytokine signaling in response to many types of environmental particles, but there have been few studies comparing identical treatments in multiple cell types or identical cells with alternative cell culture protocols. We compared soil-derived, diesel, coal fly ash, titanium dioxide, and kaolin particles along with soluble vanadium and lipopolysaccharide, applied to airway-derived cells grown in submerged culture. Cell types included A549, BEAS-2B, RAW 264.7, and primary macrophages. The cell culture models (specific combinations of cell types and culture conditions) were reproducibly different in the cytokine signaling responses to the suite of treatments. Further, Interleukin-6 (IL-6) response to the treatments changed when the same cells, BEAS-2B, were grown in KGM versus LHC-9 media or in media containing bovine serum. The effect of changing media composition was reversible over multiple changes of media type. Other variables tested included culture well size and degree of confluence. The observation that sensitivity of a cell type to environmental agonists can be manipulated by modifying culture conditions suggests a novel approach for studying biochemical mechanisms of particle toxicity. PMID:18178371

Veranth, John M.; Cutler, N. Shane; Kaser, Erin G.; Reilly, Christopher A.; Yost, Garold S.

2008-01-01

400

Mammosphere culture of metastatic breast cancer cells enriches for tumorigenic breast cancer cells  

PubMed Central

Introduction The identification of potential breast cancer stem cells is of importance as the characteristics of stem cells suggest that they are resistant to conventional forms of therapy. Several techniques have been proposed to isolate or enrich for tumorigenic breast cancer stem cells, including (a) culture of cells in non-adherent non-differentiating conditions to form mammospheres and (b) sorting of the cells by their surface phenotype (expression of CD24 and CD44). Methods We have cultured metastatic cells found in pleural effusions from breast cancer patients in non-adherent conditions without serum to form mammospheres. Dissociated cells from these mammospheres were used to determine the tumorigenicity of these cultures. Expression of CD24 and CD44 on uncultured cells and mammospheres derived from the pleural effusions was documented. Results We found that the majority (20/27) of the pleural effusions tested contained cells capable of forming mammospheres of varying sizes that could be passaged. After dissociation and plating with serum onto adherent dishes, the cells can differentiate, as determined by the increased expression of cytokeratins and MUC1. Analysis of surface expression of CD24 and CD44 on uncultured cells from 21 of the samples showed that the cells from some samples separated into two populations, but some did not. The proportion of cells that could be considered CD44+/CD24low/- was highly variable and did not appear to correlate with the ability to form the larger mammospheres. Of eight pleural effusion mammospheres tested in severe combined immunodeficiency disease (SCID) mice, four were found to induce tumours when only 5,000 or fewer cells were injected, whereas the same number of uncultured cells did not form tumours. The ability to induce tumours appeared to correlate with the ability to produce the larger mammospheres. Uncultured cells from a highly tumorigenic sample (PE14) were uniformly negative for surface expression of both CD24 and CD44. Conclusion This paper shows, for the