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Sample records for neurohypophyseal cell cultures

  1. Ghrelin-Induced Enhancement of Vasopressin and Oxytocin Secretion in Rat Neurohypophyseal Cell Cultures.

    Science.gov (United States)

    Gálfi, M; Radács, M; Molnár, Zs; Budai, I; Tóth, G; Pósa, A; Kupai, K; Szalai, Z; Szabó, R; Molnár, H A; Gardi, J; László, Ferenc A; Varga, Cs

    2016-12-01

    The effects of ghrelin on vasopressin and oxytocin secretion were studied in 13-14-day cell cultures of isolated rat neurohypophyseal tissue. The vasopressin and oxytocin contents of the supernatant were determined by radioimmunoassay after a 1- or 2-h incubation. Significantly increased levels of vasopressin and oxytocin production were detected in the cell culture media following ghrelin administration, depending on the ghrelin doses. The oxytocin level proved to be more elevated than that of vasopressin. The increase of vasopressin and oxytocin secretion could be totally blocked by previous administration of the ghrelin receptor antagonist ([D-Lys 3 ]-growth hormone-releasing peptide-6). Application of the ghrelin receptor antagonist after ghrelin administration proved ineffective. The results indicate that vasopressin and oxytocin release is influenced directly by the ghrelin system, and the effects of ghrelin on vasopressin and oxytocin secretion from the neurohypophyseal tissue in rats can occur at the level of the posterior pituitary. Our observations lend support to the view that neurohypophysis contains ghrelin receptors.

  2. Induced pluripotent stem cells derived from a patient with autosomal dominant familial neurohypophyseal diabetes insipidus caused by a variant in the AVP gene

    DEFF Research Database (Denmark)

    Toustrup, Lise Bols; Zhou, Yan; Kvistgaard, Helene

    2017-01-01

    Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) is caused by variants in the arginine vasopressin (AVP) gene. Here we report the generation of induced pluripotent stem cells (iPSCs) from a 42-year-old man carrying an adFNDI causing variant in exon 1 of the AVP gene using...

  3. Familial neurohypophyseal diabetes insipidus

    DEFF Research Database (Denmark)

    Kvistgaard, Helene

    2011-01-01

    Familial neurohypophyseal diabetes insipidus (FNDI) is characterized by severe low-solute polyuria and polydipisa. The disease is caused by a deficient neurosecretion of the antidiuretic hormone arginine vasopressin (AVP). The hormone is normally synthesized by the magnocellular neurons in the pa......Familial neurohypophyseal diabetes insipidus (FNDI) is characterized by severe low-solute polyuria and polydipisa. The disease is caused by a deficient neurosecretion of the antidiuretic hormone arginine vasopressin (AVP). The hormone is normally synthesized by the magnocellular neurons...... as one sporadic case of early-onset diabetes insipidus. Genetic testing of the sporadic case of diabetes insipidus revealed a highly unusual mosaicism for a variation in the gene encoding the AVP receptor (AVPR2). This mosaicism had resulted in a partial phenotype and initial diagnostic difficulties...

  4. Induced pluripotent stem cells derived from a patient with autosomal dominant familial neurohypophyseal diabetes insipidus caused by a variant in the AVP gene.

    Science.gov (United States)

    Toustrup, Lise Bols; Zhou, Yan; Kvistgaard, Helene; Gregersen, Niels; Rittig, Søren; Aagaard, Lars; Corydon, Thomas Juhl; Luo, Yonglun; Christensen, Jane H

    2017-03-01

    Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) is caused by variants in the arginine vasopressin (AVP) gene. Here we report the generation of induced pluripotent stem cells (iPSCs) from a 42-year-old man carrying an adFNDI causing variant in exon 1 of the AVP gene using lentivirus-mediated nuclear reprogramming. The iPSCs carried the expected variant in the AVP gene. Furthermore, the iPSCs expressed pluripotency markers; displayed in vitro differentiation potential to the three germ layers and had a normal karyotype consistent with the original fibroblasts. This iPSC line is useful in future studies focusing on the pathogenesis of adFNDI. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Induced pluripotent stem cells derived from a patient with autosomal dominant familial neurohypophyseal diabetes insipidus caused by a variant in the AVP gene

    Directory of Open Access Journals (Sweden)

    Lise Bols Toustrup

    2017-03-01

    Full Text Available Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI is caused by variants in the arginine vasopressin (AVP gene. Here we report the generation of induced pluripotent stem cells (iPSCs from a 42-year-old man carrying an adFNDI causing variant in exon 1 of the AVP gene using lentivirus-mediated nuclear reprogramming. The iPSCs carried the expected variant in the AVP gene. Furthermore, the iPSCs expressed pluripotency markers; displayed in vitro differentiation potential to the three germ layers and had a normal karyotype consistent with the original fibroblasts. This iPSC line is useful in future studies focusing on the pathogenesis of adFNDI.

  6. Neurohypophyseal hormones: novel actors of striated muscle development and homeostasis

    Directory of Open Access Journals (Sweden)

    Alessandra Costa

    2014-09-01

    Full Text Available Since the 1980's, novel functional roles of the neurohypophyseal hormones vasopressin and oxytocin have emerged. Several studies have investigated the effects of these two neurohormones on striated muscle tissues, both in vitro and in vivo. The effects of vasopressin on skeletal myogenic cells, developing muscle and muscle homeostasis have been documented. Oxytocin appears to have a greater influence on cardiomyocite differentiation and heart homeostasis. This review summarizes the studies on these novel roles of the two neurohypophyseal hormones, and open the possibility of new therapeutic approaches for diseases affecting striated muscle.

  7. Specific expression of optically active reporter gene in arginine vasopressin-secreting neurosecretory cells in the hypothalamic-neurohypophyseal system.

    Science.gov (United States)

    Ueta, Y; Fujihara, H; Dayanithi, G; Kawata, M; Murphy, D

    2008-06-01

    The anti-diuretic hormone arginine vasopressin (AVP) is synthesised in the magnocellular neurosecretory cells (MNCs) in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the hypothalamus. AVP-containing MNCs that project their axon terminals to the posterior pituitary can be identified using immunohistochemical techniques with specific antibodies recognising AVP and neurophysin II, and by virtue of their electrophysiological properties. Recently, we generated transgenic rats expressing an AVP-enhanced green fluorescent protein (eGFP) fusion gene in AVP-containing MNCs. In this transgenic rat, eGFP mRNA was observed in the PVN and the SON, and eGFP fluorescence was seen in the PVN and the SON, and also in the posterior pituitary, indicating transport of transgene protein down MNC axons to storage in nerve terminals. The expression of the AVP-eGFP transgene and eGFP fluorescence in the PVN and the SON was markedly increased after dehydration and chronic salt-loading. On the other hand, AVP-containing parvocellular neurosecretory cells in the PVN that are involved in the activation of the hypothalamic-pituitary adrenal axis exhibit robust AVP-eGFP fluorescence after bilateral adrenalectomy and intraperitoneal administration of lipopolysaccharide. In the median eminence, the internal and external layer showed strong fluorescence for eGFP after osmotic stimuli and stressful conditions, respectively, again indicating appropriate transport of transgene traslation products. Brain slices and acutely-dissociated MNCs and axon terminals also exhibited strong fluorescence, as observed under fluorescence microscopy. The AVP-eGFP transgenic animals are thus unique and provide a useful tool to study AVP-secreting cells in vivo for electrophysiology, imaging analysis such as intracellular Ca(2+) imaging, organ culture and in vivo monitoring of dynamic change in AVP secretion.

  8. MRI and CT findings of neurohypophyseal germinoma

    Energy Technology Data Exchange (ETDEWEB)

    Kanagaki, Mitsunori; Miki, Yukio E-mail: mikiy@kuhp.kyoto-u.ac.jp; Takahashi, Jun A.; Shibamoto, Yuta; Takahashi, Takahiro; Ueba, Tetsuya; Hashimoto, Nobuo; Konishi, Junji

    2004-03-01

    Objective: Magnetic resonance (MR) imaging and computed tomography (CT) findings of neurohypophyseal germinoma have not previously been described in detail. The purpose of the present study was to establish the spectrum of MR imaging and CT findings in neurohypophyseal germinomas. Materials and methods: MR and CT images of 13 consecutive patients (seven males, six females; mean age: 15 years; range: 6-31 years) with neurohypophyseal germinoma were retrospectively analyzed. The diagnosis had been made either histologically (n=8) or clinically according to established criteria (n=5). All patients had been examined using MR imaging and CT before treatment. Results: On MR imaging, infundibular thickening (up to 16 mm) was observed in all 13 cases. Hyperintensity of the posterior pituitary on T1-weighted image was absent in all 13 cases (100%) and 12 of the 13 displayed central diabetes insipidus. Ten germinomas (77%) were isointense to cerebral cortex on T1-weighted image, but variable intensities were exhibited on T2-weighted image. MR images revealed intratumoral cysts in six cases (46%), most of which demonstrated intra-third ventricular extension. Eleven of the 13 cases (85%) revealed hyperdense solid components on unenhanced CT. Calcification was absent in all cases (100%). Conclusion: Infundibular thickening, absence of the posterior pituitary high signal on T1-weighted image, lack of calcification and hyperdensity on unenhanced CT are common imaging features of neurohypophyseal germinoma.

  9. Insect Cell Culture

    NARCIS (Netherlands)

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

    2010-01-01

    Insect cell cultures are widely used in studies on insect cell physiology, developmental biology and microbial pathology. In particular, insect cell culture is an indispensable tool for the study of insect viruses. The first continuously growing insect cell cultures were established from

  10. Fish Stem Cell Cultures

    OpenAIRE

    Hong, Ni; Li, Zhendong; Hong, Yunhan

    2011-01-01

    Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is th...

  11. Cell Culture Made Easy.

    Science.gov (United States)

    Dye, Frank J.

    1985-01-01

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

  12. Magnetic resonance imaging of posterior pituitary for evaluation of the neurohypophyseal function in idiopathic and autosomal dominant neurohypophyseal diabetes insipidus

    Energy Technology Data Exchange (ETDEWEB)

    Ozata, M. [Department of Endocrinology and Metabolism, Gulhane School of Medicine, TR-06 018 Etlik-Ankara (Turkey); Tayfun, C. [Department of Radiology, Gulhane School of Medicine, TR-06 018 Etlik-Ankara (Turkey); Kurtaran, K. [Department of Radiology, Gulhane School of Medicine, TR-06 018 Etlik-Ankara (Turkey); Yetkin, I. [Department of Radiology, Gulhane School of Medicine, TR-06 018 Etlik-Ankara (Turkey); Beyhan, Z. [Department of Endocrinology and Metabolism, Gulhane School of Medicine, TR-06 018 Etlik-Ankara (Turkey); Corakci, A. [Department of Endocrinology and Metabolism, Gulhane School of Medicine, TR-06 018 Etlik-Ankara (Turkey); Caglayan, S. [Department of Endocrinology and Metabolism, Gulhane School of Medicine, TR-06 018 Etlik-Ankara (Turkey); Alemdaroglu, A. [Department of Radiology, Gulhane School of Medicine, TR-06 018 Etlik-Ankara (Turkey); Guendogan, M.A. [Department of Endocrinology and Metabolism, Gulhane School of Medicine, TR-06 018 Etlik-Ankara (Turkey)

    1997-09-01

    We investigated the role of MR imaging for evaluation of the functional status of the neurohypophyseal system in both idiopathic central diabetes insipidus (DI) and familial autosomal dominant neurohypophyseal DI. The patients and family with DI were analyzed retrospectively for the presence or absence of posterior pituitary gland hyperintense signal on MR images. A total of 19 adult patients with idiopathic central DI, 7 members of a family with autosomal dominant DI and 20 control subjects were included in the study. Diagnosis of idiopathic DI was based on the presence of central DI in the absence of any alteration that is known to be responsible for DI. The patients were studied retrospectively and the morphology and intensity of the posterior lobe by MR imaging was assessed by blinded reading. In all patients with idiopathic central DI and the affected members of the family, the posterior bright signal was absent while the stalk was normal on MR images. In contrast, normal posterior pituitary bright signal and stalk were found in unaffected members of the family and all control subjects. We conclude that MR imaging of the posterior pituitary lobe can be used to evaluate the functional status of the neurohypophyseal system in idiopathic central DI and familial autosomal dominant DI. (orig.). With 3 figs., 1 tab.

  13. Fish Stem Cell Cultures

    Science.gov (United States)

    Hong, Ni; Li, Zhendong; Hong, Yunhan

    2011-01-01

    Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on “Fish Stem Cells and Nuclear Transfer”, we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer. PMID:21547056

  14. Fish stem cell cultures.

    Science.gov (United States)

    Hong, Ni; Li, Zhendong; Hong, Yunhan

    2011-04-13

    Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on "Fish Stem Cells and Nuclear Transfer", we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer.

  15. CRC handbook of neurohypophyseal hormone analogs. Volume 2

    Energy Technology Data Exchange (ETDEWEB)

    Jost, K.; Lebl, M.; Brtnik, F.

    1987-01-01

    This book is discussed in two parts. The Part 1 discusses the: Prohormones and Hormonogens of Neuro-hypophyseal Hormones, Analogs with Inhibitory properties. Analogs with Dissociated and/or High activities. Introduction. Uterotonic Activity. Galactogogic Activity. Pressor Activity. Antidiuretic Activity. References. Part 2 discusses the Other Important Activities. CNS Activities. Corticotropin- and ..beta..-Entriuretic Action. Natriferic Action. References. Practical Use in Human and Veterinary Medicine. Introduction. Methyloxytocin. Deamino-Oxytocin. Cargutocin. Glypressin. Octapressin. Desmopressin. Analogs Clinically Tried But Not Introduced into Production and Routine Clinical Practice. References. Tables of Analogs and Index.

  16. Digital Microfluidic Cell Culture.

    Science.gov (United States)

    Ng, Alphonsus H C; Li, Bingyu Betty; Chamberlain, M Dean; Wheeler, Aaron R

    2015-01-01

    Digital microfluidics (DMF) is a droplet-based liquid-handling technology that has recently become popular for cell culture and analysis. In DMF, picoliter- to microliter-sized droplets are manipulated on a planar surface using electric fields, thus enabling software-reconfigurable operations on individual droplets, such as move, merge, split, and dispense from reservoirs. Using this technique, multistep cell-based processes can be carried out using simple and compact instrumentation, making DMF an attractive platform for eventual integration into routine biology workflows. In this review, we summarize the state-of-the-art in DMF cell culture, and describe design considerations, types of DMF cell culture, and cell-based applications of DMF.

  17. Liver Cell Culture Devices

    NARCIS (Netherlands)

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

    2010-01-01

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

  18. Cell Culturing of Cytoskeleton

    Science.gov (United States)

    2004-01-01

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

  19. Oscillating Cell Culture Bioreactor

    Science.gov (United States)

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

    2010-01-01

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

  20. Plant cell culture initiation

    NARCIS (Netherlands)

    Hall, R.D.

    2000-01-01

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

  1. Growth retardation in untreated autosomal dominant familial neurohypophyseal diabetes insipidus caused by one recurring and two novel mutations in the vasopressin-neurophysin II gene.

    Science.gov (United States)

    Brachet, Cécile; Birk, Julia; Christophe, Catherine; Tenoutasse, Sylvie; Velkeniers, Brigitte; Heinrichs, Claudine; Rutishauser, Jonas

    2011-02-01

    Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI), a disorder caused by mutations in the vasopressin (AVP)-neurophysin II (NPII) gene, manifests gradually during early childhood with progressive polyuria and polydipsia. Patients are usually treated with synthetic AVP analog. If unlimited access to water is provided, prognosis is usually good even in the absence of specific treatment. In this study, we describe three families with adFNDI, in which growth failure was a prominent complaint, on the clinical and molecular level. Histories from affected and unaffected family members were taken. Height and weight of index patients were recorded longitudinally. Patients underwent water deprivation tests, magnetic resonance imaging, and genetic analysis. One mutant was studied by heterologous expression in cell culture. A total of ten affected individuals were studied. In two of the three pedigrees, a novel mutation in the AVP-NPII gene was found. The index children in each pedigree showed growth retardation, which was the reason for referral in two. In these cases, water intake was tightly restricted by the parents in an attempt to overcome suspected psychogenic polydipsia and to improve appetite. Once the children were treated by hormone replacement, they rapidly caught up to normal weight and height. Genetic testing and appropriate parent counseling should be enforced in adFNDI families to ensure adequate treatment and avoid chronic water deprivation, which causes failure to thrive.

  2. Nitric oxide modulation of the hypothalamo-neurohypophyseal system

    Directory of Open Access Journals (Sweden)

    Kadekaro M.

    2004-01-01

    Full Text Available Nitric oxide (NO, a free radical gas produced endogenously from the amino acid L-arginine by NO synthase (NOS, has important functions in modulating vasopressin and oxytocin secretion from the hypothalamo-neurohypophyseal system. NO production is stimulated during increased functional activity of magnocellular neurons, in parallel with plastic changes of the supraoptic nucleus (SON and paraventricular nucleus. Electrophysiological data recorded from the SON of hypothalamic slices indicate that NO inhibits firing of phasic and non-phasic neurons, while L-NAME, an NOS inhibitor, increases their activity. Results from measurement of neurohypophyseal hormones are more variable. Overall, however, it appears that NO, tonically produced in the forebrain, inhibits vasopressin and oxytocin secretion during normovolemic, isosmotic conditions. During osmotic stimulation, dehydration, hypovolemia and hemorrhage, as well as high plasma levels of angiotensin II, NO inhibition of vasopressin neurons is removed, while that of oxytocin neurons is enhanced. This produces a preferential release of vasopressin over oxytocin important for correction of fluid imbalance. During late pregnancy and throughout lactation, fluid homeostasis is altered and expression of NOS in the SON is down- and up-regulated, respectively, in parallel with plastic changes of the magnocellular system. NO inhibition of magnocellular neurons involves GABA and prostaglandin synthesis and the signal-transduction mechanism is independent of the cGMP-pathway. Plasma hormone levels are unaffected by icv 1H-[1, 2, 4]oxadiazolo-[4,3-a]quinoxalin-1-one (a soluble guanylyl cyclase inhibitor or 8-Br-cGMP administered to conscious rats. Moreover, cGMP does not increase in homogenates of the neural lobe and in microdialysates of the SON when NO synthesis is enhanced during osmotic stimulation. Among alternative signal-transduction pathways, nitrosylation of target proteins affecting activity of ion

  3. Autosomal dominant neurohypophyseal diabetes insipidus in two families. Molecular analysis of the vasopressin-neurophysin II gene and functional studies of three missense mutations.

    Science.gov (United States)

    Hedrich, C M; Zachurzok-Buczynska, A; Gawlik, A; Russ, S; Hahn, G; Koehler, K; Malecka-Tendera, E; Huebner, A

    2009-01-01

    Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) is a rare disease with symptoms of polydipsia, polyuria and dehydration caused by arginine vasopressin deficiency. Disease onset is within infancy or adolescence. A variety of disease-causing mutations of the arginine vasopressin neurophysin II gene (AVP) on chromosome 20p13 have been described. Two Polish families with adFNDI were screened for mutations. Processing of wild-type (WT) and mutant AVP was monitored using immunocytochemical methods in stably transfected Neuro2A cells. AVP secretion into the cell culture supernatant was investigated with an enzyme immunoassay. In the first family a heterozygous p.G96D mutation was identified. Some patients additionally carried a novel heterozygous mutation p.A159T. The second family presented with a heterozygous mutation p.C98G. Confocal laser microscopy unveiled accumulation of p.G96D and p.C98G prohormones in the cellular bodies, whereas WT and p.A159T prohormones and/or processed products were located in the tips of cellular processes. Reduced levels of AVP in supernatant culture medium of p.G96D and p.C98G transfected cells in comparison to p.A159T and WT cells were found. We conclude that the p.G96D and p.C98G mutations cause adFNDI in the two reported families. The sequence variant p.A159T does not seem to have disease-causing effects. Copyright 2009 S. Karger AG, Basel.

  4. Perfusion based cell culture chips

    DEFF Research Database (Denmark)

    Heiskanen, Arto; Emnéus, Jenny; Dufva, Martin

    2010-01-01

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

  5. Microfluidic Cell Culture Device

    Science.gov (United States)

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

    2014-01-01

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

  6. Mechanisms Of Cell Aging in Cell Culture

    OpenAIRE

    Feit, Julia; Gorzelańczyk, Edward Jacek

    2013-01-01

    A key element in the life of cells in culture is the number of cell divisions, not their life time in culture. Serially in vivo transplanted cells also exhibit a finite lifetime, which means that the cell aging is not unique only to a cell culture. There are theories suggesting that the aging of cells in culture may be associated with the aging of the organism from which they were obtained. Cells may stop dividing because of replicative aging, which is the result of telomere shortening. The a...

  7. Mammalian Cell Tissue Culture Techniques.

    Science.gov (United States)

    Phelan, Katy; May, Kristin M

    2016-06-01

    Cultured tissues and cells are used extensively in physiological and pharmacological studies. In vitro cultures provide a means of examining cells and tissues without the complex interactions that would be present if the whole organism were studied. A number of special skills are required in order to preserve the structure, function, behavior, and biology of cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  8. Cell culture's spider silk road.

    Science.gov (United States)

    Perkel, Jeffrey

    2014-06-01

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

  9. Familial neurohypophyseal diabetes insipidus associated with a signal peptide mutation

    Energy Technology Data Exchange (ETDEWEB)

    McLeod, J.F.; Gaskill, M.B.; Bradley, G.S.; Robertson, G.L. (Northwestern Univ. Medical School, Chicago, IL (United States)); Kovacs, L. (Comenius Univ. Medical School, Bratislava (Slovakia)); Rittig, S. (Univ. of Aarhus, Aarhus (Denmark))

    1993-09-01

    The authors studied the pathophysiology, natural history, and genetic basis of familial neurohypophyseal diabetes insipidus (FNDI) in a caucasian kindred. Twelve members had polyuria and a deficiency of plasma vasopressin (AVP), which progressed in severity over time. Another had normal urine volumes and plasma AVP when first tested at age 3 yr, but developed severe FNDI a year later. For unknown reasons, one man had a normal urine volume despite severe AVP deficiency and a history of polyuria in the past. When the AVP-neurophysin-II gene was amplified and sequenced, exon 2/3 was normal, but 7 of 12 clones of exon 1 contained a base substitution (G[yields]A) predicting a substitution of threonine for alanine at the -1 position of the signal peptide. Restriction analysis found the mutation in all 14 affected members, but in none of the 41 controls of 19 adult members with normal urine volumes and plasma or urinary AVP (lod score = 5.7). The mutation was also found in 2 infants in whom AVP was normal when tested at 6 and 9 months of age. We hypothesize that a mutation in exon 1 of the AVP-neurophysin-II gene caused FNDI in this kindred by making an abnormally processed precursor that gradually destroys vasopressinergic neurons. 46 refs., 6 figs.

  10. Cell culture purity issues and DFAT cells

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Shengjuan [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); Hausman, Gary J. [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); Dodson, Michael V., E-mail: dodson@wsu.edu [Department of Animal Sciences, Washington State University, Pullman, WA 99164 (United States)

    2013-04-12

    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.

  11. Cell culture compositions

    Science.gov (United States)

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

    2014-03-18

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

  12. 9 CFR 101.6 - Cell cultures.

    Science.gov (United States)

    2010-01-01

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

  13. 3D Cell Culture: An Introduction.

    Science.gov (United States)

    Koledova, Zuzana

    2017-01-01

    3D cell culture is an invaluable tool in developmental, cell, and cancer biology. By mimicking crucial features of in vivo environment, including cell-cell and cell-extracellular matrix interactions, 3D cell culture enables proper structural architecture and differentiated function of normal tissues or tumors in vitro. Thereby 3D cell culture realistically models in vivo tissue conditions and processes, and provides in vivo like responses. Since its early days in the 1970s, 3D cell culture has revealed important insights into mechanisms of tissue homeostasis and cancer, and accelerated translational research in cancer biology and tissue engineering.

  14. Dynamized Preparations in Cell Culture

    Directory of Open Access Journals (Sweden)

    Ellanzhiyil Surendran Sunila

    2009-01-01

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

  15. Influence of neurohypophyseal hormones on human cervical smooth muscle contractility in vitro.

    Science.gov (United States)

    Bryman, I; Norström, A; Lindblom, B

    1990-02-01

    Cervical tissue strips from nonpregnant women and women in early and term pregnancy were used to study spontaneous contractile activity and the effects of oxytocin and vasopressin in vitro. Oxytocin stimulated contractions in strips from all groups of patients except for those from five term pregnant women, in which an inhibitory effect was observed at a high concentration. Vasopressin had a stimulatory effect in all groups of patients. These neurohypophyseal hormones may interact with the effect of other hormones in their regulatory influence on cervical contractility, and this interaction might be important in cervical dilatation during labor as well as in the pathophysiology of dysmenorrhea.

  16. Expanding intestinal stem cells in culture

    NARCIS (Netherlands)

    Heo, Inha; Clevers, Hans

    Culturing intestinal stem cells into 3D organoids results in heterogeneous cell populations, reflecting the in vivo cell type diversity. In a recent paper published in Nature, Wang et al. established a culture condition for a highly homogeneous population of intestinal stem cells.

  17. Best practices in cell culture: an overview.

    Science.gov (United States)

    Baust, John M; Buehring, Gertrude Case; Campbell, Lia; Elmore, Eugene; Harbell, John W; Nims, Raymond W; Price, Paul; Reid, Yvonne A; Simione, Frank

    2017-09-01

    This overview describes a series of articles to provide an unmet need for information on best practices in animal cell culture. The target audience primarily consists of entry-level scientists with minimal experience in cell culture. It also include scientists, journalists, and educators with some experience in cell culture, but in need of a refresher in best practices. The articles will be published in this journal over a six-month period and will emphasize best practices in: (1) media selection; (2) use and evaluation of animal serum as a component of cell culture medium; (3) receipt of new cells into the laboratory; (4) naming cell lines; (5) authenticating cell line identity; (6) detecting and mitigating risk of cell culture contamination; (7) cryopreservation and thawing of cells; and (8) storing and shipping viable cells.

  18. A missense mutation in the arginine-vasopressin neurophysin-II gene causes autosomal dominant neurohypophyseal diabetes insipidus in a Chinese family.

    Science.gov (United States)

    Ye, Dan; Dong, FengQin; Lu, WeiQin; Zhang, Zhe; Lu, XunLiang; Li, ChengJiang; Liu, YanNing

    2013-06-01

    Familial neurohypophyseal diabetes insipidus, an autosomal dominant disorder, is mostly caused by mutations in the genes that encode AVP or its intracellular binding protein, neurophysin-II. The mutations lead to aberrant preprohormone processing and progressive destruction of AVP-secreting cells, which gradually manifests a progressive polyuria and polydipsia during early childhood, and a disorder of water homeostasis. We characterized the clinical and biochemical features, and sequenced the AVP neurophysin-II(AVP-NPII) gene of the affected individuals with autosomal dominant neurohypophyseal diabetes insipidus(ADNDI)to determine whether this disease was genetically determined. We obtained the histories of eight affected and four unaffected family individuals. The diagnosis of ADNDI was established using a water deprivation test and exogenous AVP administration. For molecular analysis, genomic DNA was extracted and the AVP-NPII gene was amplified using polymerase chain reaction and sequenced. The eight affected individuals showed different spectra of age of onsets (7-15 years) and urine volumes (132-253 ml/kg/24 h). All affected individuals responded to vasopressin administration, with a resolution of symptoms and an increase in urine osmolality by more than 50%. The characteristic hyperintense signal in the posterior pituitary on T1-weighted magnetic resonance imaging was absent in six family members and present in one. Sequencing analysis revealed a missense heterozygous mutation 1516G > T (Gly17Val) in exon 2 of the AVP-NPII gene among the ADNDI individuals. We identified a missense mutation in the AVP-NPII gene and the same mutation showed different spectra of age of onsets and urine volumes in a new Chinese family with ADNDI. The mutation may provide a molecular basis for understanding the characteristics of NPII and add to our knowledge of the pathogenesis of ADNDI, which would allow the presymptomatic diagnosis of asymptomatic subjects. © 2012 John Wiley

  19. A novel AVP gene mutation in a Turkish family with neurohypophyseal diabetes insipidus.

    Science.gov (United States)

    Ilhan, M; Tiryakioglu, N O; Karaman, O; Coskunpinar, E; Yildiz, R S; Turgut, S; Tiryakioglu, D; Toprak, H; Tasan, E

    2016-03-01

    Familial neurohypophyseal diabetes insipidus (FNDI) is a rare, autosomal dominant, inherited disorder which is characterized by severe polydipsia and polyuria generally presenting in early childhood. In the present study, we aimed to analyze the AVP gene in a Turkish family with FNDI. Four patients with neurohypophyseal diabetes insipidus and ten healthy members of the family were studied. Diabetes insipidus was diagnosed by the water deprivation test in affected family members. Mutation analysis was performed by sequencing the whole coding region of AVP-NPII gene using DNA isolated from peripheral blood samples. Urine osmolality was low (C in all patients. c.-3A>C mutation in 5'UTR of AVP gene in this family might lead to the truncation of signal peptide, aggregation of AVP in the cytoplasm instead of targeting in the endoplasmic reticulum, thereby could disrupt AVP secretion without causing neuronal cytotoxicity, which might explain the presence of bright spot. The predicted effect of this mutation should be investigated by further in vitro molecular studies.

  20. Cell Culture as an Alternative in Education.

    Science.gov (United States)

    Nardone, Roland M.

    1990-01-01

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

  1. Filovirus contamination of cell cultures.

    Science.gov (United States)

    Peters, C J; Jahrling, P B; Ksiazek, T G; Johnson, E D; Lupton, H W

    1992-01-01

    The filoviruses Marburg and Ebola comprise a newly recognized family of viruses. The first filovirus to be isolated was Marburg virus in 1967. This virus was imported in shipments of African green monkeys from Uganda and infected several cell-culture technicians, with serious illness resulting. The rarity of Marburg and Ebola virus transmission, decreasing use of imported African monkeys, and quarantine efforts have presumably been responsible for the lack of additional episodes until 1989, when a new filovirus related to Ebola was isolated from quarantined monkeys in Reston, Virginia. This virus was imported on multiple occasions from a Philippine supplier of cynomolgus macaques as a consequence of an epidemic of acute infections in the foreign holding facility. While quarantine procedures prevented the use of any of these animals in research and the three human infections that occurred were asymptomatic, this episode emphasizes that these little understood viruses have considerable potential for mischief. The finding of antibodies reacting with Ebola viruses in many biomedically important Old World primates, including colonized monkeys in the U.S., emphasizes the need for more research to understand the specificity of the antibodies, spectrum of filovirus strains in nature, potential hosts, and true distribution of the family. The filoviruses grow well in primary and established cell strains and cell lines, and cytopathogenic effects may be absent or require several days to be manifest, leading to the possibility of occult contamination. The known viruses are readily detected by polyclonal and monoclonal antibody staining of cells and by electron microscopy; nucleic acid probes exist to develop more sensitive techniques if warranted.

  2. Culture of Cells from Amphibian Embryos.

    Science.gov (United States)

    Stanisstreet, Martin

    1983-01-01

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

  3. Spheroid Culture of Mesenchymal Stem Cells

    National Research Council Canada - National Science Library

    Cesarz, Zoe; Tamama, Kenichi

    2016-01-01

      Compared with traditional 2D adherent cell culture, 3D spheroidal cell aggregates, or spheroids, are regarded as more physiological, and this technique has been exploited in the field of oncology...

  4. Embryonic Stem Cells: Isolation, Characterization and Culture

    Science.gov (United States)

    Amit, Michal; Itskovitz-Eldor, Joseph

    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.

  5. Studying cell-cell communication in co-culture

    OpenAIRE

    Bogdanowicz, Danielle R.; Lu, Helen H.

    2013-01-01

    Heterotypic and homotypic cellular interactions are essential for biological function, and co-culture models are versatile tools for investigating these cellular interactions in vitro. Physiologically relevant co-culture models have been used to elucidate the effects of cell-cell physical contact and/or secreted factors, as well as the influence of substrate geometry and interaction scale on cell response. Identifying the relative contribution of each cell population to co-culture is often ex...

  6. New culture medium concepts for cell transplantation.

    Science.gov (United States)

    Lee, S; Kim, B Y; Yeo, J E; Nemeno, J G; Jo, Y H; Yang, W; Nam, B M; Namoto, S; Tanaka, S; Sato, M; Lee, K M; Hwang, H S; Lee, J I

    2013-10-01

    Before cell or tissue transplantation, cells or tissues have to be maintained for a certain period in vitro using culture medium and methods. Most culture media contain substances such as pH indicators and buffers. It is not known whether some of these substances are safe for subsequent application in the transplantation of cells or tissues into the human body. We investigated culture media and methods with respect to the safety of the components in future transplantation applications. A modified culture medium--medical fluid-based culture medium (FCM)--was designed by using various fluids and injectable drugs that are already currently permitted for use in clinical medicine. Medium components necessary for optimal cell growth were obtained from approved drugs. FCM was manufactured with adjusted final concentrations of the medium components similar to those in commercial Dulbecco's modified Eagle's medium (DMEM). In particular, 1029.40 mg/L amino acids, approximately 88.85 mg/L vitamins, 13,525.77 mg/L inorganic salts, and 4500 mg/L D-glucose comprise the high-glucose FCM. Next, human fat synovium-derived mesenchymal stem cells and rat H9c2 (2-1) cells were cultured under 2 conditions: (1) DMEM-high glucose (HG), an original commercial medium, and (2) optimized FCM-HG. We assessed the morphologies and proliferation rates of these cells. We observed that FCM-HG was able to induce the growth of FS-MSC and commercially available H9c2 cell. The morphologies and proliferation patterns of these cells cultured under FCM-HG showed no differences compared with cells grown in DMEM-HG. Our data suggest that FCM, which we developed for the first time according to the concept of drug repositioning, was a useful culture medium, especially in cultured cells intended for human cell transplantation. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

  7. 3D Cell Culture in Alginate Hydrogels.

    Science.gov (United States)

    Andersen, Therese; Auk-Emblem, Pia; Dornish, Michael

    2015-03-24

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

  8. Cell culture from sponges: pluripotency and immortality.

    Science.gov (United States)

    de Caralt, Sònia; Uriz, María J; Wijffels, René H

    2007-10-01

    Sponges are a source of compounds with potential pharmaceutical applications. In this article, methods of sponge cell culture for production of these bioactive compounds are reviewed, and new approaches for overcoming the problem of metabolite supply are examined. The use of embryos is proposed as a new source of sponge material for cell culture. Stem cells are present in high amounts in embryos and are more versatile and resistant to infections than adult cells. Additionally, genetic engineering and cellular research on apoptotic mechanisms are promising new fields that might help to improve cell survival in sponge-cell lines. We propose that one topic for future research should be how to reduce apoptosis, which appears to be very high in sponge cell cultures.

  9. Porcine mitral valve interstitial cells in culture.

    Science.gov (United States)

    Lester, W; Rosenthal, A; Granton, B; Gotlieb, A I

    1988-11-01

    There are connective tissue cells present within the interstitium of the heart valves. This study was designed to isolate and characterize mitral valve interstitial cells from the anterior leaflet of the mitral valve. Explants obtained from the distal part of the leaflet, having been scraped free of surface endocardial cells, were incubated in medium 199 supplemented with 10% fetal bovine serum. Cells grew out of the explant after 3 to 5 days and by 3 weeks these cells were harvested and passaged. Passages 1 to 22 were characterized in several explant sets. The cells showed a growth pattern reminiscent of fibroblasts. Growth was dependent on serum concentration. Cytoskeletal localization of actin and myosin showed prominent stress fibers. Ultrastructural studies showed many elongated cells with prominent stress fibers and some gap junctions and few adherens junctions. There were as well cells with fewer stress fibers containing prominent Golgi complex and dilated endoplasmic reticulum. In the multilayered superconfluent cultures, the former cells tended to be on the substratum of the dish or surface of the multilayered culture, whereas the latter was generally located within the layer of cells. Extracellular matrix was prominent in superconfluent cultures, often within the layers as well. Labeling of the cells with antibody HHF 35 (Tsukada T, Tippens D, Gordon D, Ross R, Gown AM: Am J Pathol 126:51, 1987), which recognizes smooth muscle cell actin, showed prominent staining of the elongated stress fiber-containing cells and much less in the secretory type cells. These studies show that interstitial mitral valve cells can be grown in culture and that either two different cell types or one cell type with two phenotypic expressions is present in culture.

  10. Methods for maintaining insect cell cultures

    OpenAIRE

    Dwight E. Lynn

    2002-01-01

    Insect cell cultures are now commonly used in insect physiology, developmental biology, pathology, and molecular biology. As the field has advanced from methods development to a standard procedure, so has the diversity of scientists using the technique. This paper describes methods that are effective for maintaining various insect cell lines. The procedures are differentiated between loosely or non-attached cell strains, attached cell strains, and strongly adherent cell strains.

  11. Advances in cell culture: anchorage dependence

    Science.gov (United States)

    Merten, Otto-Wilhelm

    2015-01-01

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

  12. Familial neurohypophyseal diabetes insipidus due to a novel mutation in the arginine vasopressin-neurophysin II gene.

    Science.gov (United States)

    de Fost, M; van Trotsenburg, A S P; van Santen, H M; Endert, E; van den Elzen, C; Kamsteeg, E J; Swaab, D F; Fliers, E

    2011-07-01

    Familial neurohypophyseal (central) diabetes insipidus (DI) is caused by mutations in the arginine vasopressin-neurophysin II (AVP-NPII) gene. The majority of cases is inherited in an autosomal dominant way. In this study, we present the clinical features of a mother and her son with autosomal dominant neurohypophyseal DI caused by a novel mutation. A thirty-four-year-old woman and her three-year-old son were evaluated because of polyuria and polydipsia since the age of 1.5 years onwards. Both patients were subjected to a water deprivation test confirming the diagnosis of central DI. Magnetic resonance imaging of the brain of the mother showed a hypothalamus without apparent abnormalities and a relatively small neurohypophysis without a hyperintense signal. Mutation analysis showed a c.322G>T (p.?/p.Glu108X) in Exon 2 of the AVP-NPII gene in both mother and son. This study reports neurohypophyseal DI in a mother and her son due to a novel mutation in Exon 2 of the AVP-NPII gene. Clinical and pathophysiological aspects of this disease are shortly reviewed and discussed.

  13. Spheroid Culture of Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Zoe Cesarz

    2016-01-01

    Full Text Available Compared with traditional 2D adherent cell culture, 3D spheroidal cell aggregates, or spheroids, are regarded as more physiological, and this technique has been exploited in the field of oncology, stem cell biology, and tissue engineering. Mesenchymal stem cells (MSCs cultured in spheroids have enhanced anti-inflammatory, angiogenic, and tissue reparative/regenerative effects with improved cell survival after transplantation. Cytoskeletal reorganization and drastic changes in cell morphology in MSC spheroids indicate a major difference in mechanophysical properties compared with 2D culture. Enhanced multidifferentiation potential, upregulated expression of pluripotency marker genes, and delayed replicative senescence indicate enhanced stemness in MSC spheroids. Furthermore, spheroid formation causes drastic changes in the gene expression profile of MSC in microarray analyses. In spite of these significant changes, underlying molecular mechanisms and signaling pathways triggering and sustaining these changes are largely unknown.

  14. Culture of Mouse Neural Stem Cell Precursors

    OpenAIRE

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

    2007-01-01

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

  15. Isolation and culture of biliary epithelial cells.

    OpenAIRE

    Joplin, R

    1994-01-01

    At one time it was thought that biliary epithelial cells simply formed the lining to the tubular conduits which constitute the biliary tract. Development of in vitro systems for culturing biliary epithelial cells has enabled functional studies which increasingly show that this is far from true, and that biliary epithelial cells do have important functional roles. Disruption of these functions may be involved in the generation of pathology. Most functional studies to date have utilised cells i...

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

    NARCIS (Netherlands)

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

    2003-01-01

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

  17. Flux analysis of mammalian cell culture

    NARCIS (Netherlands)

    Martens, D.E.; Tramper, J.

    2010-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R.; Garbe, James C.

    2016-06-28

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

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

    Science.gov (United States)

    Stampfer, Martha R; Garbe, James C

    2015-02-24

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

  20. Effects of teicoplanin on cell number of cultured cell lines

    Directory of Open Access Journals (Sweden)

    Kashkolinejad-Koohi Tahere

    2015-03-01

    Full Text Available Teicoplanin is a glycopeptide antibiotic with a wide variation in human serum half-life. It is also a valuable alternative of vancomycin. There is however no study on its effect on cultured cells. The aim of the present study was to test the effect of teicoplanin on cultured cell lines CHO, Jurkat E6.1 and MCF-7. The cultured cells were exposed to teicoplanin at final concentrations of 0–11000 μg/ml for 24 hours. To determine cell viability, the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT test was performed. At low concentrations of teicoplanin the numbers of cultured cells (due to cell proliferation were increased in the three cell lines examined. The maximum cell proliferation rates were observed at concentrations of 1000, 400, and 200 μg/ml of teicoplanin for CHO, MCF-7 and Jurkat cell lines, respectively. Cell toxicity was observed at final concentrations over 2000, 6000, and 400 μg/ml of teicoplanin for CHO, MCF-7 and Jurkat cell lines, respectively. A dose-dependent manner of cell toxicity was observed. Our present findings indicated that teicoplanin at clinically used concentrations induced cell proliferation. It should therefore be used cautiously, particularly in children, pregnant women and patients with cancer.

  1. Human ES cells: Starting Culture from Frozen Cells

    OpenAIRE

    Trish, Erin; Dimos, John; Eggan, Kevin

    2006-01-01

    Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock. First, a one to two day old ten cm plate of approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator. A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submer...

  2. Pinoresinol from Ipomoea cairica cell cultures.

    Science.gov (United States)

    Páska, Csilla; Innocenti, Gabbriella; Ferlin, Mariagrazia; Kunvári, Mónika; László, Miklós

    2002-10-01

    Ipomoea cairica cell cultures produced a tetrahydrofuran lignan, (+)-pinoresinol, identified by UV, IR, MS and NMR methods, not yet found in the intact plant, and new in the Convolvulaceae family. Pinoresinol was found to have antioxidant and Ca2+ antagonist properties. As it could be requested for its biological activity, we examined the possibility to raise the pinoresinol yield of I. cairica cultures, as well as we continued investigations on lignans' response to optimization.

  3. 21 CFR 864.2280 - Cultured animal and human cells.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cultured animal and human cells. 864.2280 Section... (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro...

  4. Cell Culture on MEMS Platforms: A Review

    Science.gov (United States)

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

    2009-01-01

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

  5. Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor

    Science.gov (United States)

    Parks, Kelsey

    2009-01-01

    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.

  6. Retinol esterification in cultured rat liver cells.

    Science.gov (United States)

    Drevon, C A; Blomhoff, R; Rasmussen, M; Kindberg, G M; Berg, T; Norum, K R

    1985-01-01

    Retinol esterification was examined in cultured hepatocytes and stellate cells from the rat. Esterification of [3H]retinol was linear for 2 h in both cell types. By increasing the concentration of retinol in the medium, there was a marked increase in retinol esterification in both cell types. The capacity for esterification of retinol was in the same order of magnitude in the two cell types at 3.5 microM-retinol in the medium. This represents a rate of retinol esterification which far exceeds that required to esterify the amount of retinol absorbed in the intestine. It was demonstrated in particulate homogenates from cultured hepatocytes that the esterification of retinol was dependent on acyl-CoA. Addition of 25-hydroxycholesterol or mevalonolactone promoted an increase in cholesterol esterification, whereas retinol esterification was unaffected, suggesting that cholesterol and retinol are esterified by two different enzymes. Some 80% of vitamin A in cultured hepatocytes is retinyl esters, mostly retinyl palmitate. By adding 87 microM-retinol in the medium the cells accumulated 100-fold free retinol and 2.5-3.0-fold retinyl esters within 1 h. When retinol-loaded cells were incubated without retinol, there was a marked decrease especially in free but also in esterified retinol. In the presence of 1 mM-oleic acid in the medium the amount of retinyl oleate was twice that in control cells. PMID:4062867

  7. Autosomal dominant familial neurohypophyseal diabetes insipidus caused by a mutation in the arginine-vasopressin II gene in four generations of a Korean family.

    Science.gov (United States)

    Kim, Myo-Jing; Kim, Young-Eun; Ki, Chang-Seok; Yoo, Jae-Ho

    2014-12-01

    Autosomal dominant neurohypophyseal diabetes insipidus is a rare form of central diabetes insipidus that is caused by mutations in the vasopressin-neurophysin II (AVP-NPII) gene. It is characterized by persistent polydipsia and polyuria induced by deficient or absent secretion of arginine vasopressin (AVP). Here we report a case of familial neurohypophyseal diabetes insipidus in four generations of a Korean family, caused by heterozygous missense mutation in exon 2 of the AVP-NPII gene (c.286G>T). This is the first report of such a case in Korea.

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

    Science.gov (United States)

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

    2000-01-01

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

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

    DEFF Research Database (Denmark)

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

    2006-01-01

    We have previously shown that a polymeric (PMMA) chip with medium perfusion and integrated heat regulation provides sufficiently precise heat regulation, pH-control and medium exchange to support cell growth for weeks. However, it was unclear how closely the cells cultured in the chip resembled...... cells cultured in the culture flask. In the current study, gene expression profiles of cells cultured in the chip were compared with gene expression profiles of cells cultured in culture flasks. The results showed that there were only two genes that were differently expressed in cells grown in the cell...... culture chip compared to cell culture flasks. The cell culture chip could without further modification support cell growth of two other cell lines. Light coming from the microscope lamp during optical recordings of the cells was the only external factor identified, that could have a negative effect...

  10. Cell Culture Microfluidic Biochips: Experimental Throughput Maximization

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  11. 3D culture for cardiac cells.

    Science.gov (United States)

    Zuppinger, Christian

    2016-07-01

    This review discusses historical milestones, recent developments and challenges in the area of 3D culture models with cardiovascular cell types. Expectations in this area have been raised in recent years, but more relevant in vitro research, more accurate drug testing results, reliable disease models and insights leading to bioartificial organs are expected from the transition to 3D cell culture. However, the construction of organ-like cardiac 3D models currently remains a difficult challenge. The heart consists of highly differentiated cells in an intricate arrangement.Furthermore, electrical “wiring”, a vascular system and multiple cell types act in concert to respond to the rapidly changing demands of the body. Although cardiovascular 3D culture models have been predominantly developed for regenerative medicine in the past, their use in drug screening and for disease models has become more popular recently. Many sophisticated 3D culture models are currently being developed in this dynamic area of life science. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

  12. Plant Cell Culture Initiation: practical tips

    NARCIS (Netherlands)

    Hall, R.D.

    2001-01-01

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

  13. Nanotechnology, Cell Culture and Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Kazutoshi Haraguchi

    2011-01-01

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

  14. Cell Culture Assay for Human Noroviruses [response

    Energy Technology Data Exchange (ETDEWEB)

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

    2007-07-01

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

  15. Recurring dominant-negative mutations in the AVP-NPII gene cause neurohypophyseal diabetes insipidus

    Energy Technology Data Exchange (ETDEWEB)

    Repaske, D.R. [Children`s Hospital Medical Center, Cincinnati, OH (United States); Phillips, J.A.; Krishnamani, M.R.S. [Vanderbilt Univ. School of Medicine, Nashville, TN (United States)] [and others

    1994-09-01

    Autosomal dominant neurohypophyseal diabetes insipidus (ADNDI) is a familial form of arginine vasopressin (or antidiuretic hormone) deficiency that is usually manifest in early childhood with polyuria, polydipsia and an antidiuretic response to exogenous vasopressin or its analogs. The phenotype is postulated to arise from gliosis and depletion of the magnocellular neurons that produce vasopressin in the supraoptic and paraventricular nuclei of the hypothalamus. ADNDI is caused by heterozygosity for a variety of mutations in the AVP-NPII gene which encodes vasopressin, its carrier protein (NPII) and a glycoprotein (copeptin) of unknown function. These mutations include: (1) Ala 19{r_arrow}Thr (G279A) in AVP`s signal peptide, (2) Gly 17{r_arrow}Val (G1740T), (3) Pro 24{r_arrow}Leu (C1761T), (4) Gly 57{r_arrow}Ser (G1859A) and (5) del Glu 47({delta}AGG 1824-26), all of which occur in NPII. In characterizing the AVP-NPII mutations in five non-related ADNDI kindreds, we have detected two kindreds having mutation 1 (G279A), two having mutation 3 (C1761T) and one having mutation 4 (G1859A) without any other allelic changes being detected. Two of these recurring mutations (G279A and G1859A) are transitions that occur at CpG dinucleotides while the third (C1761T) does not. Interestingly, families with the same mutations differed in their ethnicity or in their affected AVP-NPII allele`s associated haplotype of closely linked DNA polymorphisms. Our data indicated that at least three of five known AVP-NPII mutations causing ADNDI tend to recur but the mechanisms by which these dominant-negative mutations cause variable or progressive expression of the ADNDI phenotype remain unclear.

  16. LINE-1 Cultured Cell Retrotransposition Assay

    Science.gov (United States)

    Kopera, Huira C.; Larson, Peter A.; Moldovan, John B.; Richardson, Sandra R.; Liu, Ying; Moran, John V.

    2016-01-01

    Summary The Long INterspersed Element-1 (LINE-1 or L1) retrotransposition assay has facilitated the discovery and characterization of active (i.e., retrotransposition-competent) LINE-1 sequences from mammalian genomes. In this assay, an engineered LINE-1 containing a retrotransposition reporter cassette is transiently transfected into a cultured cell line. Expression of the reporter cassette, which occurs only after a successful round of retrotransposition, allows the detection and quantification of the LINE-1 retrotransposition efficiency. This assay has yielded insight into the mechanism of LINE-1 retrotransposition. It also has provided a greater understanding of how the cell regulates LINE-1 retrotransposition and how LINE-1 retrotransposition impacts the structure of mammalian genomes. Below, we provide a brief introduction to LINE-1 biology and then detail how the LINE-1 retrotransposition assay is performed in cultured mammalian cells. PMID:26895052

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

    Science.gov (United States)

    Cogoli, Augusto

    1992-01-01

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

  18. Human ES cells: starting culture from frozen cells.

    Science.gov (United States)

    Trish, Erin; Dimos, John; Eggan, Kevin

    2006-11-09

    Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock. First, a one to two day old ten cm plate of approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator. A frozen vial of cells from long term liquid nitrogen storage or a -80 C freezer is sourced and quickly submerged in a 37 C water bath for quick thawing. Cells in freezing media are then removed from the vial and placed in a large volume of HuES media. The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate. Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate. It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate. The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

  19. The presence and in vivo biosynthesis of fragments of CPP (the C-terminal glycopeptide of the rat vasopressin precursor) in the hypothalamo-neurohypophyseal system

    NARCIS (Netherlands)

    Seger, M.A.; Burbach, J.P.H.

    1987-01-01

    The existence and rate of formation of fragments of the 39-residue C-terminal glycopeptide of propressophysin (CPP1–39) was investigated in the hypothalamo-neurohypophyseal system. Newly-prepared antisera to CPP were used to confirm the existence of smaller C-terminal fragments derived from CPP1–39.

  20. Clinical and molecular evidence of abnormal processing and trafficking of the vasopressin preprohormone in a large kindred with familial neurohypophyseal diabetes insipidus due to a signal peptide mutation

    DEFF Research Database (Denmark)

    Siggaard, C; Rittig, S; Corydon, T J

    1999-01-01

    The autosomal dominant form of familial neurohypophyseal diabetes insipidus (adFNDI) is a rare disease characterized by postnatal onset of polyuria and a deficient neurosecretion of the antidiuretic hormone, arginine vasopressin (AVP). Since 1991, adFNDI has been linked to 31 different mutations ...

  1. A novel splice site mutation of the arginine vasopressin-neurophysin II gene identified in a kindred with autosomal dominant familial neurohypophyseal diabetes insipidus.

    Science.gov (United States)

    Tae, Hyun-Jung; Baek, Ki-Hyun; Shim, Sun-Mi; Yoo, Soon-Jib; Kang, Moo-Il; Cha, Bong-Yun; Lee, Kwang-Woo; Son, Ho-Young; Kang, Sung-Koo

    2005-01-01

    Autosomal dominant familial neurohypophyseal diabetes insipidus is an inherited deficiency of arginine vasopressin (AVP), and this is caused by mutations in the AVP-neurophysin II (AVP-NP II) gene. Most of these mutations have been located in the signal peptide or in the NP II moiety. In the present study, we have analyzed the AVP-NP II gene in a Korean family. Clinical and genetic studies were performed on three members of the family, and on a normal healthy unrelated individual. The diagnosis of neurohypophyseal diabetes insipidus was done by performing a fluid deprivation test and a vasopressin challenge. For genetic analysis, the genomic DNA was extracted and the AVP-NP II gene was amplified by polymerase chain reaction (PCR). Clinical assessment of the affected individuals confirmed the diagnosis of neurohypophyseal diabetes insipidus. Genetic analysis of the AVP-NP II gene revealed a novel deletion mutation of a single nucleotide (guanine) within the splice acceptor site of intron 2 (IVS2 +1 delG). The affected individuals were heterozygous for this mutation. We also demonstrated through RT-PCR analysis of the mutant gene that this mutation resulted in the retention of intron 2 during pre-mRNA splicing. We concluded that a novel splicing mutation in the AVP-NP II gene causes neurohypophyseal diabetes insipidus in this family.

  2. Callus and cell suspension cultures of carnation

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1972-01-01

    of growth regulators were observed to be 3 × 10−6M indoleacetic acid (JAA) combined with 3 × 10−6M benzylaminopurin (BAP) or 10−6M 2,4-dichlorophenoxy acetic acid (2,4-D) alone. IAA + BAP caused a 100 fold increase in fresh weight over 4 weeks at 25°C. Addition of casein hydrolysate increased growth further....... Cell suspension cultures worked best in media containing 2,4-D in which they had a doubling time of about 2 days. Filtered suspensions were successfully plated on agar in petri dishes, but division was never observed in single cells. The cultures initiated roots at higher concentrations of IAA or NAA...

  3. Mouse cell culture - Methods and protocols

    Directory of Open Access Journals (Sweden)

    CarloAlberto Redi

    2010-12-01

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

  4. Culturing the epiblast cells of the pig blastocyst.

    Science.gov (United States)

    Talbot, N C; Rexroad, C E; Pursel, V G; Powell, A M; Nel, N D

    1993-07-01

    Pig epiblast cells that had been separated from other early embryonic cells were cultured in vitro. A three-step dissection protocol was used to isolate the epiblast from trophectoderm and primitive endoderm before culturing. Blastocysts collected at 7 to 8 days postestrus were immunodissected to obtain the inner cell mass (ICM) and destroy trophectodermal cells. The ICM was cultured for 2 to 3 days on STO feeder cells. The epiblast was then physically dissected free of associated primitive endoderm. Epiblast-derived cells, grown on STO feeders, produced colonies of small cells resembling mouse embryonic stem cells. This primary cell morphology changed as the colonies grew and evolved into three distinct colony types (endodermlike, neural rosette, or complex). Cell cultures derived from these three colony types spontaneously differentiated into numerous specialized cell types in STO co-culture. These included fibroblasts, endodermlike cells, neuronlike cells, pigmented cells, adipogenic cells, contracting muscle cells, dome-forming epithelium, ciliated epithelium, tubule-forming epithelium, and a round amoeboid cell type resembling a plasmacyte after Wright staining. The neuronlike cells, contracting muscle cells, and tubule-forming epithelium had normal karyotypes and displayed finite or undefined life spans upon long-term STO co-culture. The dome-forming epithelium had an indefinite life span in STO co-culture and also retained a normal karyotype. These results demonstrate the in vitro pluripotency of pig epiblast cells and indicate the epiblast can be a source for deriving various specialized cell cultures or cell lines.

  5. Conversion of primordial germ cells to pluripotent stem cells: methods for cell tracking and culture conditions.

    Science.gov (United States)

    Nagamatsu, Go; Suda, Toshio

    2013-01-01

    Primordial germ cells (PGCs) are unipotent cells committed to germ lineage: PGCs can only differentiate into gametes in vivo. However, upon fertilization, germ cells acquire the capacity to differentiate into all cell types in the body, including germ cells. Therefore, germ cells are thought to have the potential for pluripotency. PGCs can convert to pluripotent stem cells in vitro when cultured under specific conditions that include bFGF, LIF, and the membrane-bound form of SCF (mSCF). Here, the culture conditions which efficiently convert PGCs to pluripotent embryonic germ (EG) cells are described, as well as methods used for identifying pluripotent candidate cells during culture.

  6. A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids.

    Science.gov (United States)

    Massai, Diana; Isu, Giuseppe; Madeddu, Denise; Cerino, Giulia; Falco, Angela; Frati, Caterina; Gallo, Diego; Deriu, Marco A; Falvo D'Urso Labate, Giuseppe; Quaini, Federico; Audenino, Alberto; Morbiducci, Umberto

    2016-01-01

    A versatile bioreactor suitable for dynamic suspension cell culture under tunable shear stress conditions has been developed and preliminarily tested culturing cancer cell spheroids. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits the laminar hydrodynamics establishing within the culture chamber enabling dynamic cell suspension in an environment favourable to mass transport, under a wide range of tunable shear stress conditions. The design phase of the device has been supported by multiphysics modelling and has provided a comprehensive analysis of the operating principles of the bioreactor. Moreover, an explanatory example is herein presented with multiphysics simulations used to set the proper bioreactor operating conditions for preliminary in vitro biological tests on a human lung carcinoma cell line. The biological results demonstrate that the ultralow shear dynamic suspension provided by the device is beneficial for culturing cancer cell spheroids. In comparison to the static suspension control, dynamic cell suspension preserves morphological features, promotes intercellular connection, increases spheroid size (2.4-fold increase) and number of cycling cells (1.58-fold increase), and reduces double strand DNA damage (1.5-fold reduction). It is envisioned that the versatility of this bioreactor could allow investigation and expansion of different cell types in the future.

  7. Sodium 22+ washout from cultured rat cells

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-10-01

    The washout of Na/sup +/ isotopes from tissues and cells is quite complex and not well defined. To further gain insight into this process, we have studied /sup 22/Na/sup +/ washout from cultured Wistar rat skin fibroblasts and vascular smooth muscle cells (VSMCs). In these preparations, /sup 22/Na/sup +/ washout is described by a general three-exponential function. The exponential factor of the fastest component (k1) and the initial exchange rate constant (kie) of cultured fibroblasts decrease in magnitude in response to incubation in K+-deficient medium or in the presence of ouabain and increase in magnitude when the cells are incubated in a Ca++-deficient medium. As the magnitude of the kie declines (in the presence of ouabain) to the level of the exponential factor of the middle component (k2), /sup 22/Na/sup +/ washout is adequately described by a two-exponential function. When the kie is further diminished (in the presence of both ouabain and phloretin) to the range of the exponential factor of the slowest component (k3), the washout of /sup 22/Na/sup +/ is apparently monoexponential. Calculations of the cellular Na/sup +/ concentrations, based on the /sup 22/Na/sup +/ activity in the cells at the initiation of the washout experiments, and the medium specific activity agree with atomic absorption spectrometry measurements of the cellular concentration of this ion. Thus, all three components of /sup 22/Na/sup +/ washout from cultured rat cells are of cellular origin. Using the exponential parameters, compartmental analyses of two models (in parallel and in series) with three cellular Na/sup +/ pools were performed. The results indicate that, independent of the model chosen, the relative size of the largest Na+ pool is 92-93% in fibroblasts and approximately 96% in VSMCs. This pool is most likely to represent the cytosol.

  8. Obtaining phenolic acids from cell cultures of various Artemisia ...

    African Journals Online (AJOL)

    The most productive cell cultures for phenolic acids were Artemisia frigida and Silybum marianum. Isochlorogenic acid and chlorogenic acid were the most abundant phenolic acids identified in the analyzed cell cultures. Cell culture of Nicotiana tabacum contained only one hydroxycinnamic acid derivative in low amounts.

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

    Science.gov (United States)

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

    2000-01-01

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

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

    Science.gov (United States)

    Kihara, Takanori; Kashitani, Kosuke; Miyake, Jun

    2017-07-14

    Cell proliferation is a key characteristic of eukaryotic cells. During cell proliferation, cells interact with each other. In this study, we developed a cellular automata model to estimate cell-cell interactions using experimentally obtained images of cultured cells. We used four types of cells; HeLa cells, human osteosarcoma (HOS) cells, rat mesenchymal stem cells (MSCs), and rat smooth muscle A7r5 cells. These cells were cultured and stained daily. The obtained cell images were binarized and clipped into squares containing about 104 cells. These cells showed characteristic cell proliferation patterns. The growth curves of these cells were generated from the cell proliferation images and we determined the doubling time of these cells from the growth curves. We developed a simple cellular automata system with an easily accessible graphical user interface. This system has five variable parameters, namely, initial cell number, doubling time, motility, cell-cell adhesion, and cell-cell contact inhibition (of proliferation). Within these parameters, we obtained initial cell numbers and doubling times experimentally. We set the motility at a constant value because the effect of the parameter for our simulation was restricted. Therefore, we simulated cell proliferation behavior with cell-cell adhesion and cell-cell contact inhibition as variables. By comparing growth curves and proliferation cell images, we succeeded in determining the cell-cell interaction properties of each cell. Simulated HeLa and HOS cells exhibited low cell-cell adhesion and weak cell-cell contact inhibition. Simulated MSCs exhibited high cell-cell adhesion and positive cell-cell contact inhibition. Simulated A7r5 cells exhibited low cell-cell adhesion and strong cell-cell contact inhibition. These simulated results correlated with the experimental growth curves and proliferation images. Our simulation approach is an easy method for evaluating the cell-cell interaction properties of cells.

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

    Science.gov (United States)

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

    2015-12-01

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

  12. PHYTOCHEMICAL STUDY OF CELL CULTURE JATROPHA CURCAS

    Directory of Open Access Journals (Sweden)

    KOMAR RUSLAN

    2011-01-01

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

  13. Good cell culture practices &in vitro toxicology.

    Science.gov (United States)

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

    2017-12-01

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

  14. Peroxisome dynamics in cultured mammalian cells.

    Science.gov (United States)

    Huybrechts, Sofie J; Van Veldhoven, Paul P; Brees, Chantal; Mannaerts, Guy P; Los, Georgyi V; Fransen, Marc

    2009-11-01

    Despite the identification and characterization of various proteins that are essential for peroxisome biogenesis, the origin and the turnover of peroxisomes are still unresolved critical issues. In this study, we used the HaloTag technology as a new approach to examine peroxisome dynamics in cultured mammalian cells. This technology is based on the formation of a covalent bond between the HaloTag protein--a mutated bacterial dehalogenase which is fused to the protein of interest--and a synthetic haloalkane ligand that contains a fluorophore or affinity tag. By using cell-permeable ligands of distinct fluorescence, it is possible to image distinct pools of newly synthesized proteins, generated from a single genetic HaloTag-containing construct, at different wavelengths. Here, we show that peroxisomes display an age-related heterogeneity with respect to their capacity to incorporate newly synthesized proteins. We also demonstrate that these organelles do not exchange their protein content. In addition, we present evidence that the matrix protein content of pre-existing peroxisomes is not evenly distributed over new organelles. Finally, we show that peroxisomes in cultured mammalian cells, under basal growth conditions, have a half-life of approximately 2 days and are mainly degraded by an autophagy-related mechanism. The implications of these findings are discussed.

  15. The corticostriatal system in dissociated cell culture

    Directory of Open Access Journals (Sweden)

    Fiona E Randall

    2011-06-01

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

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

    OpenAIRE

    Sundin, D R; Mecham, J.O.

    1989-01-01

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

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

    DEFF Research Database (Denmark)

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

    2006-01-01

    We have previously shown that a polymeric (PMMA) chip with medium perfusion and integrated heat regulation provides sufficiently precise heat regulation, pH-control and medium exchange to support cell growth for weeks. However, it was unclear how closely the cells cultured in the chip resembled...... culture chip compared to cell culture flasks. The cell culture chip could without further modification support cell growth of two other cell lines. Light coming from the microscope lamp during optical recordings of the cells was the only external factor identified, that could have a negative effect...

  18. Growth of cultured porcine retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Wiencke, A.K.; Kiilgaard, Jens Folke; Nicolini, Jair

    2003-01-01

    To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation.......To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation....

  19. Muscle cell cultures in Menkes' disease: copper accumulation in myotubes

    NARCIS (Netherlands)

    van den Berg, G. J.; Kroon, J. J.; Wijburg, F. A.; Sinjorgo, K. M.; Herzberg, N. H.; Bolhuis, P. A.

    1990-01-01

    We present 64Cu uptake studies in cultured muscle cells from a one-year-old patient with Menkes' disease. The cultured muscle cells from the patient showed a five-fold higher 64Cu uptake than control muscle cells. Copper uptake in muscle cells was of the same magnitude as that found in fibroblasts

  20. Phosphatidylinositol species of suspension cultured plant cells

    Energy Technology Data Exchange (ETDEWEB)

    Heim, S.; Wagner, K.G.

    Suspension cultured Nicotiana tabacum and Catharanthus roseus cells were labeled with (/sup 3/H)inositol, the phospholipid fraction extracted and separated by thin layer chromatography. Three different solvent systems and reference compounds were used to assign the different /sup 3/H-labeled species by autoradiography. The ratio of (/sup 3/H)inositol incorporation into PI, PIP and PIP/sub 2/ was found to be 95:4:1; with some preparations a lyso-PI band was obtained which incorporated about a tenth of the label of the PIP band. With Catharanthus roseus cells a very faint band between PI and lyso-PI was detected which could not be assigned to a reference compound.

  1. Protection of cultured mammalian cells by rebamipide

    Energy Technology Data Exchange (ETDEWEB)

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

    1997-06-01

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

  2. Polyamine Uptake in Carrot Cell Cultures 1

    Science.gov (United States)

    Pistocchi, Rossella; Bagni, Nello; Creus, José A.

    1987-01-01

    Putrescine and spermidine uptake into carrot (Daucus carota L.) cells in culture was studied. The time course of uptake showed that the two polyamines were very quickly transported into the cells, reaching a maximum absorption within 1 minute. Increasing external polyamine concentrations up to 100 millimolar showed the existence of a biphasic system with different affinities at low and high polyamine concentrations. The cellular localization of absorbed polyamines was such that a greater amount of putrescine was present in the cytoplasmic soluble fraction, while spermidine was mostly present in cell walls. The absorbed polyamines were released into the medium in the presence of increasing external concentrations of the corresponding polyamine or Ca2+. The effects of Ca2+ were different for putrescine and spermidine; putrescine uptake was slightly stimulated by 10 micromolar Ca2+ and inhibited by higher concentrations, while for spermidine uptake there was an increasing stimulation in the Ca2+ concentration range between 10 micromolar and 1 millimolar. La3+ nullified the stimulatory effect of 10 micromolar Ca2+ on putrescine uptake and that of 1 millimolar Ca2+ on spermidine uptake. La3+ at 0.5 to 1 millimolar markedly inhibited the uptake of both polyamines, suggesting that it interferes with the sites of polyamine uptake. Putrescine uptake was affected to a lesser extent by metabolic inhibitors than was spermidine uptake. It is proposed that the entry of polyamines into the cells is driven by the transmembrane electrical gradient, with a possible antiport mechanism between external and internal polyamine molecule. PMID:16665446

  3. The evolution of chicken stem cell culture methods.

    Science.gov (United States)

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

    2017-12-01

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

  4. Three-dimensional cell culturing by magnetic levitation.

    Science.gov (United States)

    Haisler, William L; Timm, David M; Gage, Jacob A; Tseng, Hubert; Killian, T C; Souza, Glauco R

    2013-10-01

    Recently, biomedical research has moved toward cell culture in three dimensions to better recapitulate native cellular environments. This protocol describes one method for 3D culture, the magnetic levitation method (MLM), in which cells bind with a magnetic nanoparticle assembly overnight to render them magnetic. When resuspended in medium, an external magnetic field levitates and concentrates cells at the air-liquid interface, where they aggregate to form larger 3D cultures. The resulting cultures are dense, can synthesize extracellular matrix (ECM) and can be analyzed similarly to the other culture systems using techniques such as immunohistochemical analysis (IHC), western blotting and other biochemical assays. This protocol details the MLM and other associated techniques (cell culture, imaging and IHC) adapted for the MLM. The MLM requires 45 min of working time over 2 d to create 3D cultures that can be cultured in the long term (>7 d).

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

    Science.gov (United States)

    Krikorian, Abraham D.

    1993-01-01

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

  6. Novel egg white-based 3-D cell culture system.

    Science.gov (United States)

    Kaipparettu, Benny Abraham; Kuiatse, Isere; Tak-Yee Chan, Bonita; Benny Kaipparettu, Meju; Lee, Adrian V; Oesterreich, Steffi

    2008-08-01

    Although three dimensional (3-D) cell culture systems have numerous advantages over traditional monolayer culture, the currently available 3-D cell culture media are cost-prohibitive for regular use by the majority of research laboratories. Here we show a simple system based on avian egg white that supports growth of cells in 3-D, at a significantly decreased cost. Specifically, we show that growth of immortalized human breast epithelial cells (MCF10A) in egg white-based medium results in formation of acini with hollow lumens, apoptotic clearance of the cells in the lumen, and apicobasal polarization comparable to what has been described using established 3-D culture media such as reconstituted basement membrane preparations (BM). There was no significant difference in MCF10A proliferation and acinar size between egg white and BM. We also cultured different established cell lines, oncogene-transformed MCF10A, and mouse mammary epithelial cells in egg white and BM, and observed similar morphology. In summary, our data convincingly argue that egg white can be used as a suitable alternative model for 3-D cell culture studies. We strongly believe that this simple and inexpensive method should allow researchers to perform 3-D cell culture experiments on a regular basis, and result in a dramatic increase of use of the 3-D cell culture in research. Thus, this finding lays the foundation for significantly increased, cost-effective use of 3-D cultures in cell biology.

  7. Establishment and characterization of American elm cell suspension cultures

    Science.gov (United States)

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

    2000-01-01

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

  8. Rapid method for culturing embryonic neuron-glial cell cocultures

    DEFF Research Database (Denmark)

    Svenningsen, Åsa Fex; Shan, Wei-Song; Colman, David R

    2003-01-01

    to cultures first treated with antimitotic agents. It also ensures that all the cells present in vivo will be present in the culture. Myelination commences after approximately 2 weeks in culture for dissociated DRG and 3-4 weeks in cerebellar cultures. In enteric cultures, glial wrapping of the enteric...... neurons is seen after 3 weeks (2 weeks in ascorbic acid), suggesting that basal lamina production is important even for glial ensheathment in the enteric nervous system. No overgrowth of fibroblasts or other nonneuronal cells was noted in any cultures, and myelination of the peripheral nervous system...

  9. Can established cultured papilloma cells harbor bovine papillomavirus?

    Science.gov (United States)

    Campos, S R C; Trindade, C; Ferraz, O P; Giovanni, D N S; Lima, A A; Caetano, H V A; Carvalho, R F; Birgel, E H; Dagli, M L Z; Mori, E; Brandão, P E; Richtzenhain, L J; Beçak, W; Stocco, R C

    2008-10-21

    Papillomaviruses have been reported to be very difficult to grow in cell culture. Also, there are no descriptions of cell cultures from lesions of bovine cutaneous papillomatosis, with identification of different bovine papilloma virus (BPV) DNA sequences. In the present report, we describe primary cell cultures from samples of cutaneous lesions (warts). We investigated the simultaneous presence of different BPV DNA sequences, comparing the original lesion to different passages of the cell cultures and to peripheral blood. BPV 1, 2 and 4 DNA sequences were found in lesion samples, and respective cell cultures and peripheral blood, supporting our previous hypothesis of the possible activity of these sequences in different samples and now also showing how they can be maintained in different passages of cell cultures.

  10. Improving alpha-tocopherol production in plant cell cultures.

    Science.gov (United States)

    Gala, Rosa; Mita, Giovanni; Caretto, Sofia

    2005-07-01

    Suspension cell cultures of Helianthus annuus L. were previously established for the production of the most active component of vitamin E, alpha-tocopherol, by optimizing medium composition and culture conditions. In the present work, the possibility of enhancing alpha-tocopherol production by the addition of jasmonic acid to the culture medium was investigated both in sunflower and Arabidopsis cell cultures. A considerable increase (49% and 66%, respectively) of alpha-tocopherol production was obtained in both, after a 72-h treatment with 5 microM jasmonic acid. The modulation of alpha-tocopherol levels in plant cell cultures can provide useful hints for a regulatory impact on tocopherol metabolism.

  11. Expression of three different mutations in the arginine vasopressin gene suggests genotype-phenotype correlation in familial neurohypophyseal diabetes insipidus kindreds

    DEFF Research Database (Denmark)

    Siggaard, Charlotte; Christensen, Jane Hvarregaard; Corydon, Thomas Juhl

    2005-01-01

    OBJECTIVE AND STUDY DESIGN: The autosomal dominant form of familial neurohypophyseal diabetes insipidus (adFNDI) is a rare disease characterized by a severe and progressive deficiency of AVP secondary to mutations in the gene encoding the AVP precursor. Whereas a number of studies have investigated....... CONCLUSION: The study suggests a genotype-phenotype correlation with regard to age of onset of diabetes insipidus symptoms and provides support by expression studies....

  12. Skeletal muscle satellite cells cultured in simulated microgravity

    Science.gov (United States)

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

    1993-01-01

    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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2010-04-01

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

  15. Usability and Applicability of Microfluidic Cell Culture Systems

    DEFF Research Database (Denmark)

    Hemmingsen, Mette

    possibilities for, for example, precise control of the chemical environment, 3D cultures, controlled co-culture of different cell types or automated, individual control of up to 96 cell culture chambers in one integrated system. Despite the great new opportunities to perform novel experimental designs......Microfluidic cell culture has been a research area with great attention the last decade due to its potential to mimic the in vivo cellular environment more closely compared to what is possible by conventional cell culture methods. Many exciting and complex devices have been presented providing......, these devices still lack general implementation into biological research laboratories. In this project, the usability and applicability of microfluidic cell culture systems have been investigated. The tested systems display good properties regarding optics and compatibility with standard laboratory equipment...

  16. THE ALKALOID CYTISINE IN THE CELL CULTURE

    Directory of Open Access Journals (Sweden)

    Gazaliev A.M.

    2012-08-01

    Full Text Available Alkaloids are vegetative establishments of complex and original structure with nitrous heterocycles in the basis. For a long time they drew researchers’ attention because of their unique and specific physiological effect on alive organisms. Not all the representatives of the globe’s flora contain these unique substances. Alkaloid cytisine is to be found mainly in the plants of the fabaceous family - Fabaceae. For the cytisine production the seeds of Thermopsis lanceolata R.Br (T. lanceolata R.Br and Cytisus laburnum (C. laburnum are used as a raw material. The object of the research is T. lanceolata cell culture. Sterile sprouts are used at the first stage of the experiment. Callus genesis is accompanied with dedifferentiation. It leads to the cellular organization simplification. Based on an important property of a plant cell, such as totipotency, there appears the formation of the “de novo” biosynthetic device. The cultivation algorithm consists of two basic stages: (i the cultivation conditions optimization of callus with a high level of the primary metabolites biosynthesis (Aspartat – lysine; (ii the research of cultivation chemical and physical factors influence on the secondary metabolite (cytisine biosynthesis and accumulation. During the cultivation the Murashige and Skoog classical recipe of nutrient medium will be used. Optimization of the cultivation conditions will concern the phytohormones, macro- and micronutrients content, as the purpose of optimization is the production of the determined high-level competence embriogenical callus. The main problem is genetic heterogeneity of a cellular population and instability of morpho-physiological processes. The correct management of higher plants cells population is possible at the synchronization of a cellular cycle phases. The references analysis has shown that it is almost impossible to synchronize cellular cycles in the culture of plant tissue. The application of chemical

  17. Isolation and culture of larval cells from C. elegans.

    Directory of Open Access Journals (Sweden)

    Sihui Zhang

    Full Text Available Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×10(4 cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81% of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology.

  18. Systems biology for organotypic cell cultures.

    Science.gov (United States)

    Grego, Sonia; Dougherty, Edward R; Alexander, Francis J; Auerbach, Scott S; Berridge, Brian R; Bittner, Michael L; Casey, Warren; Cooley, Philip C; Dash, Ajit; Ferguson, Stephen S; Fennell, Timothy R; Hawkins, Brian T; Hickey, Anthony J; Kleensang, Andre; Liebman, Michael N J; Martin, Florian; Maull, Elizabeth A; Paragas, Jason; Qiao, Guilin Gary; Ramaiahgari, Sreenivasa; Sumner, Susan J; Yoon, Miyoung

    2017-01-01

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

  19. Systems Biology for Organotypic Cell Cultures

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-08-04

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

  20. Using Tissue Culture To Investigate Plant Cell Differentiation and Dedifferentiation.

    Science.gov (United States)

    Bozzone, Donna M.

    1997-01-01

    Describes an experimental project that uses plant tissue culture techniques to examine cell differentiation in the carrot. Allows students to gain experience in some important techniques and to explore fundamental questions about cell differentiation. (DDR)

  1. Cell cultures from the symbiotic soft coral Sinularia flexibilis

    NARCIS (Netherlands)

    Khalesi, M.K.; Vera-Jimenez, N.I.; Aanen, D.K.; Beeftink, H.H.; Wijffels, R.H.

    2008-01-01

    The symbiotic octocoral Sinularia flexibilis is a producer of potential pharmaceuticals. Sustainable mass production of these corals as a source of such compounds demands innovative approaches, including coral cell culture. We studied various cell dissociation methodologies and the feasibility of

  2. Synthesis of polymer materials for use as cell culture substrates

    Energy Technology Data Exchange (ETDEWEB)

    Lakard, Sophie [Laboratoire de Chimie des Materiaux et Interfaces, University of Franche-Comte, IUT, 30 Avenue de l' Observatoire, 25009 Besancon (France)], E-mail: sophie.lakard@univ-fcomte.fr; Morrand-Villeneuve, Nadege [Laboratoire de Neurosciences, University of Franche-Comte, Place Leclerc, 25030 Besancon (France); Lesniewska, Eric [Laboratoire de Physique de l' Universite de Bourgogne, University of Bourgogne, 9 Avenue Savary, 21078 Dijon (France); Lakard, Boris [Laboratoire de Chimie des Materiaux et Interfaces, University of Franche-Comte, 16 Route de Gray, 25030 Besancon (France); Michel, Germaine [Laboratoire de Neurosciences, University of Franche-Comte, Place Leclerc, 25030 Besancon (France); Herlem, Guillaume [Laboratoire de Chimie des Materiaux et Interfaces, University of Franche-Comte, 16 Route de Gray, 25030 Besancon (France); Gharbi, Tijani [Laboratoire d' Optique P.M. Duffieux, University of Franche-Comte, 16 Route de Gray, 25030 Besancon (France); Fahys, Bernard [Laboratoire de Chimie des Materiaux et Interfaces, University of Franche-Comte, 16 Route de Gray, 25030 Besancon (France)

    2007-12-20

    Up to today, several techniques have been used to maintain cells in culture for studying many aspects of cell biology and physiology. More often, cell culture is dependent on proper anchorage of cells to the growth surface. Thus, poly-L-lysine, fibronectin or laminin are the most commonly used substrates. In this study, electrosynthesized biocompatible polymer films are proposed as an alternative to these standard substrates. The electrosynthesized polymers tested were polyethylenimine, polypropylenimine and polypyrrole. Then, the adhesion, proliferation and morphology of rat neuronal cell lines were investigated on these polymer substrates in an attempt to develop new and efficient polymer materials for cell culture. During their growth on the polymers, the evolution of the cell morphology was monitored using both confocal microscopy and immunohistochemistry, leading to the conclusion of a normal development. An estimation of the adhesion and proliferation rates of rat neuronal cell cultures indicated that polyethylenimine and polypropylenimine were the best substrates for culturing olfactory neuronal cells. A method to favour the differentiation of the neuronal cells was also developed since the final aim of this work is to develop a biosensor for odour detection using differentiated neuronal cells as transducers. Consequently, a biosensor was microfabricated using silicon technology. This microsystem allowed us to culture the cells on a silicon wafer and to position the cells on certain parts of the silicon wafer.

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

    Science.gov (United States)

    Sundin, D R; Mecham, J O

    1989-07-01

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

  4. 3D cell culture systems: advantages and applications.

    Science.gov (United States)

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

    2015-01-01

    Cell cultures are important material of study for the variety of advantages that they offer. Both established continuous cell lines and primary cell cultures continue to be invaluable for basic research and for direct applications. Technological advancements are necessary to address emerging complex challenges and the way cells are cultured in vitro is an area of intense activity. One important advancement in cell culture techniques has been the introduction of three dimensional culture systems. This area is one of the fastest growing experimental approaches in life sciences. Augmented with advancements in cell imaging and analytical systems, as well as the applications of new scaffolds and matrices, cells have been increasingly grown as three dimensional models. Such cultures have proven to be closer to in vivo natural systems, thus proving to be useful material for many applications. Here, we review the three dimensional way of culturing cells, their advantages, the scaffolds and matrices currently available, and the applications of such cultures in major areas of life sciences. © 2014 Wiley Periodicals, Inc.

  5. Induction of interdigitating cell processes in podocyte culture.

    Science.gov (United States)

    Yaoita, Eishin; Yoshida, Yutaka; Nameta, Masaaki; Takimoto, Hiroki; Fujinaka, Hidehiko

    2017-09-07

    Highly organized cell processes characterize glomerular podocytes in vivo. However, podocytes in culture have a simple morphology lacking cell processes, especially upon reaching confluence. Here, we aimed to establish culture conditions under which cultured podocytes extend cell processes at confluence. Among various culture conditions that could possibly cause phenotypic changes in podocytes, we examined the effects of heparin, all-trans retinoic acid, fetal bovine serum, and extracellular matrices on the morphology of podocytes in rat primary culture. Consequently, long arborized cell processes were observed to radiate extensively from the cell body only when cells were cultured in the presence of heparin and all-trans retinoic acid on laminin-coated dishes with decreasing concentrations of fetal bovine serum. Primary processes branching repeatedly into terminal processes and cell process insertion under adjacent cell bodies were evident by electron microscopy-based analysis. Immunostaining for podocin showed conspicuous elongations of intercellular junctions. Under these conditions, the expression levels of podocyte-specific proteins and genes were markedly upregulated. Thus, we succeeded in establishing culture conditions in which the cultured podocytes exhibit phenotypes similar to those under in vivo conditions. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  6. Establishment of sorghum cell suspension culture system for ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-03-18

    Mar 18, 2008 ... This study describes the establishment of sorghum cell suspension culture system for use in proteomics studies. ... Key words: Sorghum, proteomics, callus, cell suspension cultures, total soluble protein, secretome. INTRODUCTION ..... system, are dynamic and heterogeneous, being com- posed of a ...

  7. Establishment of the callus and cell suspension culture of ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-05

    Oct 5, 2009 ... plant growth regulators on the callus induction and accumulation of condensed tannins, and (iii) determine the optimum medium and the hormone combination for cell suspension culture of E. angustifolia. This paper presents the feasibility of condensed tannins production in callus and cell culture of E.

  8. Viable Cell Culture Banking for Biodiversity Characterization and Conservation.

    Science.gov (United States)

    Ryder, Oliver A; Onuma, Manabu

    2018-02-15

    Because living cells can be saved for indefinite periods, unprecedented opportunities for characterizing, cataloging, and conserving biological diversity have emerged as advanced cellular and genetic technologies portend new options for preventing species extinction. Crucial to realizing the potential impacts of stem cells and assisted reproductive technologies on biodiversity conservation is the cryobanking of viable cell cultures from diverse species, especially those identified as vulnerable to extinction in the near future. The advent of in vitro cell culture and cryobanking is reviewed here in the context of biodiversity collections of viable cell cultures that represent the progress and limitations of current efforts. The prospects for incorporating collections of frozen viable cell cultures into efforts to characterize the genetic changes that have produced the diversity of species on Earth and contribute to new initiatives in conservation argue strongly for a global network of facilities for establishing and cryobanking collections of viable cells.

  9. Eliminating acute lymphoblastic leukemia cells from human testicular cell cultures: a pilot study

    NARCIS (Netherlands)

    Sadri-Ardekani, Hooman; Homburg, Christa H.; van Capel, Toni M. M.; van den Berg, Henk; van der Veen, Fulco; van der Schoot, C. Ellen; van Pelt, Ans M. M.; Repping, Sjoerd

    2014-01-01

    To study whether acute lymphoblastic leukemia (ALL) cells survive in a human testicular cell culture system. Experimental laboratory study. Reproductive biology laboratory, academic medical center. Acute lymphoblastic leukemia cells from three patients and testicular cells from three other patients.

  10. Long-term maintenance of human induced pluripotent stem cells by automated cell culture system.

    Science.gov (United States)

    Konagaya, Shuhei; Ando, Takeshi; Yamauchi, Toshiaki; Suemori, Hirofumi; Iwata, Hiroo

    2015-11-17

    Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem (iPS) cells, are regarded as new sources for cell replacement therapy. These cells can unlimitedly expand under undifferentiated conditions and be differentiated into multiple cell types. Automated culture systems enable the large-scale production of cells. In addition to reducing the time and effort of researchers, an automated culture system improves the reproducibility of cell cultures. In the present study, we newly designed a fully automated cell culture system for human iPS maintenance. Using an automated culture system, hiPS cells maintained their undifferentiated state for 60 days. Automatically prepared hiPS cells had a potency of differentiation into three germ layer cells including dopaminergic neurons and pancreatic cells.

  11. PECULIARITIES OF SECONDARY METABOLITES BIOSYNTHESIS IN PLANT CELL CULTURES

    Directory of Open Access Journals (Sweden)

    A.M. NOSOV

    2014-06-01

    Full Text Available metabolites formation in plant cell cultures of Panax spp., (ginsenosides; Dioscorea deltoidea (steroid glycosides; Ajuga reptans, Serratula coronata, Rhaponticum carthamoides (ecdisteroids; Polyscias spp., (triterpene glycosides, Taxus spp. (taxoids, Stevia rebaudiana (diterpene steviol-glycosides, Stephania glabra (alkaloids. They are some regular trends of secondary metabolites synthesis in the plant cell culture:It can be noted the stable synthesis of the compound promoting cell proliferation. Indeed, cell cultures of Dioscorea deltoidea were demonstrated to accumulate only furostanol glycosides, which promoted cell division. Furostanol glycoside content of Dioscorea strain DM-0.5 was up to 6 - 12% by dry biomass.Panax ginseng and P. japonicus plant cell cultures synthesize as minimum seven triterpene glycosides (ginsenosides, the productivity of these compounds was up to 6.0 - 8.0% on dry biomass.By contrast, the detectable synthesis of diterpene steviol-glycosides in cultivated cells of Stevia rebaudiana initiated in the mixotrophic cultures during chloroplast formation only.Despite these differences, or mainly due to them, plant cell cultures have become an attractive source of phytochemicals in alternative to collecting wild plants. It provides a guideline to bioreactor-based production of isoprenoids using undifferentiated plant cell cultures

  12. The release of iron by Sertoli cells in culture

    NARCIS (Netherlands)

    Wauben-Penris, P. J.; Veldscholte, J.; van der Ende, A.; van der Donk, H. A.

    1988-01-01

    In seminiferous tubules, iron transport from the blood to the abluminal germinal cells must occur through the Sertoli cell cytoplasm. We investigated the release of previously accumulated iron by cultured Sertoli cells. We found that Sertoli cells contain easily releasable and less easily releasable

  13. Morphological and Immunohistochemical Characterization of Canine Osteosarcoma Spheroid Cell Cultures.

    Science.gov (United States)

    Gebhard, C; Gabriel, C; Walter, I

    2016-06-01

    Spheroid cell culture emerges as powerful in vitro tool for experimental tumour research. In this study, we established a scaffold-free three-dimensional spheroid system built from canine osteosarcoma (OS) cells (D17). Spheroids (7, 14 and 19 days of cultivation) and monolayer cultures (2 and 7 days of cultivation) were evaluated and compared on light and electron microscopy. Monolayer and spheroid cultures were tested for vimentin, cytokeratin, alkaline phosphatase, osteocalcin and collagen I by means of immunohistochemistry. The spheroid cell culture exhibited a distinct network of collagen I in particular after 19-day cultivation, whereas in monolayer cultures, collagen I was arranged as a lamellar basal structure. Necrotic centres of large spheroids, as observed in 14- and 19-day cultures, were characterized by significant amounts of osteocalcin. Proliferative activity as determined by Ki-67 immunoreactivity showed an even distribution in two-dimensional cultures. In spheroids, proliferation was predominating in the peripheral areas. Metastasis-associated markers ezrin and S100A4 were shown to be continuously expressed in monolayer and spheroid cultures. We conclude that the scaffold-free spheroid system from canine OS cells has the ability to mimic the architecture of the in vivo tumour, in particular cell-cell and cell-matrix interactions. © 2015 The Authors. Anatomia, Histologia, Embryologia Published by Blackwell Verlag GmbH.

  14. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Tissue culture media for human ex vivo tissue and... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell culture...

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

    Science.gov (United States)

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

    2014-01-01

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

  16. A kinetic model for flavonoid production in tea cell culture.

    Science.gov (United States)

    Shibasaki-Kitakawa, Naomi; Iizuka, Yasuhiro; Takahashi, Atsushi; Yonemoto, Toshikuni

    2017-02-01

    As one of the strategies for efficient production of a metabolite from cell cultures, a kinetic model is very useful tool to predict productivity under various culture conditions. In this study, we propose a kinetic model for flavonoid production in tea cell culture based on the cell life cycle and expression of PAL, the gene encoding phenylalanine ammonia-lyase (PAL)-the key enzyme in flavonoid biosynthesis. The flavonoid production rate was considered to be related to the amount of active PAL. Synthesis of PAL was modelled based on a general gene expression/translation mechanism, including the transcription of DNA encoding PAL into mRNA and the translation of PAL mRNA into the PAL protein. The transcription of DNA was assumed to be promoted at high light intensity and suppressed by a feedback regulatory mechanism at high flavonoid concentrations. In the model, mRNA and PAL were considered to self-decompose and to be lost by cell rupture. The model constants were estimated by fitting the experimental results obtained from tea cell cultures under various light intensities. The model accurately described the kinetic behaviors of dry and fresh cell concentrations, glucose concentration, cell viability, PAL specific activity, and flavonoid content under a wide range of light intensities. The model simulated flavonoid productivity per medium under various culture conditions. Therefore, this model will be useful to predict optimum culture conditions for maximum flavonoid productivity in cultured tea cells.

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

    Science.gov (United States)

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

    2015-07-10

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

  18. [Experiences with the demonstration of Mycoplasma in cell cultures].

    Science.gov (United States)

    Nicklas, W; Mauter, P

    1988-03-01

    Over an eight years period about 6200 cell cultures, sera, cell culture media and supernatants were routinely monitored for contamination with mycoplasmas, bacteria and fungi. Mycoplasmas were detected in 24.0% of 4443 samples which were checked for possible contamination. In 1742 samples from a laboratory, known to have only mycoplasma free cultures, 2 were positive, both samples having an external origin. The value of routine monitoring to prevent the introduction of mycoplasma was confirmed. Culture and direct fluorescent assay using the fluorochrome bisbenzimide (Hoechst 33258) yielded comparable results. The applicability and significance of both methods is discussed. In spite of a few disadvantages the culture method is considered to be superior to the fluorescence assay, but both methods should be employed in order to obtain sufficiently reliable results. The importance of appropriate methods for the detection of mycoplasmas is stressed because of their potential influence on experimental results. The probable sources of cell culture contamination are also discussed.

  19. Uptake of dehydroascorbic acid and ascorbic acid to isolated nerve terminals and secretory granules from ox neurohypophyses

    DEFF Research Database (Denmark)

    Thorn, N A; Nielsen, F S; Jeppesen, C K

    1986-01-01

    When uptake of L-[14C]ascorbic acid ([14C]AA) to various organs in guinea-pigs was studied after intracardiac injection, the adenohypophysis, pars intermedia, and the neurohypophysis had an uptake per milligramme protein which was about half of the uptake to the adrenals. Adrenal uptake was 20...... labelled dehydroascorbic acid ([14C]DHA), the uptake was much slower than when the medium contained labelled ascorbic acid. The uptake of [14C]DHA showed a linear dependence on concentration, and was not influenced by addition of Mg2+ and ATP. Addition of Mg2+ + ATP, omission of Ca2+ and Mg2+ or exchange...... of Na+ in the medium with K+ had no effect on the uptake of ascorbic acid. When isolated secretory granules from ox neurohypophyses were incubated with a medium containing [14C]DHA, uptake was considerably faster than the uptake when they were incubated in a medium containing [14C]AA. The uptake...

  20. Type III Bartter-like syndrome in an infant boy with Gitelman syndrome and autosomal dominant familial neurohypophyseal diabetes insipidus.

    Science.gov (United States)

    Brugnara, Milena; Gaudino, Rossella; Tedeschi, Silvana; Syrèn, Marie-Louise; Perrotta, Silverio; Maines, Evelina; Zaffanello, Marco

    2014-09-01

    We report the case of an infant boy with polyuria and a familial history of central diabetes insipidus. Laboratory blood tests disclosed hypokalemia, metabolic alkalosis, hyperreninemia, and hyperaldosteronism. Plasma magnesium concentration was slightly low. Urine analysis showed hypercalciuria, hyposthenuria, and high excretion of potassium. Such findings oriented toward type III Bartter syndrome (BSIII). Direct sequencing of the CLCNKB gene revealed no disease-causing mutations. The water deprivation test was positive. Magnetic resonance imaging showed a lack of posterior pituitary hyperintensity. Finally, direct sequencing of the AVP-NPII gene showed a point mutation (c.1884G>A) in a heterozygous state, confirming an autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI). This condition did not explain the patient's phenotype; thus, we investigated for Gitelman syndrome (GS). A direct sequencing of the SLC12A3 gene showed c.269A>C and c.1205C>A new mutations. In conclusion, the patient had a genetic combination of GS and adFNDI with a BSIII-like phenotype.

  1. Cell culture arrays using magnetic force-based cell patterning for dynamic single cell analysis.

    Science.gov (United States)

    Ino, Kosuke; Okochi, Mina; Konishi, Nao; Nakatochi, Masahiro; Imai, Rentaro; Shikida, Mitsuhiro; Ito, Akira; Honda, Hiroyuki

    2008-01-01

    In order to understand the behavior of individual cells, single cell analyses have attracted attention since most cell-based assays provide data with values averaged across a large number of cells. Techniques for the manipulation and analysis of single cells are crucial for understanding the behavior of individual cells. In the present study, we have developed single cell culture arrays using magnetic force and a pin holder, which enables the allocation of the magnetically labeled cells on arrays, and have analyzed their dynamics. The pin holder was made from magnetic soft iron and contained more than 6000 pillars on its surface. The pin holder was placed on a magnet to concentrate the magnetic flux density above the pillars. NIH/3T3 fibroblasts that were labeled with magnetite cationic liposomes (MCLs) were seeded into a culture dish, and the dish was placed over the pin holder with the magnet. The magnetically labeled cells were guided on the surface where the pillars were positioned and allocated on the arrays with a high resolution. Single-cell patterning was achieved by adjusting the number of cells seeded, and the target cell was collected by a micromanipulator after removing the pin holder with the magnet. Furthermore, change in the morphology of magnetically patterned cells was analyzed by microscopic observation, and cell spreading on the array was observed with time duration. Magnetic force-based cell patterning on cell culture arrays would be a suitable technique for the analysis of cell behavior in studies of cell-cell variation and cell-cell interactions.

  2. Glial cells aneuploid from culture of equine neonatal spinal cord

    OpenAIRE

    Maia, Leandro [UNESP; Mota, Ligia Souza Lima de Oliveira da [UNESP; Alvarenga, Fernanda da Cruz Landrim e [UNESP; Amorim, Renée Laufer [UNESP; Vita, Bruna de [UNESP; Moraes, Carolina Nogueira de [UNESP; Amorim, Rogério Martins [UNESP

    2012-01-01

    The aim of this communication is to report the occurrence of glia cells aneuploid obtained from the culture of spinal cord of a newborn horse. Cells were maintained in culture until the sixth passage characterized by imunocytochemistry technique prior to cytogenetic analysis. Karyotype analysis showed loss or gain of one or more chromosomes in glial cells analyzed, when compared with the normal karyotype for equine specie. The occurrence of aneuploidy may be considered a normal finding in you...

  3. Multizone Paper Platform for 3D Cell Cultures

    Science.gov (United States)

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

    2011-01-01

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

  4. Adenosine formation in contracting primary rat skeletal muscle cells and endothelial cells in culture

    DEFF Research Database (Denmark)

    Hellsten, Ylva; Frandsen, Ulrik

    1997-01-01

    1. The present study examined the capacity for adenosine formation, uptake and metabolism in contracting primary rat muscle cells and in microvascular endothelial cells in culture. 2. Strong and moderate electrical simulation of skeletal muscle cells led to a significantly greater increase...... in the extracellular adenosine concentration (421 +/- 91 and 235 +/- 30 nmol (g protein)-1, respectively; P muscle cells (161 +/- 20 nmol (g protein)-1). The ATP concentration was lower (18%; P muscle cells....... 3. Addition of microvascular endothelial cells to the cultured skeletal muscle cells enhanced the contraction-induced accumulation of extracellular adenosine (P cells in culture alone did not cause extracellular accumulation of adenosine. 4. Skeletal muscle cells were...

  5. A de Novo mutation in the coding sequence for neurophysin-II (Pro{sup 24} {yields} Leu) is associated with onset and transmission of autosomal dominant neurohypophyseal diabetes insipidus

    Energy Technology Data Exchange (ETDEWEB)

    Repaske, D.R.; Browning, J.E. [Children`s Hospital Medical Center, Cincinnati, OH (United States)

    1994-08-01

    The molecular basis of autosomal dominant neurohypophyseal diabetes insipidus, a hereditary deficiency of vasopressin, was determined by nucleotide sequence analysis of the arginine vasopressin-neurophysin-II gene. A C{yields}T mutation at nucleotide 1761 was detected in one allele of this gene in each affected individual in three generations of one family. This mutant gene encodes a normal arginine vasopressin peptide, but predicts a substitution of leucine for proline at amino acid 24 of neurophysin-II, the arginine vasopressin carrier protein. This mutation was not detected in 50 control individuals, thus demonstrating that it is not a common silent genetic polymorphism. The disease arose in the second generation of the studied family, and the chromosome 20 carrying this new mutation was identified by polymorphic CA microsatellite haplotype analysis. The first affected individual inherited this chromosome segment from her mother, who had neither the disease nor this mutation in her somatic cell DNA. Third generation individuals who subsequently inherited this mutation were affected. These data demonstrate that this amino acid substitution in neurophysin-II causes this disease. Two possibilities to explain the mechanism by which clinical deficiency of arginine vasopressin develops even in the presence of one normal arginine vasopressin-neurophysin-II allele are discussed. 40 refs., 4 figs., 2 tabs.

  6. Novel culturing platform for brain slices and neuronal cells

    DEFF Research Database (Denmark)

    Svendsen, Winnie Edith; Al Atraktchi, Fatima Al-Zahraa; Bakmand, Tanya

    2015-01-01

    In this paper we demonstrate a novel culturing system for brain slices and neuronal cells, which can control the concentration of nutrients and the waste removal from the culture by adjusting the fluid flow within the device. The entire system can be placed in an incubator. The system has been te...... tested successfully with brain slices and PC12 cells. The culture substrate can be modified using metal electrodes and/or nanostructures for conducting electrical measurements while culturing and for better mimicking the in vivo conditions....

  7. A practical guide to hydrogels for cell culture.

    Science.gov (United States)

    Caliari, Steven R; Burdick, Jason A

    2016-04-28

    There is growing appreciation of the role that the extracellular environment plays in regulating cell behavior. Mechanical, structural, and compositional cues, either alone or in concert, can drastically alter cell function. Biomaterials, and particularly hydrogels, have been developed and implemented to present defined subsets of these cues for investigating countless cellular processes as a means of understanding morphogenesis, aging, and disease. Although most scientists concede that standard cell culture materials (tissue culture plastic and glass) do a poor job of recapitulating native cellular milieus, there is currently a knowledge barrier for many researchers in regard to the application of hydrogels for cell culture. Here, we introduce hydrogels to those who may be unfamiliar with procedures to culture and study cells with these systems, with a particular focus on commercially available hydrogels.

  8. Controlling the diversity of cell populations in a stem cell culture

    NARCIS (Netherlands)

    Heo, Inha; Clevers, Hans

    2015-01-01

    Culturing intestinal stem cells into 3D organoids results in heterogeneous cell populations, reflecting the in vivo cell type diversity. In a recent paper published in Nature, Wang et al. established a culture condition for a highly homogeneous population of intestinal stem cells.

  9. [Stimulation of cholinogenesis in the human fetal nerve cells culture].

    Science.gov (United States)

    Tsymbaliuk, V I; Vasyl'ieva, I H; Oleksenko, N P; Chopyk, N H; Tsiubko, O I; Halanta, O S

    2013-01-01

    The aim of the research was to establish cultured population of nerve cells reached by cholinergic neurons and their determinative precursors. The most effective combination of neuroinductors which stimulated cholinergic cells differentiation from the nerve stem cells was retinoic acid and acetylcholine. During the period of culturing the amount of ChAT+ cells reliably increased from 5.3 +/- 2.9% to 21.1 +/- 6.2%. At the same time in the control samples their concentration was 9.1 +/- 4.8% of total cell count. Enrichment of cell population by cholinergic neurons and their determinative precursors correlated with increasing of AChE-activity level. So, addition of retinoic acid and acetylcholine stimulate both neurogenesis and cholinogenesis in the culture of human fetal nerve cells.

  10. Quantitative volumetric Raman imaging of three dimensional cell cultures

    Science.gov (United States)

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

    2017-03-01

    The ability to simultaneously image multiple biomolecules in biologically relevant three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell-material interactions. Here, we present a computational framework for label-free quantitative volumetric Raman imaging (qVRI). We apply qVRI to a selection of biological systems: human pluripotent stem cells with their cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy.

  11. Nylon-3 polymers that enable selective culture of endothelial cells.

    Science.gov (United States)

    Liu, Runhui; Chen, Xinyu; Gellman, Samuel H; Masters, Kristyn S

    2013-11-06

    Substrates that selectively encourage the growth of specific cell types are valuable for the engineering of complex tissues. Some cell-selective peptides have been identified from extracellular matrix proteins; these peptides have proven useful for biomaterials-based approaches to tissue repair or regeneration. However, there are very few examples of synthetic materials that display selectivity in supporting cell growth. We describe nylon-3 polymers that support in vitro culture of endothelial cells but do not support the culture of smooth muscle cells or fibroblasts. These materials may be promising for vascular biomaterials applications.

  12. Three-Dimensional Cell Cultures in Drug Discovery and Development

    Science.gov (United States)

    Fang, Ye; Eglen, Richard M.

    2017-01-01

    The past decades have witnessed significant efforts toward the development of three-dimensional (3D) cell cultures as systems that better mimic in vivo physiology. Today, 3D cell cultures are emerging, not only as a new tool in early drug discovery but also as potential therapeutics to treat disease. In this review, we assess leading 3D cell culture technologies and their impact on drug discovery, including spheroids, organoids, scaffolds, hydrogels, organs-on-chips, and 3D bioprinting. We also discuss the implementation of these technologies in compound identification, screening, and development, ranging from disease modeling to assessment of efficacy and safety profiles. PMID:28520521

  13. Surface modified alginate microcapsules for 3D cell culture

    Science.gov (United States)

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

    2016-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Davood Mehrabani

    2014-01-01

    Full Text Available 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.

  15. Air pollutant production by algal cell cultures

    Science.gov (United States)

    Fong, F.; Funkhouser, E. A.

    1982-01-01

    The production of phytotoxic air pollutants by cultures of Chlorella vulgaris and Euglena gracilis is considered. Algal and plant culture systems, a fumigation system, and ethylene, ethane, cyanide, and nitrogen oxides assays are discussed. Bean, tobacco, mustard green, cantaloupe and wheat plants all showed injury when fumigated with algal gases for 4 hours. Only coleus plants showed any resistance to the gases. It is found that a closed or recycled air effluent system does not produce plant injury from algal air pollutants.

  16. Enhanced casein kinase II activity in human tumour cell cultures

    DEFF Research Database (Denmark)

    Prowald, K; Fischer, H; Issinger, O G

    1984-01-01

    Casein kinase II (CKII) activity is enhanced as much as 2-3 fold in established and 4-5-fold in transformed human cell lines when compared to that of fibroblasts and primary human tumour cell cultures where CKII activity never exceeded a basic level. The high activity of CKII in transformed cells...

  17. Cultured human muscle cells and respiratory chain deficiencies

    NARCIS (Netherlands)

    Herzberg, N. H.; Bolhuis, P. A.; van den Bogert, C.; Barth, P. G.

    1994-01-01

    Cultured muscle cells are useful in the study of respiratory chain disorders. Muscle tissue is affected in most cases and muscle biopsies are often taken for diagnostic purposes. Small samples of the biopsies can provide large numbers of muscle cells. In contrast with most other cell types, the

  18. Improved Method for Culturing Guinea-Pig Macrophage Cells

    Science.gov (United States)

    Savage, J.

    1982-01-01

    Proper nutrients and periodic changes in culture medium maintain cell viability for a longer period. New method uses a thioglycolate solution, instead of mineral oil, to induce macrophage cells in guinea pigs and also uses an increased percent of fetal-calf bovine serum in cultivation medium. Macrophage cells play significant roles in the body's healing and defense systems.

  19. [Cytotoxicity studies on T-3262 in cultured Chinese hamster cells].

    Science.gov (United States)

    Yoneda, T; Nakamura, S; Nojima, Y; Nishio, Y

    1989-04-01

    T-3262 is an antibacterial drug which belongs to the group of pyridonecarboxylic acids. In this study, we investigated cytotoxicity of T-3262 for inhibition of cell growth and effects on viability of, and morphological changes in cultured Chinese hamster cells (V79 cells). The following results were obtained. 1. The 50% inhibition dose of T-3262 for cell growth (ID50, cultured for 48 hours) was 12 micrograms/ml, showing that the inhibitory effect of T-3262 on the cell growth was stronger than that of enoxacin (ENX: ID50 44 micrograms/ml), norfloxacin (NFLX: ID50 105 micrograms/ml) or ofloxacin (OFLX: ID50 145 micrograms/ml). 2. The number of cells increased and dead cells were scarcely seen at the highest concentration tested in culture medium (40 micrograms/ml of T-3262 for 48 hours). At this concentration, degeneration of cytoplasm (atrophy and round shape) and decrease of mitotic cells were observed. These morphological changes were similar to those of the cells treated 400 micrograms/ml of NFLX or OFLX for 48 hours. 3. After the removal of T-3262 from culture medium, the cells began to grow actively and recovered from the morphological changes. The similar phenomenon was observed with ENX treated cells but not with fluorouracil or mitomycin C treated cells.

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

    Science.gov (United States)

    Pandurangan, Muthuraman; Hwang, Inho

    2014-09-01

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

  1. Expansion of Endothelial Progenitor Cells in High Density Dot Culture of Rat Bone Marrow Cells

    Science.gov (United States)

    Wang, Ling; Kretlow, James D.; Zhou, Guangdong; Cao, Yilin; Liu, Wei; Zhang, Wen Jie

    2014-01-01

    In vitro expansion of endothelial progenitor cells (EPCs) remains a challenge in stem cell research and its application. We hypothesize that high density culture is able to expand EPCs from bone marrow by mimicking cell-cell interactions of the bone marrow niche. To test the hypothesis, rat bone marrow cells were either cultured in high density (2×105 cells/cm2) by seeding total 9×105 cells into six high density dots or cultured in regular density (1.6×104 cells/cm2) with the same total number of cells. Flow cytometric analyses of the cells cultured for 15 days showed that high density cells exhibited smaller cell size and higher levels of marker expression related to EPCs when compared to regular density cultured cells. Functionally, these cells exhibited strong angiogenic potentials with better tubal formation in vitro and potent rescue of mouse ischemic limbs in vivo with their integration into neo-capillary structure. Global gene chip and ELISA analyses revealed up-regulated gene expression of adhesion molecules and enhanced protein release of pro-angiogenic growth factors in high density cultured cells. In summary, high density cell culture promotes expansion of bone marrow contained EPCs that are able to enhance tissue angiogenesis via paracrine growth factors and direct differentiation into endothelial cells. PMID:25254487

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

    Science.gov (United States)

    Chun, Song-I; Mun, Chi-Woong

    2015-01-01

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

  3. The Multiparametric Effects of Hydrodynamic Environments on Stem Cell Culture

    Science.gov (United States)

    Kinney, Melissa A.; Sargent, Carolyn Y.

    2011-01-01

    Stem cells possess the unique capacity to differentiate into many clinically relevant somatic cell types, making them a promising cell source for tissue engineering applications and regenerative medicine therapies. However, in order for the therapeutic promise of stem cells to be fully realized, scalable approaches to efficiently direct differentiation must be developed. Traditionally, suspension culture systems are employed for the scale-up manufacturing of biologics via bioprocessing systems that heavily rely upon various types of bioreactors. However, in contrast to conventional bench-scale static cultures, large-scale suspension cultures impart complex hydrodynamic forces on cells and aggregates due to fluid mixing conditions. Stem cells are exquisitely sensitive to environmental perturbations, thus motivating the need for a more systematic understanding of the effects of hydrodynamic environments on stem cell expansion and differentiation. This article discusses the interdependent relationships between stem cell aggregation, metabolism, and phenotype in the context of hydrodynamic culture environments. Ultimately, an improved understanding of the multifactorial response of stem cells to mixed culture conditions will enable the design of bioreactors and bioprocessing systems for scalable directed differentiation approaches. PMID:21491967

  4. Retinoids regulate gonadotropin action in cultured rat Sertoli cells.

    Science.gov (United States)

    Galdieri, M; Nisticò, L

    1994-01-01

    The effect of retinoids on cultured rat Sertoli cells was studied by evaluation of cAMP and estradiol production after gonadotropin stimulation in the presence or absence of the retinoid. Sertoli cells cultured in the presence of FSH produce a high amount of cAMP and increase their aromatase activity. The addition of retinol alone has no effect on cAMP and estradiol production; however, the presence of retinol in the culture medium exerts an inhibitory effect on Sertoli cell response to FSH stimulation. In particular, FSH-induced cAMP production of rat Sertoli cells was significantly reduced (50-60% decrease) both by retinol and by retinoic acid. This effect was observable during the first ten days of culture and was also evident when Sertoli cells were cultured in the presence of retinol and methylisobutylxanthine, an inhibitor of phosphodiesterase activity. Cholera toxin-stimulated cAMP levels were reduced by retinol, whereas forskolin-induced elevation of cAMP levels was not affected by vitamin treatment. The inhibitory effect of retinoids on FSH-stimulated aromatase activity of Sertoli cells, which is cAMP mediated, was also evident. In conclusion, the present study demonstrates that retinoids modulate FSH action on cultured rat Sertoli cells and decrease cAMP production.

  5. Maintenance of fetal human pancreatic beta cells in tissue culture.

    Science.gov (United States)

    McEvoy, R C; Thomas, N M; Bowers, C; Ginsberg-Fellner, F

    1986-01-01

    Large quantities of viable human islet tissue (beta cells) are required for transplant and for investigations of the autoimmune basis of Type I diabetes. Fetal pancreas offers a potential advantage over other possible sources of beta cells in that it retains some capacity for growth in vitro. We have cultured a total of 45 human pancreata from fetuses of gestational ages from 18 to 23 weeks. Each pancreas was obtained within minutes after delivery and usually cultured within 30 minutes. Pancreata were dispersed and cultured for up to 32 days. Maintenance and growth of the beta cells was assessed by the content of insulin in extracts of cultured tissue. As has been reported by others, fetal human beta cells survived in vitro for over 4 weeks. In three experiments in which a direct comparison was made, collagenase digestion of the fetal pancreas resulted in a significantly greater loss of insulin content compared to minced tissue cultured without digestion. Storage of three pancreata in medium overnight at 4 degrees C significantly reduced the insulin content of the pancreas compared to pancreata cultured immediately. During culture, the majority of the beta cells (based on insulin content) were found in small, macroscopic clumps attached to the surface of the culture dish, and surrounded by a nearly confluent monolayer of fibroblastoid cells. There was a marked decrease in the insulin content of the tissue during culture, most of it (to less than 25% of the original) occurring over the first 4-6 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Culturing of PC12 Cells, Neuronal Cells, Astrocytes Cultures and Brain Slices in an Open Microfluidic System

    DEFF Research Database (Denmark)

    Al Atraktchi, Fatima Al-Zahraa; Bakmand, Tanya; Rømer Sørensen, Ane

    The brain is the center of the nervous system, where serious neurodegenerative diseases such as Parkinson’s, Alzheimer’s and Huntington’s are products of functional loss in the neural cells (1). Typical techniques used to investigate these diseases lack precise control of the cellular surroundings...... cells, neuronal cells, astrocytes cultures and brain slices. The microfluidic system provides efficient nutrient delivery, waste removal, access to oxygen, fine control over the neurochemical environment and access to modern microscopy. Additionally, the setup consists of an in vitro culturing...... and electrochemical sensor system that enables real time detection of metabolites, e.g. dopamine from cell cultures and brain slices. In summary we present results on culturing of brain slices and cells in the microfluidic system as well as on the incorporation of an electrochemical sensor system for characterization...

  7. Quantitative volumetric Raman imaging of three dimensional cell cultures

    KAUST Repository

    Kallepitis, Charalambos

    2017-03-22

    The ability to simultaneously image multiple biomolecules in biologically relevant three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell–material interactions. Here, we present a computational framework for label-free quantitative volumetric Raman imaging (qVRI). We apply qVRI to a selection of biological systems: human pluripotent stem cells with their cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy.

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

    Directory of Open Access Journals (Sweden)

    Bo-jiang Li

    2015-08-01

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

  9. Radiosensitivity of cultured insect cells: II. Diptera

    Energy Technology Data Exchange (ETDEWEB)

    Koval, T.M.

    1983-10-01

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

  10. In vitro regeneration, flowering, and cell culture of Centaurea species

    African Journals Online (AJOL)

    Yomi

    2012-01-31

    Jan 31, 2012 ... This study was conducted to establish a protocol for in vitro flowering of Centaurea cyanus and cell cultures of Centaurea montana. In four weeks, 50 to 60 adventitious shoots developed on leaf explants cultured in MS medium supplemented with 2.0 mg/L benzylaminopurine (BAP) and 0.1 mg/L indole-3-.

  11. Establishment of sorghum cell suspension culture system for ...

    African Journals Online (AJOL)

    Total soluble proteins (TSP) and culture filtrate (CF) proteins were extracted from the cell culture system and solubilised in urea buffer (9 M urea, 2 M thiourea and 4% CHAPS). Both onedimensional (1D) and two-dimensional (2D) gel analysis of these two proteomes show that the TSP and CF proteomes have different ...

  12. Cell/Tissue Culture Radiation Exposure Facility Project

    Data.gov (United States)

    National Aeronautics and Space Administration — We propose to develop a Cell/Tissue Culture Radiation Exposure Facility (CTC-REF) to enable radiobiologists to investigate the real-time radiation effects on...

  13. Impact of cell culture on recombinant monoclonal antibody product heterogeneity.

    Science.gov (United States)

    Liu, Hongcheng; Nowak, Christine; Shao, Mei; Ponniah, Gomathinayagam; Neill, Alyssa

    2016-09-01

    Recombinant monoclonal antibodies are commonly expressed in mammalian cell culture and purified by several steps of filtration and chromatography. The resulting high purity bulk drug substance still contains product variants differing in properties such as charge and size. Posttranslational modifications and degradations occurring during cell culture are the major sources of heterogeneity in bulk drug substance of recombinant monoclonal antibodies. The focus of the current review is the impact of cell culture conditions on the types and levels of various modifications and degradations of recombinant monoclonal antibodies. Understanding the relationship between cell culture and product variants can help to make consistently safe and efficacious products. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1103-1112, 2016. © 2016 American Institute of Chemical Engineers.

  14. Enhancement of Diosgenin Production in Plantlet and Cell Cultures ...

    African Journals Online (AJOL)

    HSCCC), Sephadex LH-20 chromatography and preparative high performance liquid chromatography (HPLC). The biomass of the plantlet and cell cultures of D. zingiberensis as well as their diosgenin content and yield were analyzed after ...

  15. Human hematopoietic cell culture, transduction, and analyses

    DEFF Research Database (Denmark)

    Bonde, Jesper; Wirthlin, Louisa; Kohn, Donald B

    2008-01-01

    This unit provides methods for introducing genes into human hematopoietic progenitor cells. The Basic Protocol describes isolation of CD34(+) cells, transduction of these cells with a retroviral vector on fibronectin-coated plates, assaying the efficiency of transduction, and establishing long-te...

  16. Generation of a patterned co-culture system composed of adherent cells and immobilized nonadherent cells.

    Science.gov (United States)

    Yamazoe, Hironori; Ichikawa, Takashi; Hagihara, Yoshihisa; Iwasaki, Yasuhiko

    2016-02-01

    Patterned co-culture is a promising technique used for fundamental investigation of cell-cell communication and tissue engineering approaches. However, conventional methods are inapplicable to nonadherent cells. In this study, we aimed to establish a patterned co-culture system composed of adherent and nonadherent cells. Nonadherent cells were immobilized on a substrate using a cell membrane anchoring reagent conjugated to a protein, in order to incorporate them into the co-culture system. Cross-linked albumin film, which has unique surface properties capable of regulating protein adsorption, was used to control their spatial localization. The utility of our approach was demonstrated through the fabrication of a patterned co-culture consisting of micropatterned neuroblastoma cells surrounded by immobilized myeloid cells. Furthermore, we also created a co-culture system composed of cancer cells and immobilized monocytes. We observed that monocytes enhanced the drug sensitivity of cancer cells and its influence was limited to cancer cells located near the monocytes. Therefore, the incorporation of nonadherent cells into a patterned co-culture system is useful for creating culture systems containing immune cells, as well as investigating the influence of these immune cells on cancer drug sensitivity. Various methods have been proposed for creating patterned co-culture systems, in which multiple cell types are attached to a substrate with a desired pattern. However, conventional methods, including our previous report published in Acta Biomaterialia (2010, 6, 526-533), are unsuitable for nonadherent cells. Here, we developed a novel method that incorporates nonadherent cells into the co-culture system, which allows us to precisely manipulate and study microenvironments containing nonadherent and adherent cells. Using this technique, we demonstrated that monocytes (nonadherent cells) could enhance the drug sensitivity of cancer cells and that their influence had a

  17. Impact of cell culture process changes on endogenous retrovirus expression.

    Science.gov (United States)

    Brorson, Kurt; De Wit, Christina; Hamilton, Elizabeth; Mustafa, Mehnaz; Swann, Patrick G; Kiss, Robert; Taticek, Ron; Polastri, Gian; Stein, Kathryn E; Xu, Yuan

    2002-11-05

    Cell culture process changes (e.g., changes in scale, medium formulation, operational conditions) and cell line changes are common during the development life cycle of a therapeutic protein. To ensure that the impact of such process changes on product quality and safety is minimal, it is standard practice to compare critical product quality and safety attributes before and after the changes. One potential concern introduced by cell culture process improvements is the possibility of increased endogenous retrovirus expression to a level above the clearance capability of the subsequent purification process. To address this, retrovirus expression was measured in scaled down and full production scaled Chinese hamster ovary (CHO) cell cultures of four monoclonal antibodies and one recombinant protein before and after process changes. Two highly sensitive, quantitative (Q)-PCR-based assays were used to measure endogenous retroviruses. It is shown that cell culture process changes that primarily alter media components, nutrient feed volume, seed density, cell bank source (i.e., master cell bank vs. working cell bank), and vial size, or culture scale, singly or in combination, do not impact the rate of retrovirus expression to an extent greater than the variability of the Q-PCR assays (0.2-0.5 log(10)). Cell culture changes that significantly alter the metabolic state of the cells and/or rates of protein expression (e.g., pH and temperature shifts, NaButyrate addition) measurably impact the rate of retrovirus synthesis (up to 2 log(10)). The greatest degree of variation in endogenous retrovirus expression was observed between individual cell lines (up to 3 log(10)). These data support the practice of measuring endogenous retrovirus output for each new cell line introduced into manufacturing or after process changes that significantly increase product-specific productivity or alter the metabolic state, but suggest that reassessment of retrovirus expression after other

  18. Exposure to Music Alters Cell Viability and Cell Motility of Human Nonauditory Cells in Culture

    Directory of Open Access Journals (Sweden)

    Nathalia R. Lestard

    2016-01-01

    Full Text Available Although music is part of virtually all cultures in the world, little is known about how it affects us. Since the beginning of this century several studies suggested that the response to music, and to sound in general, is complex and might not be exclusively due to emotion, given that cell types other than auditory hair cells can also directly react to audible sound. The present study was designed to better understand the direct effects of acoustic vibrations, in the form of music, in human cells in culture. Our results suggest that the mechanisms of cell growth arrest and/or cell death induced by acoustic vibrations are similar for auditory and nonauditory cells.

  19. Convoluted cells as a marker for maternal cell contamination in CVS cultures

    DEFF Research Database (Denmark)

    Hertz, Jens Michael; Jensen, P K; Therkelsen, A J

    1987-01-01

    In order to identify cells of maternal origin in CVS cultures, tissue from 1st trimester abortions were cultivated and the cultures stained in situ for X-chromatin. Convoluted cells and maternal fibroblasts were found to be positive. By chromosome analysis of cultures from 105 diagnostic placenta...... biopsies, obtained by the transabdominal route, metaphases of maternal origin were found in nine cases. In eight of these cases colonies of convoluted cells were observed. We conclude that convoluted cells are of maternal origin and are a reliable marker for maternal cell contamination in CVS cultures....

  20. Isolation and Culture of Satellite Cells from Mouse Skeletal Muscle.

    Science.gov (United States)

    Musarò, Antonio; Carosio, Silvia

    2017-01-01

    Skeletal muscle tissue is characterized by a population of quiescent mononucleated myoblasts, localized between the basal lamina and sarcolemma of myofibers, known as satellite cells. Satellite cells play a pivotal role in muscle homeostasis and are the major source of myogenic precursors in mammalian muscle regeneration.This chapter describes protocols for isolation and culturing satellite cells isolated from mouse skeletal muscles. The classical procedure, which will be discussed extensively in this chapter, involves the enzymatic dissociation of skeletal muscles, while the alternative method involves isolation of satellite cells from isolated myofibers in which the satellite cells remain in their in situ position underneath the myofiber basal lamina.In particular, we discuss the technical aspect of satellite cell isolation, the methods necessary to enrich the satellite cell fraction and the culture conditions that optimize proliferation and myotube formation of mouse satellite cells.

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

    Science.gov (United States)

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

    2017-01-01

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

  2. Topological defects control collective dynamics in neural progenitor cell cultures

    Science.gov (United States)

    Kawaguchi, Kyogo; Kageyama, Ryoichiro; Sano, Masaki

    2017-04-01

    Cultured stem cells have become a standard platform not only for regenerative medicine and developmental biology but also for biophysical studies. Yet, the characterization of cultured stem cells at the level of morphology and of the macroscopic patterns resulting from cell-to-cell interactions remains largely qualitative. Here we report on the collective dynamics of cultured murine neural progenitor cells (NPCs), which are multipotent stem cells that give rise to cells in the central nervous system. At low densities, NPCs moved randomly in an amoeba-like fashion. However, NPCs at high density elongated and aligned their shapes with one another, gliding at relatively high velocities. Although the direction of motion of individual cells reversed stochastically along the axes of alignment, the cells were capable of forming an aligned pattern up to length scales similar to that of the migratory stream observed in the adult brain. The two-dimensional order of alignment within the culture showed a liquid-crystalline pattern containing interspersed topological defects with winding numbers of +1/2 and -1/2 (half-integer due to the nematic feature that arises from the head-tail symmetry of cell-to-cell interaction). We identified rapid cell accumulation at +1/2 defects and the formation of three-dimensional mounds. Imaging at the single-cell level around the defects allowed us to quantify the velocity field and the evolving cell density; cells not only concentrate at +1/2 defects, but also escape from -1/2 defects. We propose a generic mechanism for the instability in cell density around the defects that arises from the interplay between the anisotropic friction and the active force field.

  3. Growth and Plating of Cell Suspension Cultures of Datura Innoxia

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1974-01-01

    Suspension cultures of Datura innoxia Mill, were successfully grown on a modified Murashige and Skoog medium with 2,4–D, NAA or BAP as growth substances, provided the micronutrient levels were reduced to 1/10. Normal amounts of micronutrients were toxic. Attempts to identify the toxic elements did...... malate) or on NO3−-N alone. Dry weight yield was proportional to the amount of nitrate-N added (47 mg/mg N). Filtered suspension cultures containing single cells (plating cultures) could be grown in agar in petri dishes when NAA or 2,4-D were used as growth substances. Cells grew at densities above 500...

  4. Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Met, Ozcan

    2011-01-01

    in water instead of phosphate-buffered saline. Passively adsorbed IL-4 was observed to induce differentiation to dendritic cells, but analysis of cell culture supernatants revealed that leakage of IL-4 into solution could account for the differentiation observed. Covalent attachment resulted in bound IL-4...... at similar concentrations to the passive adsorption process, as measured by enzyme-linked immunosorbent assays, and the bound IL-4 did not leak into solution to any measurable extent during cell culture. However, covalently bound IL-4 was incapable of inducing monocyte differentiation. This may be caused......Standard cell culture plastic was surface modified by passive adsorption or covalent attachment of interleukin (IL)-4 and investigated for its ability to induce differentiation of human monocytes into mature dendritic cells, a process dose-dependently regulated by IL-4. Covalent attachment of IL-4...

  5. Learning about Cells as Dynamic Entities: An Inquiry-Driven Cell Culture Project

    Science.gov (United States)

    Palombi, Peggy Shadduck; Jagger, Kathleen Snell

    2008-01-01

    Using cultured fibroblast cells, undergraduate students explore cell division and the responses of cultured cells to a variety of environmental changes. The students learn new research techniques and carry out a self-designed experiment. Through this project, students enhance their creative approach to scientific inquiry, learn time-management and…

  6. EXPLANTATION OF MESANGIAL CELL HILLOCKS - A METHOD FOR OBTAINING HUMAN MESANGIAL CELLS IN CULTURE

    NARCIS (Netherlands)

    MULLER, EW; KIM, Y; MICHAEL, AF; VERNIER, RL; VANDERHEM, GK; VANDERWOUDE, FJ

    A simple method is presented for selective cell culture of human mesangial cells using explanatation of mesangial cell hillocks. Glomeruli which had been incubated with collagenase were explanted on plastic tissue culture flasks. Three to 6 weeks after explantation, a rapidly growing multilayer of

  7. Lacrimal gland primary acinar cell culture: the role of insulin

    Directory of Open Access Journals (Sweden)

    Leonardo Tannus Malki

    2016-04-01

    Full Text Available ABSTRACT Purpose: The goal of the present study was to establish a protocol for primary culture of lacrimal gland acinar cells (LGACs and to assess the effect of adding insulin to the culture media. Methods: LGACs were isolated and cultured from lacrimal glands of Wistar male rats. The study outcomes included cell number, viability, and peroxidase release over time and in response to three concentrations of insulin (0.5, 5.0, and 50.0 μg/mL. Results: In LGAC primary culture, cells started to form clusters by day 3. There was a time-response pattern of peroxidase release, which rose by day 6, in response to carbachol. Culture viability lasted for 12 days. An insulin concentration of 5.0 μg/mL in the culture medium resulted in higher viability and secretory capacity. Conclusions: The present method simplifies the isolation and culture of LGACs. The data confirmed the relevance of adding insulin to maintain LGACs in culture.

  8. Introducing Mammalian Cell Culture and Cell Viability Techniques in the Undergraduate Biology Laboratory.

    Science.gov (United States)

    Bowey-Dellinger, Kristen; Dixon, Luke; Ackerman, Kristin; Vigueira, Cynthia; Suh, Yewseok K; Lyda, Todd; Sapp, Kelli; Grider, Michael; Crater, Dinene; Russell, Travis; Elias, Michael; Coffield, V McNeil; Segarra, Verónica A

    2017-01-01

    Undergraduate students learn about mammalian cell culture applications in introductory biology courses. However, laboratory modules are rarely designed to provide hands-on experience with mammalian cells or teach cell culture techniques, such as trypsinization and cell counting. Students are more likely to learn about cell culture using bacteria or yeast, as they are typically easier to grow, culture, and manipulate given the equipment, tools, and environment of most undergraduate biology laboratories. In contrast, the utilization of mammalian cells requires a dedicated biological safety cabinet and rigorous antiseptic techniques. For this reason, we have devised a laboratory module and method herein that familiarizes students with common cell culture procedures, without the use of a sterile hood or large cell culture facility. Students design and perform a time-efficient inquiry-based cell viability experiment using HeLa cells and tools that are readily available in an undergraduate biology laboratory. Students will become familiar with common techniques such as trypsinizing cells, cell counting with a hemocytometer, performing serial dilutions, and determining cell viability using trypan blue dye. Additionally, students will work with graphing software to analyze their data and think critically about the mechanism of death on a cellular level. Two different adaptations of this inquiry-based lab are presented-one for non-biology majors and one for biology majors. Overall, these laboratories aim to expose students to mammalian cell culture and basic techniques and help them to conceptualize their application in scientific research.

  9. Study on enzymatic browning in suspension cultures of licorice cells

    Directory of Open Access Journals (Sweden)

    Yali Li

    2016-03-01

    Full Text Available Enzymatic browning is one of the main obstacles encountered in the establishment of suspension systems of licorice cells. Browning of cells may result in decreased viability, poor growth and even death. The present study investigated the mechanism of browning reactions and the effective controlling methods. The results showed that the cell viability and membrane permeabilization obviously changed when the cells were transferred to liquid medium. The transformation caused rapid increase in the levels of polyphenol oxidase activity and in the production of polyphenols. Osmotic and hydrodynamic stresses arising from liquid culture were regarded as the major causes of enzymatic browning. Ascorbic acid and L-cysteine were found to be the most significant anti-browning agents that could decrease the degree of browning with 55.8% and 52.2%, respectively, at the end of the suspension culture's lag phase. When cultured with a cycle of 21 days, the maximum biomass of the cells cultured with ascorbic acid and L-cysteine increased with 31.1% and 26.5%, respectively, when compared to the control. These findings may be essential for the development of licorice cell cultures devoted to browning prevention and cell viability maintaining.

  10. Recombinant spider silk matrices for neural stem cell cultures.

    Science.gov (United States)

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

    2012-11-01

    Neural stem cells (NSCs) have the capacity to differentiate into neurons, astrocytes, and oligodendrocytes. Accordingly, NSCs hold great promise in drug screening and treatment of several common diseases. However, a major obstacle in applied stem cell research is the limitation of synthetic matrices for culturing stem cells. The objective of this study was to evaluate the suitability of recombinant spider silk (4RepCT) matrices for growth of NSCs. NSCs isolated from the cerebral cortices of mid-gestation rat embryos were cultured on either 4RepCT matrices or conventional poly-L-ornithine and fibronectin (P + F) coated polystyrene plates. From 48 h of culture, no significant differences in cell proliferation or viability were detected in NSC cultures on 4RepCT compared to control matrices (polystyrene plates coated with P + F). The NSCs retained an undifferentiated state, displaying low or no staining for markers of differentiated cells. Upon stimulation NSCs grown on 4RepCT differentiated efficiently into neuronal and astrocytic cells to virtually the same degree as control cultures, but a slightly less efficient oligodendrocyte differentiation was noted. We suggest that recombinant spider silk matrices provide a functional microenvironment and represent a useful tool for the development of new strategies in neural stem cell research. Copyright © 2012. Published by Elsevier Ltd.

  11. Enhancement of vitamin E production in sunflower cell cultures.

    Science.gov (United States)

    Caretto, Sofia; Bray Speth, Elena; Fachechi, Christian; Gala, Rosa; Zacheo, Giuseppe; Giovinazzo, Giovanna

    2004-09-01

    The most biologically active component of vitamin E, alpha-tocopherol, is synthesized in its most effective stereoisomeric form only by photosynthetic organisms. Using sunflower cell cultures, a suitable in vitro production system of natural alpha-tocopherol was established. The most efficient medium was found to be MS basal medium with naphthaleneacetic acid and 6-benzylaminopurine with the addition of casaminoacids and myo-inositol. Culture feeding experiments using biosynthetic precursors showed that alpha-tocopherol production improved by 30% when homogentisic acid was used. Interestingly, time-course experiments with sunflower suspension cultures showed a possible increase of 78% in alpha-tocopherol production when using cultures of longer subculture intervals. Compared to the starting plant tissue, an overall 100% increase of alpha-tocopherol was reached by these sunflower cell cultures.

  12. Advances in culture and manipulation of human pluripotent stem cells.

    Science.gov (United States)

    Qian, X; Villa-Diaz, L G; Krebsbach, P H

    2013-11-01

    Recent advances in the understanding of pluripotent stem cell biology and emerging technologies to reprogram somatic cells to a stem cell-like state are helping bring stem cell therapies for a range of human disorders closer to clinical reality. Human pluripotent stem cells (hPSCs) have become a promising resource for regenerative medicine and research into early development because these cells are able to self-renew indefinitely and are capable of differentiation into specialized cell types of all 3 germ layers and trophoectoderm. Human PSCs include embryonic stem cells (hESCs) derived from the inner cell mass of blastocyst-stage embryos and induced pluripotent stem cells (hiPSCs) generated via the reprogramming of somatic cells by the overexpression of key transcription factors. The application of hiPSCs and the finding that somatic cells can be directly reprogrammed into different cell types will likely have a significant impact on regenerative medicine. However, a major limitation for successful therapeutic application of hPSCs and their derivatives is the potential xenogeneic contamination and instability of current culture conditions. This review summarizes recent advances in hPSC culture and methods to induce controlled lineage differentiation through regulation of cell-signaling pathways and manipulation of gene expression as well as new trends in direct reprogramming of somatic cells.

  13. Cell culture supernatants for detection perforin ELISA

    African Journals Online (AJOL)

    Najwa

    2014-02-19

    Feb 19, 2014 ... cytic leukemia's are derived from B or T cell precursors. Four types of leukemia are classified, Chronic Lympho- cytic Leukemia (CLL), chronic myelogenous leukemia. (CML), acutelymphocyticleukemia (ALL) and acutemyelo- genous leukemia (AML). The development of a malignant cell clone is due to the ...

  14. Stability of resazurin in buffers and mammalian cell culture media

    DEFF Research Database (Denmark)

    Rasmussen, Eva; Nicolaisen, G.M.

    1999-01-01

    The utility of a ferricyanide/ferrocyanide system used in the AlamarBlue(TM) (Serotec, Oxford, UK) vital. dye to inhibit the reduction of resazurin by mammalian cell culture media is questioned. Resazurin was found to be relatively stable when dissolved in phosphate-buffered saline (PBS). The use...... of HEPES resulted in a huge immediate dye reduction, which was significantly enhanced by exposure to diffuse light from fluorescent tubes in the laboratory 8 h per day. The reduction of resazurin by various cell culture media was time and temperature dependent, and it was significantly enhanced......'s nutrient mixture F-10 and F-12. Fetal calf serum (5-20%) slightly decreased resazurin reduction during the first 2 days of incubation. The reduction of resazurin by mammalian cell culture media do not appear to be problematic under normal culture conditions, and it is primarily dependent upon the presence...

  15. Culture temperature modulates monoclonal antibody charge variation distribution in Chinese hamster ovary cell cultures.

    Science.gov (United States)

    Zhang, Xintao; Sun, Ya-Ting; Tang, Hongping; Fan, Li; Hu, Dongdong; Liu, Jintao; Liu, Xuping; Tan, Wen-Song

    2015-11-01

    To investigate the effect of lowering culture temperature on monoclonal antibody charge variation distribution in Chinese hamster ovary cell cultures. In both batch and fed-batch cultures, lowering the culture temperature decreased the antibody acidic variant levels. The acidic variant levels (defined as variants eluting earlier than the main peak of an antibody during HPLC) at 32 °C were about 10 % lower than those at 37 °C at the end of both batch and fed-batch cultures. Additionally, lowering the culture temperature increased the lysine variant level, which further increased basic variant level. The lysine variant levels at 32 °C were about 8 % (batch culture) and 3 % (fed-batch culture) higher than those at 37 °C at the end of cultures. Real-time PCR results suggests that the decrease in carboxypeptidase B transcription level might be partially responsible for the increased lysine variant level at sub-physiological temperatures. Culture temperature exhibits noticeable impact on antibody charge variation distribution, especially the acidic variants and lysine variants.

  16. Genome Editing of Erythroid Cell Culture Model Systems.

    Science.gov (United States)

    Yik, Jinfen J; Crossley, Merlin; Quinlan, Kate G R

    2018-01-01

    Genome editing to introduce specific mutations or to knock out genes in model cell systems has become an efficient platform for research in the fields of molecular biology, genetics, and cell biology. With recent rapid improvements in genome editing techniques, bench-top manipulation of the genome in cell culture has become progressively easier. The application of this knowledge to erythroid cell culture systems now allows the rapid analysis of the downstream effects of virtually any engineered gene disruption or modification in cell systems. Here, we describe a CRISPR/Cas9-based approach to making genomic modifications in erythroid lineage cells which we have successfully used in both murine (MEL) and human (K562) erythroleukaemia immortalized cell lines.

  17. Detection of multiple mycoplasma infection in cell cultures by PCR

    Directory of Open Access Journals (Sweden)

    J. Timenetsky

    2006-07-01

    Full Text Available A total of 301 cell cultures from 15 laboratories were monitored for mycoplasma (Mollicutes using PCR and culture methodology. The infection was detected in the cell culture collection of 12 laboratories. PCR for Mollicutes detected these bacteria in 93 (30.9% samples. Although the infection was confirmed by culture for 69 (22.9% samples, PCR with generic primers did not detect the infection in five (5.4%. Mycoplasma species were identified with specific primers in 91 (30.2% of the 98 samples (32.6% considered to be infected. Mycoplasma hyorhinis was detected in 63.3% of the infected samples, M. arginini in 59.2%, Acholeplasma laidlawii in 20.4%, M. fermentans in 14.3%, M. orale in 11.2%, and M. salivarium in 8.2%. Sixty (61.2% samples were co-infected with more than one mycoplasma species. M. hyorhinis and M. arginini were the microorganisms most frequently found in combination, having been detected in 30 (30.6% samples and other associations including up to four species were detected in 30 other samples. Failure of the treatments used to eliminate mycoplasmas from cell cultures might be explained by the occurrence of these multiple infections. The present results indicate that the sharing of non-certified cells among laboratories may disseminate mycoplasma in cell cultures.

  18. Automation of 3D cell culture using chemically defined hydrogels.

    Science.gov (United States)

    Rimann, Markus; Angres, Brigitte; Patocchi-Tenzer, Isabel; Braum, Susanne; Graf-Hausner, Ursula

    2014-04-01

    Drug development relies on high-throughput screening involving cell-based assays. Most of the assays are still based on cells grown in monolayer rather than in three-dimensional (3D) formats, although cells behave more in vivo-like in 3D. To exemplify the adoption of 3D techniques in drug development, this project investigated the automation of a hydrogel-based 3D cell culture system using a liquid-handling robot. The hydrogel technology used offers high flexibility of gel design due to a modular composition of a polymer network and bioactive components. The cell inert degradation of the gel at the end of the culture period guaranteed the harmless isolation of live cells for further downstream processing. Human colon carcinoma cells HCT-116 were encapsulated and grown in these dextran-based hydrogels, thereby forming 3D multicellular spheroids. Viability and DNA content of the cells were shown to be similar in automated and manually produced hydrogels. Furthermore, cell treatment with toxic Taxol concentrations (100 nM) had the same effect on HCT-116 cell viability in manually and automated hydrogel preparations. Finally, a fully automated dose-response curve with the reference compound Taxol showed the potential of this hydrogel-based 3D cell culture system in advanced drug development.

  19. Suspension culture of pluripotent stem cells: effect of shear on stem cell fate.

    Science.gov (United States)

    Keller, Kevin C; Rodrigues, Beatriz; zur Nieden, Nicole I

    2014-01-01

    Despite significant promise, the routine usage of suspension cell culture to manufacture stem cell-derived differentiated cells has progressed slowly. Suspension culture is an innovative way of either expanding or differentiating cells and sometimes both are combined into a single bioprocess. Its advantages over static 2D culturing include a homogeneous and controllable culture environment and producing a large quantity of cells in a fraction of time. This feature makes suspension cell culture ideal for use in stem cell research and eventually ideal in the large-scale production of differentiated cells for regenerative medicine. Because of their tremendous differentiation capacities and unlimited growth properties, pluripotent stem cells (PSCs) in particular are considered potential sources for future cell-replacement therapies. Currently, expansion of PSCs is accomplished in 2D, which only permits a limited amount of cell growth per culture flask before cells need to be passaged. However, before stem cells can be applied clinically, several aspects of their expansion, such as directed growth, but also differentiation, need to be better controlled. This review will summarize recent advantages in suspension culture of PSCs, while at the same time highlighting current challenges.

  20. Radiosensitivity of cultured insect cells: I. Lepidoptera

    Energy Technology Data Exchange (ETDEWEB)

    Koval, T.M.

    1983-10-01

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

  1. Characterisation and germline transmission of cultured avian primordial germ cells.

    Science.gov (United States)

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

    2010-11-29

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

  2. Characterisation and germline transmission of cultured avian primordial germ cells.

    Directory of Open Access Journals (Sweden)

    Joni Macdonald

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

  3. A Simple Hydrophilic Treatment of SU-8 Surfaces for Cell Culturing and Cell Patterning

    DEFF Research Database (Denmark)

    Wang, Zhenyu; Stangegaard, Michael; Dufva, Hans Martin

    2005-01-01

    SU-8, an epoxy-based photoresist, widely used in constitution different mTAS systems, is incompatible with mammalian cell adhesion and culture in its native form. Here, we demonstrate a simple, cheap and robust two-step method to render a SU-8 surface hydrophilic and compatible with cell culture........ The contact angle of SU-8 surface was significantly reduced from 90° to 25° after the surface modification. The treated SU-8 surfaces provided a cell culture environment that was comparable with cell culture flask surface in terms of generation time and morphology....

  4. Imprinting of confining sites for cell cultures on thermoplastic substrates

    Science.gov (United States)

    Cone, C. D.; Fleenor, E. N.

    1969-01-01

    Prevention of test cell migration beyond the field of observation involves confining cells or cultures in microlagoons made in either a layer of grease or a thermoplastic substrate. Thermoplastic films or dishes are easily imprinted with specifically designed patterns of microlagoons.

  5. Animal-cell culture in aqueous two-phase systems

    NARCIS (Netherlands)

    Zijlstra, G.M.

    1998-01-01

    In current industrial biotechnology, animal-cell culture is an important source of therapeutic protein products. The conventional animal-cell production processes, however, include many unit operations as part of the fermentation and downstream processing strategy. The research described in

  6. Endothelial cell cultures as a tool in biomaterial research

    NARCIS (Netherlands)

    Kirkpatrick, CJ; Otto, M; van Kooten, T; Krump, [No Value; Kriegsmann, J; Bittinger, F

    1999-01-01

    Progress in biocompatibility and tissue engineering would today be inconceivable without the aid of in vitro techniques. Endothelial cell cultures represent a valuable tool not just in haemocompatibility testing, but also in the concept of designing hybrid organs. In the past endothelial cells (EC)

  7. Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Met, Özcan

    2011-01-01

    Standard cell culture plastic was surface modified by passive adsorption or covalent attachment of interleukin (IL)-4 and investigated for its ability to induce differentiation of human monocytes into mature dendritic cells, a process dose-dependently regulated by IL-4. Covalent attachment of IL-4...

  8. Fabrication of a thermoresponsive cell culture dish: a key technology for cell sheet tissue engineering

    Directory of Open Access Journals (Sweden)

    Jun Kobayashi and Teruo Okano

    2010-01-01

    Full Text Available This article reviews the properties and characterization of an intelligent thermoresponsive surface, which is a key technology for cell sheet-based tissue engineering. Intelligent thermoresponsive surfaces grafted with poly(N-isopropylacrylamide exhibit hydrophilic/hydrophobic alteration in response to temperature change. Cultured cells are harvested on thermoresponsive cell culture dishes by decreasing the temperature without the use of digestive enzymes or chelating agents. Our group has developed cell sheet-based tissue engineering for therapeutic uses with single layer or multilayered cell sheets, which were recovered from the thermoresponsive cell culture dish. Using surface derivation techniques, we developed a new generation of thermoresponsive cell culture dishes to improve culture conditions. We also designed a new methodology for constructing well-defined organs using microfabrication techniques.

  9. [Serum-free culture of umbilical cord mesenchymal stem cells].

    Science.gov (United States)

    Zhou, Ping; Li, Dan; Chen, Guang-Hua; Wang, Yi

    2013-10-01

    This study was purposed to observe the culture of umbilical cord mesenchymal stem cells (UC-MSC) with serum-free medium, and compared it with the medium containing 10% fetal bovine serum (FBS). The normal umbilical cords were acquired during cesarean section, and then were cultured with MesenCult-XF serum-free medium or medium containing 10% fetal bovine serum (FBS). The morphology, immunophenotype, cell cycle, proliferation and differentiation potential of mesenchymal stem cells and the inhibition of mixed lymphocyte reaction were observed through different medium culture method. The results showed that the MSC cultured with serum-free MesenCult(-)XF medium could transfer and multiply for average of 6.57 ± 0.7 times, and the serum medium-cultured MSC could transfer and multiply for average of 4.59 ± 0.45 times (P cultured MSC all expressed CD44, CD90, CD73, CD105 antigen, but did not expressed CD31, CD45, HLA-DR and CD34 antigen, and their expression levels were not significantly different. The serum-free medium-cultured MSC (65 ± 5.2%) were all at Go/G1 phase, and the serum-contained medium-cultured MSC (62+3.1%) were at Go/G1 phase(P > 0.05); the 2 kinds of media-cultured MSC all could differentiate into fat and ossification; when serum-free medium cultured umbilical cord MSC were inoculated at the the density of 10(3), 5×10(3), 10(4), and 2×10(4) cells/well, then co-cultured with the reactant and stimulating cells, the CPM were (6.43 ± 0.47)×10(4), (4.30 ± 0.38)×10(4), (1.97 ± 0.13)×10(4) and (0.24 ± 0.03)×10(4), respectively, and the serum-containing medium-cultured MSC were incubated with different density of mixed lymphocyte, displaying CPM that were (7.85 ± 0.07)×10(4), (5.64 ± 0.12)×10(4), (3.09 ± 0.18)×10(4) and (1.73 ± 0.05)×10(4). It is concluded that the serum-free medium has been confirmed to culture MSC, which have potential of transfer and differentiation with count for clinical application, and can avoid foreign protein

  10. Cytopathogenicity of Naegleria for cultured neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Fulford, D.E.

    1985-01-01

    The cytopathic activity of live Naegleria amoebae and cell-free lysates of Naegleria for B-103 rat neuroblastoma cells was investigated using a /sup 51/Cr release assay. Live amoebae and cell-free lysates of N. fowleri, N. australiensis, N. lovaniensis, and N. gruberi all induced sufficient damage to radiolabeled B-103 cells to cause a significant release of chromium. The cytotoxic activity present in the cell-free lysates of N. fowleri can be recovered in the supernatant fluid following centrifugation at 100,000xg and precipitation of the 100,000xg supernatant fluid with ammonium sulfate. Initial characterization of the cytotoxic factor indicates that it is a heat labile, pH sensitive, soluble protein. The cytotoxic activity is abolished by either extraction, unaffected by repeated freeze-thawing, and is not sensitive to inhibitors of proteolytic enzymes. Phospholipase A activity was detected in the cytotoxic ammonium sulfate precipitable material, suggesting that this enzyme activity may have a role in the cytotoxic activity of the cell-free lysates.

  11. Hydrodynamic effects on cells in agitated tissue culture reactors

    Science.gov (United States)

    Cherry, R. S.; Papoutsakis, E. T.

    1986-01-01

    The mechanisms by which hydrodynamic forces can affect cells grown on microcarrier beads in agitated cell culture reactors were investigated by analyzing the motion of microcarriers relative to the surrounding fluid, to each other, and to moving or stationary solid surfaces. It was found that harmful effects on cell cultures that have been previously attributed to shear can be better explained as the effects of turbulence (of a size scale comparable to the microcarriers or the spacing between them) or collisions. The primary mechanisms of cell damage involve direct interaction between microcarriers and turbulent eddies, collisions between microcarriers in turbulent flow, and collisions against the impeller or other solid surfaces. The implications of these analytical results for the design of tissue culture reactors are discussed.

  12. Recent applications of fish cell culture to biomedical research.

    Science.gov (United States)

    Hightower, L E; Renfro, J L

    1988-12-01

    Tissues of the fishes are as amenable to the techniques of modern cell culture as mammalian tissues and organs, and yet this vast resource, comprising thousands of vertebrate species, remains largely unexplored. The model systems that have been developed demonstrate the utility of fish cells as sources of special adaptations and exaggerated physiological systems. In this review, we briefly describe several of the successful models along with recent developments in fish cell culture with the hope of stimulating increased interest in the lower vertebrates as useful complements to mammalian cell culture in biomedical research. The topics covered include epithelial ion transport, endocrinological studies, the cellular stress (heat shock) response, thermotolerance, cancer biology, and environmental toxicology.

  13. Cloning higher plants from aseptically cultured tissues and cells

    Science.gov (United States)

    Krikorian, A. D.

    1982-01-01

    A review of aseptic culture methods for higher plants is presented, which focuses on the existing problems that limit or prevent the full realization of cloning plants from free cells. It is shown that substantial progress in clonal multiplication has been made with explanted stem tips or lateral buds which can be stimulated to produce numerous precocious axillary branches. These branches can then be separated or subdivided and induced to root in order to yield populations of genetically and phenotypically uniorm plantlets. Similarly, undifferentiated calluses can sometimes be induced to form shoots and/or roots adventitiously. Although the cell culture techniques required to produce somatic embryos are presently rudimentary, steady advances are being made in learning how to stimulate formation of somatic or adventive embryos from totipotent cells grown in suspension cultures. It is concluded that many problems exist in the producing and growing of totipotent or morphogenetically competent cell suspensions, but the potential benefits are great.

  14. Miniature Bioreactor System for Long-Term Cell Culture

    Science.gov (United States)

    Gonda, Steve R.; Kleis, Stanley J.; Geffert, Sandara K.

    2010-01-01

    A prototype miniature bioreactor system is designed to serve as a laboratory benchtop cell-culturing system that minimizes the need for relatively expensive equipment and reagents and can be operated under computer control, thereby reducing the time and effort required of human investigators and reducing uncertainty in results. The system includes a bioreactor, a fluid-handling subsystem, a chamber wherein the bioreactor is maintained in a controlled atmosphere at a controlled temperature, and associated control subsystems. The system can be used to culture both anchorage-dependent and suspension cells, which can be either prokaryotic or eukaryotic. Cells can be cultured for extended periods of time in this system, and samples of cells can be extracted and analyzed at specified intervals. By integrating this system with one or more microanalytical instrument(s), one can construct a complete automated analytical system that can be tailored to perform one or more of a large variety of assays.

  15. Differential heat shock response of primary human cell cultures and established cell lines

    DEFF Research Database (Denmark)

    Richter, W W; Issinger, O G

    1986-01-01

    degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized...

  16. CD34 marks angiogenic tip cells in human vascular endothelial cell cultures

    NARCIS (Netherlands)

    Siemerink, Martin J.; Klaassen, Ingeborg; Vogels, Ilse M. C.; Griffioen, Arjan W.; van Noorden, Cornelis J. F.; Schlingemann, Reinier O.

    2012-01-01

    The functional shift of quiescent endothelial cells into tip cells that migrate and stalk cells that proliferate is a key event during sprouting angiogenesis. We previously showed that the sialomucin CD34 is expressed in a small subset of cultured endothelial cells and that these cells extend

  17. Mesenchymal origin of multipotent human testis-derived stem cells in human testicular cell cultures

    NARCIS (Netherlands)

    Chikhovskaya, J. V.; van Daalen, S. K. M.; Korver, C. M.; Repping, S.; van Pelt, A. M. M.

    2014-01-01

    In contrast to mouse germ cell-derived pluripotent stem cells, the pluripotent state of human testis-derived embryonic stem cell (ESC)-like that spontaneously arise in primary testicular cell cultures remains controversial. Recent studies have shown that these cells closely resemble multipotent

  18. Establishing a stem cell culture laboratory for clinical trials

    Directory of Open Access Journals (Sweden)

    Elíseo Joji Sekiya

    2012-01-01

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

  19. Establishing a stem cell culture laboratory for clinical trials

    Science.gov (United States)

    Sekiya, Elíseo Joji; Forte, Andresa; Kühn, Telma Ingrid Borges de Bellis; Janz, Felipe; Bydlowski, Sérgio Paulo; Alves, Adelson

    2012-01-01

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

  20. Apoptosis in CHO cell batch cultures: examination by flow cytometry.

    Science.gov (United States)

    Moore, A; Donahue, C J; Hooley, J; Stocks, D L; Bauer, K D; Mather, J P

    1995-02-01

    Chinese hamster ovary cells grown under conditions which are optimal for the production of a genetically engineered protein in batch culture, lose significant viability shortly after entering the stationary phase. This cell death was investigated morphologically and was found to be almost exclusively via apoptosi. Furthermore, cells were analyzed by flow cytometry using a fluorescent DNA end-labeling assay to label apoptotic cells, in conjunction with cell cycle analysis using propidium iodide. Apoptotic cells could be detected by this method, and by the radioactive end-labeling of extracted DNA, on all days of culture from day 1 to day 7; however, the degree of apoptotic cell death increased dramatically when the cells entered the stationary phase, rising to 50-60% of the total cell number at the termination of the culture. Flow cytometric analysis showed that the majority of cells underwent apoptosis whilst in G(1)/G(0) and formed an apoptotic population with high DNA FITC end-labeling and hypodiploid propidium iodide binding. Additionally, the ability or inability to secrete specific protein products did not appear to interfere with the development of the apoptotic population with time.

  1. Clinical and molecular analysis of a Chinese family with autosomal dominant neurohypophyseal diabetes insipidus associated with a novel missense mutation in the vasopressin-neurophysin II gene.

    Science.gov (United States)

    Luo, Yongfeng; Wang, Binbin; Qiu, Yu; Zhang, Chuan; Jin, Chengluo; Zhao, Yakun; Zhu, Qingguo; Ma, Xu

    2012-08-01

    The objective of this study is to identify the genetic defects in a Chinese family with autosomal dominant familial neurohypophyseal diabetes insipidus. Complete physical examination, fluid deprivation, and DDAVP tests were performed in three affected and three healthy members of the family. Genomic DNA was extracted from leukocytes of venous blood of these individuals for polymerase chain reaction amplification and direct sequencing of all three coding exons of arginine vasopressin-neurophysin II (AVP-NPII) gene. Seven members of this family were suspected to have symptomatic vasopressin-deficient diabetes insipidus. The water deprivation test in all the patients confirmed the diagnosis of vasopressin-deficient diabetes insipidus, with the pedigree demonstrating an autosomal dominant inheritance. Direct sequence analysis revealed a novel mutation (c.193T>A) and a synonymous mutation (c.192C>A) in the AVP-NPII gene. The missense mutation resulted in the substitution of cysteine by serine at a highly conserved codon 65 of exon 2 of the AVP-NPII gene in all affected individuals, but not in unaffected members. We concluded that a novel missense mutation in the AVP-NPII gene caused neurohypophyseal diabetes insipidus in this family, due to impaired neurophysin function as a carrier protein for AVP. The Cys65 is essential for NPII in the formation of a salt bridge with AVP. Presence of this mutation suggests that the portion of the neurophysin peptide encoded by this sequence is important for the normal expression of vasopressin.

  2. Dilatative uropathy as a manifestation of neurohypophyseal diabetes insipidus due to a novel mutation in the arginine vasopressin-neurophysin-II gene.

    Science.gov (United States)

    Lindenthal, V; Mainberger, A; Morris-Rosendahl, D J; Löning, L; Mayer, W; Müller, H L

    2013-12-01

    Polydypsia and polyuria are frequent symptoms in patients with sellar masses caused by neurohypophyseal diabetes insipidus. Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI), a disorder caused by mutations in the arginine vasopressin (AVP) -neurophysin II (NPII) gene, should be considered as a rare differential diagnosis. A delayed diagnosis bears the risk of life-threatening electrolyte imbalances and permanent urinary tract damage, leading to impaired quality of life.We present a Caucasian kindred of at least 4 generations with FNDI.Clinical histories, endocrine para-meters, and results of molecular analyses of the AVP gene are presented with a review of the literature on diabetes insipidus (DI) related urinary tract dilatation.Polyuria and polydipsia were only reported based on explicit and thorough interrogation after more than 4 years of clinical follow-up. A novel heterozygous mutation in the AVP gene was found in all examined symptomatic subjects (c.1-33_c.4del37nt). A literature review revealed that non-obstructive hydronephrosis (NOH) is a rare but known complication of DI.Since increased fluid intake is often a typical familial pattern in adFNDI, it is frequently missed as being pathologic in affected patients, therefore a detailed clinical history of drinking volumes is of critical importance. AVP gene testing is an important component in the confirmation of the diagnosis. Otherwise unexplainable NOH should lead to further investigations and evaluation of rare diseases like FNDI. © Georg Thieme Verlag KG Stuttgart · New York.

  3. Differentiation of oligodendrocyte progenitor cells from dissociated monolayer and feeder-free cultured pluripotent stem cells.

    Science.gov (United States)

    Yamashita, Tomoko; Miyamoto, Yuki; Bando, Yoshio; Ono, Takashi; Kobayashi, Sakurako; Doi, Ayano; Araki, Toshihiro; Kato, Yosuke; Shirakawa, Takayuki; Suzuki, Yutaka; Yamauchi, Junji; Yoshida, Shigetaka; Sato, Naoya

    2017-01-01

    Oligodendrocytes myelinate axons and form myelin sheaths in the central nervous system. The development of therapies for demyelinating diseases, including multiple sclerosis and leukodystrophies, is a challenge because the pathogenic mechanisms of disease remain poorly understood. Primate pluripotent stem cell-derived oligodendrocytes are expected to help elucidate the molecular pathogenesis of these diseases. Oligodendrocytes have been successfully differentiated from human pluripotent stem cells. However, it is challenging to prepare large amounts of oligodendrocytes over a short amount of time because of manipulation difficulties under conventional primate pluripotent stem cell culture methods. We developed a proprietary dissociated monolayer and feeder-free culture system to handle pluripotent stem cell cultures. Because the dissociated monolayer and feeder-free culture system improves the quality and growth of primate pluripotent stem cells, these cells could potentially be differentiated into any desired functional cells and consistently cultured in large-scale conditions. In the current study, oligodendrocyte progenitor cells and mature oligodendrocytes were generated within three months from monkey embryonic stem cells. The embryonic stem cell-derived oligodendrocytes exhibited in vitro myelinogenic potency with rat dorsal root ganglion neurons. Additionally, the transplanted oligodendrocyte progenitor cells differentiated into myelin basic protein-positive mature oligodendrocytes in the mouse corpus callosum. This preparative method was used for human induced pluripotent stem cells, which were also successfully differentiated into oligodendrocyte progenitor cells and mature oligodendrocytes that were capable of myelinating rat dorsal root ganglion neurons. Moreover, it was possible to freeze, thaw, and successfully re-culture the differentiating cells. These results showed that embryonic stem cells and human induced pluripotent stem cells maintained in a

  4. Tympanic membrane organ culture using cell culture well inserts engrafted with tympanic membrane tissue explants.

    Science.gov (United States)

    Liew, Lawrence J; Day, Richard M; Dilley, Rodney J

    2017-03-01

    Tissue engineering approaches using growth factors and various materials for repairing chronic perforations of the tympanic membrane are being developed, but there are surprisingly few relevant tissue culture models available to test new treatments. Here, we present a simple three-dimensional model system based on micro-dissecting the rat tympanic membrane umbo and grafting it into the membrane of a cell culture well insert. Cell outgrowth from the graft produced sufficient cells to populate a membrane of similar surface area to the human tympanic membrane within 2 weeks. Tissue grafts from the annulus region also showed cell outgrowth but were not as productive. The umbo organoid supported substantial cell proliferation and migration under the influence of keratinocyte growth medium. Cells from umbo grafts were enzymatically harvested from the polyethylene terephthalate (PET) membrane for expansion in routine culture and cells could be harvested consecutively from the same graft over multiple cycles. We used harvested cells to test cell migration properties and to engraft a porous silk scaffold material as proof-of-principle for tissue engineering applications. This model is simple enough to be widely adopted for tympanic membrane regeneration studies and has promise as a tissue-equivalent model alternative to animal testing.

  5. Animal-cell culture media: History, characteristics, and current issues.

    Science.gov (United States)

    Yao, Tatsuma; Asayama, Yuta

    2017-04-01

    Cell culture technology has spread prolifically within a century, a variety of culture media has been designed. This review goes through the history, characteristics and current issues of animal-cell culture media. A literature search was performed on PubMed and Google Scholar between 1880 and May 2016 using appropriate keywords. At the dawn of cell culture technology, the major components of media were naturally derived products such as serum. The field then gradually shifted to the use of chemical-based synthetic media because naturally derived ingredients have their disadvantages such as large batch-to-batch variation. Today, industrially important cells can be cultured in synthetic media. Nevertheless, the combinations and concentrations of the components in these media remain to be optimized. In addition, serum-containing media are still in general use in the field of basic research. In the fields of assisted reproductive technologies and regenerative medicine, some of the medium components are naturally derived in nearly all instances. Further improvements of culture media are desirable, which will certainly contribute to a reduction in the experimental variation, enhance productivity among biopharmaceuticals, improve treatment outcomes of assisted reproductive technologies, and facilitate implementation and popularization of regenerative medicine.

  6. Therapeutic touch stimulates the proliferation of human cells in culture.

    Science.gov (United States)

    Gronowicz, Gloria A; Jhaveri, Ankur; Clarke, Libbe W; Aronow, Michael S; Smith, Theresa H

    2008-04-01

    Our objective was to assess the effect of Therapeutic Touch (TT) on the proliferation of normal human cells in culture compared to sham and no treatment. Several proliferation techniques were used to confirm the results, and the effect of multiple 10-minute TT treatments was studied. Fibroblasts, tendon cells (tenocytes), and bone cells (osteoblasts) were treated with TT, sham, or untreated for 2 weeks, and then assessed for [(3)H]-thymidine incorporation into the DNA, and immunocytochemical staining for proliferating cell nuclear antigen (PCNA). The number of PCNA-stained cells was also quantified. For 1 and 2 weeks, varying numbers of 10-minute TT treatments were administered to each cell type to determine whether there was a dose-dependent effect. TT administered twice a week for 2 weeks significantly stimulated proliferation of fibroblasts, tenocytes, and osteoblasts in culture (p = 0.04, 0.01, and 0.01, respectively) compared to untreated control. These data were confirmed by PCNA immunocytochemistry. In the same experiments, sham healer treatment was not significantly different from the untreated cultures in any group, and was significantly less than TT treatment in fibroblast and tenocyte cultures. In 1-week studies involving the administration of multiple 10-minute TT treatments, four and five applications significantly increased [(3)H]-thymidine incorporation in fibroblasts and tenocytes, respectively, but not in osteoblasts. With different doses of TT for 2 weeks, two 10-minute TT treatments per week significantly stimulated proliferation in all cell types. Osteoblasts also responded to four treatments per week with a significant increase in proliferation. Additional TT treatments (five per week for 2 weeks) were not effective in eliciting increased proliferation compared to control in any cell type. A specific pattern of TT treatment produced a significant increase in proliferation of fibro-blasts, osteoblasts, and tenocytes in culture. Therefore, TT may

  7. Selective culture of different types of human parotid gland cells.

    Science.gov (United States)

    Chan, Yen-Hui; Huang, Tsung-Wei; Young, Tai-Horng; Lou, Pei-Jen

    2011-03-01

    Advances in salivary gland tissue engineering can benefit patients diagnosed with xerostomia. Complexity of the gland explains the urgent demand for a reliable protocol to isolate and expand various gland cells that can be used for further study. Three cells with different morphologies were isolated from the same human parotid glands using different culture medium systems and then were identified by the expressions from mRNA to the protein level. Among the 34 specimens, parotid gland acinar cells, myoepithelial cells, and fibroblasts expressing specific markers that belonged to individual cell types, were successfully isolated and expanded from 30 specimens without a complex mechanical process and expensive flow technique. The proposed protocol is simple with a high success rate to culture various gland cells, making it highly promising for use in future tissue engineering studies. Copyright © 2010 Wiley Periodicals, Inc.

  8. The replacement of serum by hormones in cell culture media.

    Science.gov (United States)

    Sato, G; Hayashi, I

    1976-12-01

    The replacement of serum by hormones in cell culture media. (Reemplazo del suero por hormonas en el medio de cultivo de células). Arch. Biol. Med. Exper. 10: 120-121, 1976. The serum used in cell culture media can be replaced by a mixture of hormones and some accesory blood factors. The pituitary cell line GH3 can be grown in a medium in which serum is replaced by triiodothyronine, transferrin, parathormone, tyrotrophin releasing hormone and somatomedins. Hela and BHK cell strains can also be grown in serum free medium supplemented with hormones. Each cell type appears to have different hormonal requirements yet it may found that some hormones are required for most cell types.

  9. Culture and Characterization of Circulating Endothelial Progenitor Cells in Patients with Renal Cell Carcinoma.

    Science.gov (United States)

    Gu, Wenyu; Sun, Wei; Guo, Changcheng; Yan, Yang; Liu, Min; Yao, Xudong; Yang, Bin; Zheng, Junhua

    2015-07-01

    Although emerging evidence demonstrates increased circulating endothelial progenitor cells in patients with solid tumors, to our knowledge it is still unknown whether such cells can be cultured from patients with highly angiogenic renal cell carcinoma. We cultured and characterized circulating endothelial progenitor cells from patients with renal cell carcinoma. The circulating endothelial progenitor cell level (percent of CD45(-)CD34(+) VEGF-R2(+) cells in total peripheral blood mononuclear cells) was quantified in 47 patients with renal cell carcinoma and 40 healthy controls. Peripheral blood mononuclear cells were then isolated from 33 patients with renal cell carcinoma and 30 healthy controls to culture and characterize circulating endothelial progenitor cells. The circulating endothelial progenitor cell level was significantly higher in patients with renal cell carcinoma than in healthy controls (0.276% vs 0.086%, p cells first emerged significantly earlier in patient than in control preparations (6.72 vs 14.67 days, p culture success rate (87.8% vs 40.0% of participants) and the number of colonies (10.06 vs 1.83) were significantly greater for patients than for controls (each p cell level correlated positively with the number of patient colonies (r = 0.762, p Cells cultured from patients and controls showed a similar growth pattern, immunophenotype, ability to uptake Ac-LDL and bind lectin, and form capillary tubes in vitro. However, significantly more VEGF-R2(+) circulating endothelial progenitor cells were found in preparations from patients with renal cell carcinoma than from healthy controls (21.1% vs 13.4%, p cell colonies, a higher cell culture success rate and more colonies were found for patients with renal cell carcinoma than for healthy controls. Results indicate the important significance of VEGF-R2(+) circulating endothelial progenitors in patients with renal cell carcinoma. Copyright © 2015 American Urological Association Education and Research

  10. Histopathology and cell culture characteristics of liver cells from grc- and grc+ rats given diethylnitrosamine.

    Science.gov (United States)

    Smith, G J; Kunz, H W; Dunsford, H A; Gill, T J

    1990-04-01

    The histopathological response and cell culture characteristics of liver cells from the R16 (grc-) strain of rats, which carries an MHC-linked deletion, were examined one week after a single intraperitoneal injection of 200 mg/kg body weight diethylnitrosamine (DEN) and were compared with the response of liver cells from wild type (grc+) rats. The DEN exposure induced hydropic/vacuolar changes in the parenchymal cells and a limited proliferation of oval cells in the periportal areas of the livers of both grc+ and grc- rats. Primary culture of collagenase-digested livers consisted of parenchymal, bile ductular and oval-related cells as determined by cell-specific immunohistochemistry. Subpassaged cells from grc+ rats exhibited oval cell ultrastructural morphology, inducible histochemical staining for gamma-glutamyl transpeptidase (GGT), and DEN-associated onset of anchorage-independent growth. Primary cultures of liver cells from R16 rats consistently failed to form cell strains upon subpassage.

  11. Laboratory scaleup of cell cultures (0.5-50 liters).

    Science.gov (United States)

    Mather, J P

    1998-01-01

    With the modern tools of molecular and cell biology now available, many researchers find the need to scale up cell culture in order to produce large quantities of cells or conditioned medium for the further purification of proteins or subcellular fractions. The method used for scaleup will depend on the properties of the cell being used, the amount of material desired, the number of times the process is to be run, and the resources available. Roller bottles, microcarrier cultures, and hollow fiber cultures provide appropriate and scaleable growth systems for attachment-dependent cells. However, the most efficient production of material, especially if the large-scale production is to be repeated several times, is obtained by suspension adapting the cells and growing them in suspension. This can be done in spinners or in fermenters, which range in size from 0.1 to 12,000 liters in volume. This chapter describes methods for choosing the optimal production system and medium for scaling up to different levels of production and for suspension adapting cells for scaleup of suspension culture.

  12. Isolation, culture and characterization of primary mouse RPE cells.

    Science.gov (United States)

    Fernandez-Godino, Rosario; Garland, Donita L; Pierce, Eric A

    2016-07-01

    Mouse models are powerful tools for the study of ocular diseases. Alterations in the morphology and function of the retinal pigment epithelium (RPE) are common features shared by many ocular disorders. We report a detailed protocol to collect, seed, culture and characterize RPE cells from mice. We describe a reproducible method that we previously developed to collect and culture murine RPE cells on Transwells as functional polarized monolayers. The collection of RPE cells takes ∼3 h, and the cultures mimic in vivo RPE cell features within 1 week. This protocol also describes methods to characterize the cells on Transwells within 1-2 weeks by transmission and scanning electron microscopy (TEM and SEM, respectively), immunostaining of vibratome sections and flat mounts, and measurement of transepithelial electrical resistance. The RPE cell cultures are suitable to study the biology of the RPE from wild-type and genetically modified strains of mice between the ages of 10 d and 12 months. The RPE cells can also be manipulated to investigate molecular mechanisms underlying the RPE pathology in the numerous mouse models of ocular disorders. Furthermore, modeling the RPE pathology in vitro represents a new approach to testing drugs that will help accelerate the development of therapies for vision-threatening disorders such as macular degeneration (MD).

  13. Mycoplasma Removal from Cell Culture Using Antimicrobial Photodynamic Therapy

    OpenAIRE

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

    2013-01-01

    Objective: The objective of this research was to determine the effectiveness of antimicrobial photodynamic therapy (aPDT) in the removal of mycoplasmas from contaminated cells. Background data: Mycoplasmas often contaminate cell cultures. The cell-contaminating mycoplasmas are removed by antibiotics, but the use of antibiotics usually induces antibiotic-resistant bacteria. aPDT is expected to be a possible alternative to antibiotic treatments for suppressing infections. Materials and Methods:...

  14. Isolation and Culture of Postnatal Stem Cells from Deciduous Teeth

    OpenAIRE

    Olávez, Daniela; Facultad de Odontología Universidad de Los Andes; Salmen, Siham; Instituto de Inmunología Clínica, Universidad de Los Andes.; Padrón, Karla; Facultad de Odontología. Univerisdad de Los Andes.; Lobo, Carmine; Facultad de Odontología. Univerisdad de Los Andes.; Díaz, Nancy; Facultad de Odontología, Universidad de Los Andes.; Berrueta, Lisbeth; Doctora en Inmunología por Instituto Venezolano de Investigaciones Científicas (IVIC). Instituto de Inmunología Clínica, Facultad de Medicina, Universidad de Los Andes, Venezuela.; Solorzanio, Eduvigis; Facultad de Odontología, Universidad de Los Andes.

    2014-01-01

    Background: Currently, degenerative diseases represent a public health problem; therefore, the development and implementation of strategies to fully or partially recover of damaged tissues has a special interest in the biomedical field. Therapeutic strategies based on mesenchymal stem cells transplantation from dental pulp have been proposed as an alternative. Purpose: To develop a mesenchymal stem cells culture isolated from dental pulp of deciduous teeth. Methods: The mesenchymal stem cells...

  15. Effects of epiplakin-knockdown in cultured corneal epithelial cells

    OpenAIRE

    Kokado, Masahide; Okada, Yuka; Miyamoto, Takeshi; Yamanaka, Osamu; Saika, Shizuya

    2016-01-01

    Background To investigate effects of knockdown of epiplakin gene expression on the homeostasis of cultured corneal epithelial cell line. We previously reported acceleration of corneal epithelial wound healing in an epiplakin-null mouse. Methods Gene expression of epiplakin was knockdowned by employing siRNA transfection in SV40-immortalized human corneal epithelial cell line. Protein expression of E-cadherin, keratin 6 and vimentin was examined by western blotting. Cell migration and prolifer...

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

    Science.gov (United States)

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

    2017-12-07

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

  17. Human dendritic cell culture and bacterial infection.

    Science.gov (United States)

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

    2012-01-01

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

  18. Tocopherol biosynthesis is enhanced in photomixotrophic sunflower cell cultures.

    Science.gov (United States)

    Fachechi, Christian; Nisi, Rossella; Gala, Rosa; Leone, Antonella; Caretto, Sofia

    2007-04-01

    Alpha-tocopherol is the most biologically active component of vitamin E and is synthesized only by photosynthetic organisms. Two heterotrophic cell lines of sunflower (Helianthus annuus L.) of differing alpha-tocopherol biosynthetic capability, three-fold higher in the high synthesizing cell line, HT, than in the low synthesizing one, LT, were previously identified. To investigate the relationship between alpha-tocopherol biosynthesis and photomixotrophic culture conditions, a new photomixotrophic sunflower cell line HS3 was established by selecting HT cells able to grow in the presence of a ten-fold reduced sucrose concentration in the culture medium. The photosynthetic properties of HS3 cells were characterized in comparison with HT and LT cells, revealing an increase in chlorophyll content, chloroplast number, and level of the photosynthesis related enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Furthermore, an enhanced expression of the gene encoding for the tocopherol biosynthetic enzyme geranyl-geranylpyrophosphate synthase (GGPPS) was observed in HS3 cells. HS3 cells also revealed a 25% and a more than three-fold higher tocopherol level than HT and LT, respectively, indicating a positive correlation between alpha-tocopherol biosynthesis of sunflower cell cultures and their photosynthetic properties. These findings can be useful for improving the tocopherol yields of the sunflower in vitro production system.

  19. Uptake of T-2 Mycotoxin in Cultured Cells. Relationship to Sodium Fluoride and Cell Type

    Science.gov (United States)

    1986-10-20

    woC)) -13-7i3 , UFILE CORY UPTAKE OF T-2 MYCOTOXIN IN CULTURED CELLS. RELATIONSHIP TO SODIUM FLUORIDE AND CELL TYPE. S~DTIC’ Lynn R. Trusal and Lee...NUMBER &. TITLE (and SubettI.) 13&i.’TYPE Of RE PORT A PERIOD COVERED Uptake of T-2 Mycotoxin in Cultured Cells. Relationship to Sodium Fluoride and Cell...necees.m and ident~il by block member) T-2 mycotoxin cultured cells sodium fluoride 24k ANITRACT’ (Cmet i 0 09 6401D Nid urnews 001 edast~ biY10 block

  20. Initiation of primary cell culture from amphioxus Branchiostoma belcheri tsingtauense

    Science.gov (United States)

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

    2009-02-01

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

  1. Specimen Sample Preservation for Cell and Tissue Cultures

    Science.gov (United States)

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

    1996-01-01

    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.

  2. cultured cells under phenylethanoid glycosides (PEG) 6000

    African Journals Online (AJOL)

    akpobome

    2013-03-13

    Mar 13, 2013 ... This study was carried out to investigate the effect of the intracellular signaling molecule nitric oxide. (NO) on osmoregulation of tobacco cells under osmotic stress caused by phenylethanoid glycosides. 6000 (PEG 6000). The results ... is one of the major environmental factors limiting plant productivity and ...

  3. Primary mouse small intestinal epithelial cell cultures

    NARCIS (Netherlands)

    Sato, T.; Clevers, H.

    2013-01-01

    The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently shown that Lgr5 (Leucine-rich repeat-containing G protein-coupled receptor) is expressed in intestinal stem cells by an in vivo genetic lineage tracing strategy. In the past, extensive efforts have

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

    Science.gov (United States)

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

    2011-12-01

    Treatment of skeletal muscle loss due to trauma or tumor ablation therapy still lacks a suitable clinical approach. Creation of functional muscle tissue in vitro using the differentiation potential of human satellite cells (myoblasts) is a promising new research field called tissue engineering. Strong differentiation stimuli, which can induce formation of myofibers after cell expansion, have to be identified and evaluated in order to create sufficient amounts of neo-tissue. The objective of this study was to determine the influence of static magnetic fields (SMF) on human satellite cell cultures as one of the preferred stem cell sources in skeletal muscle tissue engineering. Experiments were performed using human satellite cells with and without SMF stimulation after incubation with a culture medium containing low [differentiation medium (DM)] or high [growth medium (GM)] concentrations of growth factors. Proliferation analysis using the alamarBlue assay revealed no significant influence of SMF on cell division. Real-time RT-PCR of the following marker genes was investigated: myogenic factor 5 (MYF5), myogenic differentiation antigen 1 (MYOD1), myogenin (MYOG), skeletal muscle α1 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

  5. 21 CFR 864.2240 - Cell and tissue culture supplies and equipment.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cell and tissue culture supplies and equipment... SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2240 Cell and tissue culture supplies and equipment. (a) Identification. Cell and tissue culture...

  6. Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma.

    Science.gov (United States)

    Palmini, Gaia; Zonefrati, Roberto; Mavilia, Carmelo; Aldinucci, Alessandra; Luzi, Ettore; Marini, Francesca; Franchi, Alessandro; Capanna, Rodolfo; Tanini, Annalisa; Brandi, Maria Luisa

    2016-10-14

    The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed "cellular spheres". Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

  7. Mammary Gland Cell Culture of Macaca fascicularis as a Reservoir for Stem Cells

    Directory of Open Access Journals (Sweden)

    Silmi Mariya

    2017-07-01

    Full Text Available The mammary gland contains adult stem cells that are capable of self-renewal and are likely target for neoplastic transformation leading to breast cancer. In this study, we developed a cell culture derived from the mammary glands of cynomolgus monkeys (Macaca fascicularis (MfMC and furthermore identified the expression of markers for stemness and estrogen receptor-associated activities. We found that the primary culture can be successfully subcultured to at least 3 passages, primarily epithelial-like in morphology, the cultured cells remained heterogenous in phenotype as they expressed epithelial cell markers CD24, CK18, and marker for fibroblast S1004A. Importantly, the cell population also consistently expressed the markers of mammary stem cells (ITGB1 or CD29 and ITGA6 or CD49f, mesenchymal stem cells (CD73 and CD105 and pluripotency (NANOG, OCT4, SOX2. In addition to this, the cells were also positive for Estrogen Receptor (ER, and ER-activated marker Trefoil Factor 1, suggesting an estrogen responsiveness of the culture model. These results indicate that our cell culture model is a reliable model for acquiring a population of cells with mammary stem cell properties and that these cultures may also serve as a reservoir from which more purified populations of stem cell populations can be isolated in the future.

  8. [In vitro cell culture technology in cosmetology research].

    Science.gov (United States)

    Gojniczek, Katarzyna; Garncarczyk, Agnieszka; Pytel, Agata

    2005-01-01

    For ages the humanity has been looking for all kind of active substances, which could be used in improving the health and the appearance of our skin. People try to find out how to protect the skin from harmful, environmental factors. Every year a lot of new natural and synthetic, chemical substances are discovered. All of them potentially could be used as a cosmetic ingredient. In cosmetology research most of new xenobiotics were tested in vivo on animals. Alternative methods to in vivo tests are in vitro tests with skin cell culture system. The aim of this work was to describe two-dimensional and tree-dimensional skin cell cultures. Additionally, in this work we wanted to prove the usefulness of in vitro skin cell cultures in cosmetology research.

  9. Batch variation between branchial cell cultures: An analysis of variance

    DEFF Research Database (Denmark)

    Hansen, Heinz Johs. Max; Grosell, M.; Kristensen, L.

    2003-01-01

    We present in detail how a statistical analysis of variance (ANOVA) is used to sort out the effect of an unexpected batch-to-batch variation between cell cultures. Two separate cultures of rainbow trout branchial cells were grown on permeable filtersupports ("inserts"). They were supposed...... and introducing the observed difference between batches as one of the factors in an expanded three-dimensional ANOVA, we were able to overcome an otherwisecrucial lack of sufficiently reproducible duplicate values. We could thereby show that the effect of changing the apical medium was much more marked when...... the radioactive lipid precursors were added on the apical, rather than on the basolateral, side. Theinsert cell cultures were obviously polarized. We argue that it is not reasonable to reject troublesome experimental results, when we do not know a priori that something went wrong. The ANOVA is a very useful...

  10. Identification and quantitation of morphological cell types in electrophoretically separated human embryonic kidney cell cultures

    Science.gov (United States)

    Williams, K. B.; Kunze, M. E.; Todd, P. W.

    1985-01-01

    Four major cell types were identified by phase microscopy in early passage human embryonic kidney cell cultures. They are small and large epithelioid, domed, and fenestrated cells. Fibroblasts are also present in some explants. The percent of each cell type changes with passage number as any given culture grows. As a general rule, the fraction of small epithelioid cells increases, while the fraction of fenestrated cells, always small, decreases further. When fibroblasts are present, they always increase in percentage of the total cell population. Electrophoretic separation of early passage cells showed that the domed cells have the highest electrophoretic mobility, fibroblasts have an intermediate high mobility, small epithelioid cells have a low mobility, broadly distributed, and fenestrated cells have the lowest mobility. All cell types were broadly distributed among electrophoretic subfractions, which were never pure but only enriched with respect to a given cell type.

  11. Extracellular transport of cell-size particles and tumor cells by dendritic cells in culture.

    Science.gov (United States)

    Thacker, Robert I; Retzinger, Andrew C; Cash, James G; Dentler, Michael D; Retzinger, Gregory S

    2013-12-01

    Many particulate materials of sizes approximating that of a cell disseminate after being introduced into the body. While some move about within phagocytic inflammatory cells, others appear to move about outside of, but in contact with, such cells. In this report, we provide unequivocal photomicroscopic evidence that cultured, mature, human dendritic cells can transport in extracellular fashion over significant distances both polymeric beads and tumor cells. At least in the case of polymeric beads, both fibrinogen and the β2-integrin subunit, CD18, appear to play important roles in the transport process. These discoveries may yield insight into a host of disease-related phenomena, including and especially tumor cell invasion and metastasis. © 2013. Published by Elsevier Inc. All rights reserved.

  12. Lingual Epithelial Stem Cells and Organoid Culture of Them

    Directory of Open Access Journals (Sweden)

    Hiroko Hisha

    2016-01-01

    Full Text Available As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP, were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine.

  13. Effects of rotational culture on morphology, nitric oxide production and cell cycle of endothelial cells.

    Science.gov (United States)

    Tang, Chaojun; Wu, Xue; Ye, Linqi; Xie, Xiang; Wang, Guixue

    2012-12-01

    Devices for the rotational culture of cells and the study of biological reactions have been widely applied in tissue engineering. However, there are few reports exploring the effects of rotational culture on cell morphology, nitric oxide (NO) production, and cell cycle of the endothelial cells from human umbilical vein on the stent surface. This study focuses on these parameters after the cells are seeded on the stents. Results showed that covering of stents by endothelial cells was improved by rotational culture. NO production decreased within 24 h in both rotational and static culture groups. In addition, rotational culture significantly increased NO production by 37.9% at 36 h and 28.9% at 48 h compared with static culture. Flow cytometry showed that the cell cycle was not obviously influenced by rotational culture. Results indicate that rotational culture may be helpful for preparation of cell-seeded vascular grafts and intravascular stents, which are expected to be the most frequently implanted materials in the future.

  14. A Simple Hydrophilic Treatment of SU-8 Surfaces for Cell Culturing and Cell Patterning

    DEFF Research Database (Denmark)

    Wang, Zhenyu; Stangegaard, Michael; Dufva, Hans Martin

    2005-01-01

    SU-8, an epoxy-based photoresist, widely used in constitution different mTAS systems, is incompatible with mammalian cell adhesion and culture in its native form. Here, we demonstrate a simple, cheap and robust two-step method to render a SU-8 surface hydrophilic and compatible with cell culture...

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

    Science.gov (United States)

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

    2016-09-01

    In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described. © 2016 by the Society for Experimental Biology and Medicine.

  16. Biological Effects of Culture Substrates on Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Yohei Hayashi

    2016-01-01

    Full Text Available In recent years, as human pluripotent stem cells (hPSCs have been commonly cultured in feeder-free conditions, a number of cell culture substrates have been applied or developed. However, the functional roles of these substrates in maintaining hPSC self-renewal remain unclear. Here in this review, we summarize the types of these substrates and their effect on maintaining hPSC self-renewal. Endogenous extracellular matrix (ECM protein expression has been shown to be crucial in maintaining hPSC self-renewal. These ECM molecules interact with integrin cell-surface receptors and transmit their cellular signaling. We discuss the possible effect of integrin-mediated signaling pathways on maintaining hPSC self-renewal. Activation of integrin-linked kinase (ILK, which transmits ECM-integrin signaling to AKT (also known as protein kinase B, has been shown to be critical in maintaining hPSC self-renewal. Also, since naïve pluripotency has been widely recognized as an alternative pluripotent state of hPSCs, we discuss the possible effects of culture substrates and integrin signaling on naïve hPSCs based on the studies of mouse embryonic stem cells. Understanding the role of culture substrates in hPSC self-renewal and differentiation enables us to control hPSC behavior precisely and to establish scalable or microfabricated culture technologies for regenerative medicine and drug development.

  17. Dynamic cell culture system: a new cell cultivation instrument for biological experiments in space

    Science.gov (United States)

    Gmunder, F. K.; Nordau, C. G.; Tschopp, A.; Huber, B.; Cogoli, A.

    1988-01-01

    The prototype of a miniaturized cell cultivation instrument for animal cell culture experiments aboard Spacelab is presented (Dynamic cell culture system: DCCS). The cell chamber is completely filled and has a working volume of 200 microliters. Medium exchange is achieved with a self-powered osmotic pump (flowrate 1 microliter h-1). The reservoir volume of culture medium is 230 microliters. The system is neither mechanically stirred nor equipped with sensors. Hamster kidney (Hak) cells growing on Cytodex 3 microcarriers were used to test the biological performance of the DCCS. Growth characteristics in the DCCS, as judged by maximal cell density, glucose consumption, lactic acid secretion and pH, were similar to those in cell culture tubes.

  18. [Culture and control of cells producing bovine leukemia virus].

    Science.gov (United States)

    Granátová, M

    1987-10-01

    In the field surveys of the occurrence of enzootic bovine leucosis caused by the bovine leucosis virus (BLV), the identification of positive animals is based on the detection of specific antiviral antibodies by serological methods. The reliability of these tests (particularly their sensitivity and specificity) depends on the quality of the virus antigen. The preparation of the antigen is based on the cultivation of BLV virus in cultures of the FLS cell line. A modified procedure of preparing the BLV antigen in the FLS cell culture is described, along with the control of its production by the immunoperoxidase test.

  19. A single-cell and feeder-free culture system for monkey embryonic stem cells.

    Science.gov (United States)

    Ono, Takashi; Suzuki, Yutaka; Kato, Yosuke; Fujita, Risako; Araki, Toshihiro; Yamashita, Tomoko; Kato, Hidemasa; Torii, Ryuzo; Sato, Naoya

    2014-01-01

    Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.

  20. The role of Vimentin in Regulating Cell Invasive Migration in Dense Cultures of Breast Carcinoma Cells

    Science.gov (United States)

    Messica, Yonatan; Laser-Azogui, Adi; Volberg, Tova; Elisha, Yair; Lysakovskaia, Kseniia; Eils, Roland; Gladilin, Evgeny; Geiger, Benjamin; Beck, Roy

    2017-11-01

    Cell migration and mechanics are tightly regulated by the integrated activities of the various cytoskeletal networks. In cancer cells, cytoskeletal modulations have been implicated in the loss of tissue integrity, and acquisition of an invasive phenotype. In epithelial cancers, for example, increased expression of the cytoskeletal filament protein vimentin correlates with metastatic potential. Nonetheless, the exact mechanism whereby vimentin affects cell motility remains poorly understood. In this study, we measured the effects of vimentin expression on the mechano-elastic and migratory properties of the highly invasive breast carcinoma cell line MDA231. We demonstrate here that vimentin stiffens cells and enhances cell migration in dense cultures, but exerts little or no effect on the migration of sparsely plated cells. These results suggest that cell-cell interactions play a key role in regulating cell migration, and coordinating cell movement in dense cultures. Our findings pave the way towards understanding the relationship between cell migration and mechanics, in a biologically relevant context.

  1. Prediction of cell culture media performance using fluorescence spectroscopy.

    Science.gov (United States)

    Ryan, Paul W; Li, Boyan; Shanahan, Michael; Leister, Kirk J; Ryder, Alan G

    2010-02-15

    Cell culture media used in industrial mammalian cell culture are complex aqueous solutions that are inherently difficult to analyze comprehensively. The analysis of media quality and variance is of utmost importance in efficient manufacturing. We are exploring the use of rapid "holistic" analytical methods that can be used for routine screening of cell culture media used in industrial biotechnology. The application of rapid fluorescence spectroscopic techniques to the routine analysis of cell culture media (Chinese hamster ovary cell-based manufacture) was investigated. We have developed robust methods which can be used to identify compositional changes and ultimately predict the efficacy of individual fed batch media in terms of downstream protein product yield with an accuracy of +/-0.13 g/L. This is achieved through the implementation of chemometric methods such as multiway robust principal component analysis (MROBPCA), and n-way partial least-squares-discriminant analysis and regression (NPLS-DA and NPLS). This ability to observe compositional changes and predict product yield before media use has enormous potential and should permit the effective elimination of one of the major process variables leading to more consistent product quality and improved yield. These robust and reliable methods have the potential to become an important part of upstream biopharmaceutical quality control and analysis.

  2. Clock genes of Mammalian cells: practical implications in tissue culture.

    Science.gov (United States)

    Kaeffer, Bertrand; Pardini, Lissia

    2005-01-01

    The clock genes family is expressed by all the somatic cells driving central and peripheral circadian rhythms through transcription/translation feedback loops. The circadian clock provides a local time for a cell and a way to integrate the normal environmental changes to smoothly adapt the cellular machinery to new conditions. The central circadian rhythm is retained in primary cultures by neurons of the suprachiasmatic nuclei. The peripheral circadian rhythms of the other somatic cells are progressively dampened down up to loss unless neuronal signals of the central clock are provided for re-entrainment. Under typical culture conditions (obscurity, 37 +/- 1 degrees C, 5-7% CO(2)), freshly explanted peripheral cells harbor chaotic expression of clock genes for 12-14 h and loose, coordinated oscillating patterns of clock components. Cells of normal or cancerous phenotypes established in culture harbor low levels of clock genes idling up to the re-occurrence of new synchronizer signals. Synchronizers are physicochemical cues (like thermic oscillations, short-term exposure to high concentrations of serum or single medium exchange) able to re-induce molecular oscillations of clock genes. The environmental synchronizers are integrated by response elements located in the promoter region of period genes that drive the central oscillator complex (CLOCK:BMAL1 and NPAS2:BMAL1 heterodimers). Only a few cell lines from different species and lineages have been tested for the existence or the functioning of a circadian clockwork. The best characterized cell lines are the immortalized SCN2.2 neurons of rat suprachiasmatic nuclei for the central clock and the Rat-1 fibroblasts or the NIH/3T3 cells for peripheral clocks. Isolation methods of fragile cell phenotypes may benefit from research on the biological clocks to design improved tissue culture media and new bioassays to diagnose pernicious consequences for health of circadian rhythm alterations.

  3. Cell culture media impact on drug product solution stability.

    Science.gov (United States)

    Purdie, Jennifer L; Kowle, Ronald L; Langland, Amie L; Patel, Chetan N; Ouyang, Anli; Olson, Donald J

    2016-07-08

    To enable subcutaneous administration of monoclonal antibodies, drug product solutions are often needed at high concentrations. A significant risk associated with high drug product concentrations is an increase in aggregate level over the shelf-life dating period. While much work has been done to understand the impact of drug product formulation on aggregation, there is limited understanding of the link between cell culture process conditions and soluble aggregate growth in drug product. During cell culture process development, soluble aggregates are often measured at harvest using cell-free material purified by Protein A chromatography. In the work reported here, cell culture media components were evaluated with respect to their impact on aggregate levels in high concentration solution drug product during accelerated stability studies. Two components, cysteine and ferric ammonium citrate, were found to impact aggregate growth rates in our current media (version 1) leading to the development of new chemically defined media and concentrated feed formulations. The new version of media and associated concentrated feeds (version 2) were evaluated across four cell lines producing recombinant IgG4 monoclonal antibodies and a bispecific antibody. In all four cell lines, the version 2 media reduced aggregate growth over the course of a 12 week accelerated stability study compared with the version 1 media, although the degree to which aggregate growth decreased was cell line dependent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:998-1008, 2016. © 2016 American Institute of Chemical Engineers.

  4. Testing of serum atherogenicity in cell cultures: questionable data published

    Directory of Open Access Journals (Sweden)

    Sergei V. Jargin

    2012-01-01

    Full Text Available In a large series of studies was reported that culturing of smooth muscle cells with serum from atherosclerosis patients caused intracellular lipid accumulation, while serum from healthy controls had no such effect. Cultures were used for evaluation of antiatherogenic drugs. Numerous substances were reported to lower serum atherogenicity: statins, trapidil, calcium antagonists, garlic derivatives etc. On the contrary, beta-blockers, phenothiazines and oral hypoglycemics were reported to be pro-atherogenic. Known antiatherogenic agents can influence lipid metabolism and cholesterol synthesis, intestinal absorption or endothelium-related mechanisms. All these targets are absent in cell monocultures. Inflammatory factors, addressed by some antiatherogenic drugs, are also not reproduced. In vivo, relationship between cholesterol uptake by cells and atherogenesis must be inverse rather than direct: in familial hypercholesterolemia, inefficient clearance of LDL-cholesterol by cells predisposes to atherosclerosis. Accordingly, if a pharmacological agent reduces cholesterol uptake by cells in vitro, it should be expected to elevate cholesterol in vivo. Validity of clinical recommendations, based on serum atherogenicity testing in cell monocultures, is therefore questionable. These considerations pertain also to the drugs developed on the basis of the cell culture experiments.

  5. High-Throughput Cancer Cell Sphere Formation for 3D Cell Culture.

    Science.gov (United States)

    Chen, Yu-Chih; Yoon, Euisik

    2017-01-01

    Three-dimensional (3D) cell culture is critical in studying cancer pathology and drug response. Though 3D cancer sphere culture can be performed in low-adherent dishes or well plates, the unregulated cell aggregation may skew the results. On contrary, microfluidic 3D culture can allow precise control of cell microenvironments, and provide higher throughput by orders of magnitude. In this chapter, we will look into engineering innovations in a microfluidic platform for high-throughput cancer cell sphere formation and review the implementation methods in detail.

  6. Cannabinoids induce incomplete maturation of cultured human leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-08-01

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

  7. Culture of Oral Mucosal Epithelial Cells for the Purpose of Treating Limbal Stem Cell Deficiency.

    Science.gov (United States)

    Utheim, Tor Paaske; Utheim, Øygunn Aass; Khan, Qalb-E-Saleem; Sehic, Amer

    2016-03-01

    The cornea is critical for normal vision as it allows allowing light transmission to the retina. The corneal epithelium is renewed by limbal epithelial cells (LEC), which are located in the periphery of the cornea, the limbus. Damage or disease involving LEC may lead to various clinical presentations of limbal stem cell deficiency (LSCD). Both severe pain and blindness may result. Transplantation of cultured autologous oral mucosal epithelial cell sheet (CAOMECS) represents the first use of a cultured non-limbal autologous cell type to treat this disease. Among non-limbal cell types, CAOMECS and conjunctival epithelial cells are the only laboratory cultured cell sources that have been explored in humans. Thus far, the expression of p63 is the only predictor of clinical outcome following transplantation to correct LSCD. The optimal culture method and substrate for CAOMECS is not established. The present review focuses on cell culture methods, with particular emphasis on substrates. Most culture protocols for CAOMECS used amniotic membrane as a substrate and included the xenogeneic components fetal bovine serum and murine 3T3 fibroblasts. However, it has been demonstrated that tissue-engineered epithelial cell sheet grafts can be successfully fabricated using temperature-responsive culture surfaces and autologous serum. In the studies using different substrates for culture of CAOMECS, the quantitative expression of p63 was generally poorly reported; thus, more research is warranted with quantification of phenotypic data. Further research is required to develop a culture system for CAOMECS that mimics the natural environment of oral/limbal/corneal epithelial cells without the need for undefined foreign materials such as serum and feeder cells.

  8. Influence of three laser wavelengths on human fibroblasts cell culture.

    Science.gov (United States)

    Crisan, Bogdan; Soritau, Olga; Baciut, Mihaela; Campian, Radu; Crisan, Liana; Baciut, Grigore

    2013-02-01

    Although experimental studies in vitro and vivo have been numerous, the effect of laser wavelength irradiation on human fibroblast cell culture is poorly understood. This emphasizes the need of additional cellular and molecular research into laser influence with low energy and power. The aim of this study was to assess the influence of three different laser wavelengths on the human skin fibroblasts cell culture. We wanted to evaluate if near infrared lasers had any influence in healing of wounds by stimulating mitochondrial activity of fibroblasts. The cells were irradiated using 830-, 980- and 2,940-nm laser wavelengths. The irradiated cells were incubated and their mitochondrial activity was assessed by the MTT assay at 24, 48 and 72 h. Simultaneously, an apoptosis assay was assessed on the irradiated fibroblasts. It can be concluded that laser light of the near-infrared region (830 and 980 nm) influences fibroblasts mitochondrial activity compared to the 2,940-nm wavelength which produces apoptosis.

  9. In vitro culture of Keratinocytes from human umbilical cord blood mesenchymal stem cells: the Saigonese culture.

    Science.gov (United States)

    Tran, Cong Toai; Huynh, Duy Thao; Gargiulo, Ciro; Nguyen, Phuong Thao; Tran, Thi Thanh Thuy; Huynh, Minh Tuan; Nguyen, Thanh Tung; Filgueira, Luis; Strong, D Micheal

    2011-05-01

    There have been many attempts to acquire and culture human keratinocytes for clinical purposes including from keratotome slices in media with fetal calf serum (FCS) or pituitary extract (PE), from skin specimens in media with feeder layers, from suction blister epidermal roofs' in serum-free culture and from human umbilical cord blood (hUCB) mesenchymal stem cells (MSCs) in media with skin feeder layers. Conversely this study was designed to investigate whether keratinocytes could be obtained directly from hUCB MSCs in vitro. It is widely established that mesenchymal stem cells from human umbilical cord blood have multipotent capacity and the ability to differentiate into disparate cell lineages hUCB MSCs were directly induced to differentiate into keratinocytes by using a specific medium composed of primary culture medium (PCM) and serum free medium (SFM) in a ratio 1:9 for a period of 7 days and tested by immunostain p63 and K1-K10. Cells thus cultured were positive in both tests, confirming the possibility to directly obtain keratinocytes from MSCs hUCB in vitro.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    Microfluidic biochips offer a promising alternative to a conventional biochemical laboratory. There are two technologies for the microfluidic biochips: droplet-based and flow-based. In this paper we are interested in flow-based microfluidic biochips, where the liquid flows continuously through pre......-defined micro-channels using valves and pumps. We present an approach to the system-level modeling and simulation of a cell culture microfluidic biochip called ProCell, Programmable Cell Culture Chip. ProCell contains a cell culture chamber, which is envisioned to run 256 simultaneous experiments (viewed...... and a comprehensive fault model that captures permanent faults occurring during chip operation. Using the proposed modeling and simulation framework, we perform an architectural level evaluation of two cell culture chamber implementations. A qualitative success metric is also proposed to evaluate chip performance...

  11. Cell therapy with human renal cell cultures containing erythropoietin-positive cells improves chronic kidney injury.

    Science.gov (United States)

    Yamaleyeva, Liliya M; Guimaraes-Souza, Nadia K; Krane, Louis S; Agcaoili, Sigrid; Gyabaah, Kenneth; Atala, Anthony; Aboushwareb, Tamer; Yoo, James J

    2012-05-01

    New therapeutic strategies for chronic kidney disease (CKD) are necessary to offset the rising incidence of CKD and donor shortage. Erythropoietin (EPO), a cytokine produced by fibroblast-like cells in the kidney, has recently emerged as a renoprotective factor with anti-inflammatory, antioxidant properties. This study (a) determined whether human renal cultures (human primary kidney cells [hPKC]) can be enriched in EPO-positive cells (hPKC(F+)) by using magnetic-bead sorting; (b) characterized hPKC(F+) following cell separation; and (c) established that intrarenal delivery of enriched hPKC(F+) cells would be more beneficial in treatment of renal injury, inflammation, and oxidative stress than unsorted hPKC cultures in a chronic kidney injury model. Fluorescence-activated cell sorting analysis revealed higher expression of EPO (36%) and CD73 (27%) in hPKC(F+) as compared with hPKC. After induction of renal injury, intrarenal delivery of hPKC(F+) or hPKC significantly reduced serum creatinine, interstitial fibrosis in the medulla, and abundance of CD68-positive cells in the cortex and medulla (p renal cortex and decreased urinary albumin (3.5-fold) and urinary tubular injury marker kidney injury molecule 1 (16-fold). hPKC(F+) also significantly reduced levels of renal cortical monocyte chemotactic protein 1 (1.8-fold) and oxidative DNA marker 8-hydroxy-deoxyguanosine (8-OHdG) (2.4-fold). After 12 weeks, we detected few injected cells, which were localized mostly to the cortical interstitium. Although cell therapy with either hPKC(F+) or hPKC improved renal function, the hPKC(F+) subpopulation provides greater renoprotection, perhaps through attenuation of inflammation and oxidative stress. We conclude that hPKC(F+) may be used as components of cell-based therapies for degenerative kidney diseases.

  12. Isolation and Culture of Single Cell Types from Rat Liver.

    Science.gov (United States)

    Zhang, Qidi; Qu, Ying; Li, Zhenghong; Zhang, Qingqing; Xu, Mingyi; Cai, Xiaobo; Li, Fei; Lu, Lungen

    2016-01-01

    There have been few reports on the simultaneous isolation of multiple liver cell populations thus far. As such, this study was aimed at establishing a protocol for the simultaneous separation of hepatocytes (HCs), hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) from the rat liver and assessing the in vitro culture of these cells. Single-cell suspensions from the liver were obtained by ethylene glycol tetraacetic acid/collagenase perfusion. After low-speed centrifugal separation of HCs, pronase was added to the nonparenchymal cell fraction to eliminate the remaining HCs. Subsequently, HSCs, LSECs and KCs were purified by two steps of density gradient centrifugation using Nycodenz and Percoll in addition to selective attachment. Pronase treatment increased the HSC yield (1.5 ± 0.2 vs. 0.7 ± 0.3 cells/g liver, p cultured in vitro. LSEC apoptosis began on day 3 and reached a maximum on day 7. A few surviving LSECs began proliferating and split to form a cobblestone, sheet-like appearance on day 14. The LSECs on day 14 lost fenestrations but retained scavenger function. Thus, viable and purified liver cells were obtained with a high yield from the rat liver using the developed method, which may be useful for studying the physiology and pathology of the liver in the future. © 2016 S. Karger AG, Basel.

  13. Carbohydrates-chitosan composite carrier for Vero cell culture.

    Science.gov (United States)

    Lin, Ya-Ching; Chen, Guan-Ting; Wu, Sheng-Chi

    2016-12-01

    In this study, carbohydrate-chitosan composite including glucose-chitosan, sucrose-chitosan and starch-chitosan with varied carbohydrate concentrations were prepared as carriers for Vero cell culture. Our results show that among these composites, 30 % starch-chitosan composite (STC) were the best carriers for the growth of Vero cells. The initial number of attached cells on the surface of composite carriers did not have any significant effect on subsequent cell production. A higher glucose level in the growth medium during the exponential phase of cell growth, however, played an important factor for cell production. Vero cells on the STC carriers were able to convert starch inside the composite carriers into glucose and further utilized the glucose for their growth. Moreover, by crosslink with serum the STC carriers supported an even better cell production in the normal medium without adding fetal bovine serum, as well as a good extracellular virus production. The STC composite is therefore a promising alternative carrier for Vero cell culture.

  14. Microtable Arrays for Culture and Isolation of Cell Colonies

    Science.gov (United States)

    Pai, Jeng-Hao; Xu, Wei; Sims, Christopher E.; Allbritton, Nancy L.

    2010-01-01

    Cell microarrays with culture sites composed of individually removable microstructures or micropallets have proven benefits for isolation of cells from a mixed population. The laser energy required to selectively remove these micropallets with attached cells from the array depends on the microstructure surface area in contact with the substrate. Laser energies sufficient to release micropallets greater than 100 μm resulted in loss of cell viability. A new 3-dimensional culture site similar in appearance to a table was designed and fabricated using a simple process that relied on a differential sensitivity of two photoresists to UV-mediated photopolymerization. With this design, the larger culture area rests on four small supports to minimize the surface area in contact with the substrate. Microtables up to 250 × 250 μm were consistently released with single 10 μJ pulses to each of the 4 support structures. In contrast, microstructures with a 150 × 150 μm surface area in contact with the substrate could not be reliably released at pulse energies up to 212 μJ. Cassie-Baxter wetting is required to provide a barrier of air to localize and sequester cells to the culture sites. A second asset of the design was an increased retention of this air barrier under conditions of decreased surface tension and after prolonged culture of cells. The improved air retention was due to the hydrophobic cavity created beneath the table and above the substrate which entrapped air when an aqueous solution was added to the array. The microtables proved an efficient method for isolating colonies from the array with 100% of selected colonies competent to expand following release from the array. PMID:20644916

  15. Effects of viscoelastic ophthalmic solutions on cell cultures

    Directory of Open Access Journals (Sweden)

    Madhavan Hajib

    1998-01-01

    Full Text Available The development of mild but significant inflammation probably attributable to viscoelastic ophthalmic solutions in cataract surgery was recently brought to the notice of the authors, and hence a study of the effects of these solutions available in India, on cell cultures was undertaken. We studied the effects of 6 viscoelastic ophthalmic solutions (2 sodium hyaluronate designated as A and B, and 4 hydroxypropylmethylcellulose designated as C, D, E and F on HeLa, Vero and BHK-21 cell lines in tissue culture microtitre plates using undiluted, 1:10 and 1:100 dilutions of the solutions, and in cover slip cultures using undiluted solutions. Phase contrast microscopic examination of the solutions was also done to determine the presence of floating particles. The products D and F produced cytotoxic changes in HeLa cell line and these products also showed the presence of floating particles under phase contrast microscopy. Other products did not have any adverse effects on the cell lines nor did they show floating particles. The viscoelastic ophthalmic pharmaceutical products designated D and F have cytotoxic effects on HeLa cell line which appears to be a useful cell line for testing these products for their toxicity. The presence of particulate materials in products D and F indicates that the methods used for purification of the solution are not effective.

  16. Three-dimensional cultures of mouse mammary epithelial cells.

    Science.gov (United States)

    Mroue, Rana; Bissell, Mina J

    2013-01-01

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

  17. A Novel Counter Sheet-flow Sandwich Cell Culture Device for Mammalian Cell Growth in Space

    Science.gov (United States)

    Sun, Shujin; Gao, Yuxin; Shu, Nanjiang; Tang, Zemei; Tao, Zulai; Long, Mian

    2008-08-01

    Cell culture and growth in space is crucial to understand the cellular responses under microgravity. The effects of microgravity were coupled with such environment restrictions as medium perfusion, in which the underlying mechanism has been poorly understood. In the present work, a customer-made counter sheet-flow sandwich cell culture device was developed upon a biomechanical concept from fish gill breathing. The sandwich culture unit consists of two side chambers where the medium flow is counter-directional, a central chamber where the cells are cultured, and two porous polycarbonate membranes between side and central chambers. Flow dynamics analysis revealed the symmetrical velocity profile and uniform low shear rate distribution of flowing medium inside the central culture chamber, which promotes sufficient mass transport and nutrient supply for mammalian cell growth. An on-orbit experiment performed on a recovery satellite was used to validate the availability of the device.

  18. The molecularly crowded cytoplasm of bacterialcCells : Dividing cells contrasted with viable but non-culturable (VBNC) bacterial cells

    NARCIS (Netherlands)

    Trevors, J. T.; van Elsas, J. D.; Bej, A. K.

    2013-01-01

    In this perspective, we discuss the cytoplasm in actively growing bacterial cells contrasted with viable but non-culturable (VBNC) cells. Actively growing bacterial cells contain a more molecularly crowded and organized cytoplasm, and are capable of completing their cell cycle resulting in cell

  19. Cell Stratification, Spheroid Formation and Bioscaffolds Used to Grow Cells in Three Dimensional Cultures

    Directory of Open Access Journals (Sweden)

    Hana Hrebíková

    2015-12-01

    Full Text Available The cell culture became an invaluable tool for studying cell behaviour, development, function, gene expression, toxicity of compounds and efficacy of novel drugs. Although most results were obtained from cell cultivation in two-dimensional (2D systems, in which cells are grown in a monolayer, three-dimensional (3D cultures are more promising as they correspond closely to the native arrangement of cells in living tissues. In our study, we focused on three types of 3D in vitro systems used for cultivation of one cell type. Cell morphology, their spatial distribution inside of resulting multicellular structures and changes in time were analysed with histological examination of samples harvested at different time periods. In multilayered cultures of WRL 68 hepatocytes grown on semipermeable membranes and non-passaged neurospheres generated by proliferation of neural progenitor cells, the cells were tightly apposed, showed features of cell differentiation but also cell death that was observable in short-term cultures. Biogenic scaffolds composed of extracellular matrix of the murine tibial anterior muscle were colonized with C2C12 myoblasts in vitro. The recellularized scaffolds did not reach high cell densities comparable with the former systems but supported well cell anchorage and migration without any signs of cell regression.

  20. CYTOTOXICITY TESTING OF WOUND DRESSINGS USING METHYLCELLULOSE CELL-CULTURE

    NARCIS (Netherlands)

    VANLUYN, MJA; VANWACHEM, PB; NIEUWENHUIS, P; JONKMAN, MF

    1992-01-01

    Wound dressings may induce cytotoxic effects. In this study, we check several, mostly commercially available, wound dressings for cytotoxicity. We used our previously described, newly developed and highly sensitive 7 d methylcellulose cell culture with fibroblasts as the test system. Cytotoxicity is

  1. Biotransformation of Dydrogesterone by Cell Suspension Cultures of Azadirachta indica

    OpenAIRE

    KHAN, Saifullah; CHOUDHARY, Muhammad Iqbal

    2008-01-01

    Biotransformation of dydrogesterone (1) by using cell suspension cultures of Azadirachta indica yielded a metabolite 20R-hydroxy-9b ,10a-pregna-4,6-diene-3-one (2). The structure of this compound was deduced on the basis of various spectroscopic techniques.

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

    Science.gov (United States)

    Majumdar, Shyamal K.; Castellano, John M.

    1977-01-01

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

  3. Biodegradable Mg corrosion and osteoblast cell culture studies

    Energy Technology Data Exchange (ETDEWEB)

    Yun, Yeo Heung, E-mail: yunyg@email.uc.edu [University of Cincinnati, Cincinnati, OH, 45221 (United States); Dong Zhongyun; Yang Dianer; Schulz, Mark J.; Shanov, Vesselin N. [University of Cincinnati, Cincinnati, OH, 45221 (United States); Yarmolenko, Sergey; Xu Zhigang [North Carolina A and T SU, Greensboro, NC, 27358 (United States); Kumta, Prashant; Sfeir, Charles [University of Pittsburgh, Pittsburgh, PA, 15261 (United States)

    2009-08-01

    Magnesium (Mg) is a biodegradable metal that has significant potential advantages as an implant material. In this paper, corrosion and cell culture experiments were performed to evaluate the biocompatibility of Mg. The corrosion current and potential of a Mg disk were measured in different physiological solutions including deionized (DI) water, phosphate-buffered saline (PBS), and McCoy's 5A culture medium. The corrosion currents in the PBS and in the McCoy's 5A-5% FBS media were found to be higher than in DI water, which is expected because corrosion of Mg occurs faster in a chloride solution. Weight loss, open-circuit potential, and electrochemical impedance spectroscopy measurements were also performed. The Mg specimens were also characterized using an environmental scanning electron microscope and energy-dispersive X-ray analysis (EDAX). The X-ray analysis showed that in the cell culture media a passive interfacial layer containing oxygen, chloride, phosphate, and potassium formed on the samples. U2OS cells were then co-cultured with a Mg specimen for up to one week. Cytotoxicity results of magnesium using MTT assay and visual observation through cell staining were not significantly altered by the presence of the corroding Mg sample. Further, bone tissue formation study using von Kossa and alkaline phosphatase staining indicates that Mg may be suitable as a biodegradable implant material.

  4. Disposable Bioreactors for Plant Micropropagation and Mass Plant Cell Culture

    Science.gov (United States)

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

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

  5. Metabotropic glutamate receptors in cultured cerebellar granule cells: developmental profile

    NARCIS (Netherlands)

    Aronica, E.; Condorelli, D. F.; Nicoletti, F.; Dell'Albani, P.; Amico, C.; Balázs, R.

    1993-01-01

    Excitatory amino acid (EAA)-induced polyphosphoinositide (PPI) hydrolysis was studied during the development in culture of cerebellar granule cells. The developmental pattern was similar using metabotropic glutamate (Glu) receptor (mGluR) agonists, including L-Glu, quisqualate, and

  6. Enhancement of Diosgenin Production in Plantlet and Cell Cultures ...

    African Journals Online (AJOL)

    Purpose: To study the effect of palmarumycin C13, an elicitor from the endophytic fungus Berkleasmium sp. Dzf12, on growth and diosgenin production in plantlet or cell cultures of its host plant, Dioscorea zingiberensis. Methods: Palmarumycin C13 was isolated from the ethyl acetate extract of the endophytic fungus.

  7. Plant Cell Cultures as Source of Cosmetic Active Ingredients

    Directory of Open Access Journals (Sweden)

    Ani Barbulova

    2014-04-01

    Full Text Available The last decades witnessed a great demand of natural remedies. As a result, medicinal plants have been increasingly cultivated on a commercial scale, but the yield, the productive quality and the safety have not always been satisfactory. Plant cell cultures provide useful alternatives for the production of active ingredients for biomedical and cosmetic uses, since they represent standardized, contaminant-free and biosustainable systems, which allow the production of desired compounds on an industrial scale. Moreover, thanks to their totipotency, plant cells grown as liquid suspension cultures can be used as “biofactories” for the production of commercially interesting secondary metabolites, which are in many cases synthesized in low amounts in plant tissues and differentially distributed in the plant organs, such as roots, leaves, flowers or fruits. Although it is very widespread in the pharmaceutical industry, plant cell culture technology is not yet very common in the cosmetic field. The aim of the present review is to focus on the successful research accomplishments in the development of plant cell cultures for the production of active ingredients for cosmetic applications.

  8. Establishment of the callus and cell suspension culture of ...

    African Journals Online (AJOL)

    The objective of this work was the optimization of the conditions of callus and cell suspension culture of Elaeagnus angustifolia for the production of condensed tannins. The effects of different conditions on the callus growth and the production of condensed tannins were researched. The leaf tissue part of E. angustifolia was ...

  9. In vitro production of azadirachtin from cell suspension cultures of ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR G

    In vitro production of azadirachtin from cell suspension cultures of Azadirachta indica. 113. J. Biosci. 33(1), March 2008. 1. Introduction. Chemical pesticides, once considered a boon for increasing the yield of food crops, have now become a bane owing to the numerous cases of pesticide poisoning. Alternative pesticides ...

  10. DIVERSITY OF ARSENIC METABOLISM IN CULTURED HUMAN CANCER CELL LINES

    Science.gov (United States)

    Diversity of arsenic metabolism in cultured human cancer cell lines. Arsenic has been known to cause a variety of malignancies in human. Pentavalent As (As 5+) is reduced to trivalent As (As3+) which is further methylated by arsenic methyltransferase(s) to monomethylarson...

  11. Mixed cultures of Kimchi lactic acid bacteria show increased cell ...

    African Journals Online (AJOL)

    ufuoma

    solo culture, L. sakei 171 was superior in cell growth, lactate production and the release of amino acids .... The bacteria were grown under anaerobic conditions in the MRS broth containing glucose. The medium composition per liter was as follows: (a) 10 g peptone, 10 g beef extract, 5 g yeast extract, 3 g diammonium.

  12. In vitro plant regeneration from embryogenic cell suspension culture ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-05-02

    May 2, 2008 ... In vitro plant regeneration was achieved from embryogenic cell suspension culture of Astragalus chrysochlorus. When 30-day-old aseptically ... previous study, cytotoxic activities of stem and root ex-. *Corresponding author. E-mail: ... For callus induction, 30-day-old mesocotyl parts of seedlings were used.

  13. CD90 Expression on human primary cells and elimination of contaminating fibroblasts from cell cultures.

    Science.gov (United States)

    Kisselbach, Lynn; Merges, Michael; Bossie, Alexis; Boyd, Ann

    2009-01-01

    Cluster Differentiation 90 (CD90) is a cell surface glycoprotein originally identified on mouse thymocytes. Although CD90 has been identified on a variety of stem cells and at varying levels in non-lymphoid tissues such as on fibroblasts, brain cells, and activated endothelial cells, the knowledge about the levels of CD90 expression on different cell types, including human primary cells, is limited. The goal of this study was to identify CD90 as a human primary cell biomarker and to develop an efficient and reliable method for eliminating unwanted or contaminating fibroblasts from human primary cell cultures suitable for research pursuant to cell based therapy technologies.

  14. T cell resistance to activation by dendritic cells requires long-term culture in simulated microgravity

    Science.gov (United States)

    Bradley, Jillian H.; Stein, Rachel; Randolph, Brad; Molina, Emily; Arnold, Jennifer P.; Gregg, Randal K.

    2017-11-01

    Immune impairment mediated by microgravity threatens the success of space exploration requiring long-duration spaceflight. The cells of most concern, T lymphocytes, coordinate the host response against microbial and cancerous challenges leading to elimination and long-term protection. T cells are activated upon recognition of specific microbial peptides bound on the surface of antigen presenting cells, such as dendritic cells (DC). Subsequently, this engagement results in T cell proliferation and differentiation into effector T cells driven by autocrine interleukin-2 (IL-2) and other cytokines. Finally, the effector T cells acquire the weaponry needed to destroy microbial invaders and tumors. Studies conducted on T cells during spaceflight, or using Earth-based culture systems, have shown reduced production of cytokines, proliferation and effector functions as compared to controls. This may account for the cases of viral reactivation events and opportunistic infections associated with astronauts of numerous missions. This work has largely been based upon the outcome of T cell activation by stimulatory factors that target select T cell signaling pathways rather than the complex, signaling events related to the natural process of antigen presentation by DC. This study tested the response of an ovalbumin peptide-specific T cell line, OT-II TCH, to activation by DC when the T cells were cultured 24-120 h in a simulated microgravity (SMG) environment generated by a rotary cell culture system. Following 72 h culture of T cells in SMG (SMG-T) or control static (Static-T) conditions, IL-2 production by the T cells was reduced in SMG-T cells compared to Static-T cells upon stimulation by phorbol 12-myristate 13-acetate (PMA) and ionomycin. However, when the SMG-T cells were stimulated with DC and peptide, IL-2 was significantly increased compared to Static-T cells. Such enhanced IL-2 production by SMG-T cells peaked at 72 h SMG culture time and decreased thereafter. When

  15. Electrolytic Valving Isolation for Cell Co-Culture Microenvironment with Controlled Cell Pairing Ratios

    Science.gov (United States)

    Chen, Yu-Chih; Ingram, Patrick; Yoon, Euisik

    2016-01-01

    Cancer-stromal interaction is a critical process in tumorigenesis. Conventional dish-based co-culture assays simply mix two cell types in the same dish; thus, they are deficient in controlling cell locations and precisely tracking single cell behavior from heterogeneous cell populations. Microfluidic technology can provide a good spatial temporal control of microenvironments, but the control has been typically realized by using external pumps, making long-term cultures cumbersome and bulky. In this work, we present a cell-cell interaction microfluidic platform that can accurately control co-culture microenvironment by using a novel electrolytic cell isolation scheme without using any valves or pneumatic pumps. The proposed microfluidic platform can also precisely control the number of interacting cells and pairing ratios to emulate cancer niches. More than 80% of the chambers captured the desired number of cells. The duration of cell isolation can be adjusted by electrolytic bubble generation and removal. We verified that electrolytic process has a negligible effect on cell viability and proliferation in our platform. To the best of our knowledge, this work is the first attempt to incorporate electrolytic bubble generation as a cell isolation method in microfluidics. For proof of feasibility, we performed cell-cell interaction assays between prostate cancer (PC3) cells and myoblast (C2C12) cells. The preliminary results demonstrated the potential of using electrolysis for micro-environmental control during cell culture. Also, the ratio controlled cell-cell interaction assays was successfully performed showing that the cell pairing ratios of PC3 to C2C12 affected the proliferation rate of myoblast cells due to increased secretion of growth factors from prostate cancer cells. PMID:25118341

  16. Electrolytic valving isolation of cell co-culture microenvironment with controlled cell pairing ratios.

    Science.gov (United States)

    Chen, Yu-Chih; Ingram, Patrick; Yoon, Euisik

    2014-12-21

    Cancer-stromal interaction is a critical process in tumorigenesis. Conventional dish-based co-culture assays simply mix two cell types in the same dish; thus, they are deficient in controlling cell locations and precisely tracking single cell behavior from heterogeneous cell populations. Microfluidic technology can provide a good spatial-temporal control of microenvironments, but the control has been typically realized by using external pumps, making long-term cultures cumbersome and bulky. In this work, we have presented a cell-cell interaction microfluidic platform that can accurately control the co-culture microenvironment by using a novel electrolytic cell isolation scheme without using any valves or pneumatic pumps. The proposed microfluidic platform can also precisely control the number of interacting cells and pairing ratios to emulate cancer niches. More than 80% of the chambers captured the desired number of cells. The duration of cell isolation can be adjusted by electrolytic bubble generation and removal. We have verified that the electrolytic process has a negligible effect on cell viability and proliferation in our platform. To the best of our knowledge, this work is the first attempt to incorporate electrolytic bubble generation as a cell isolation method in microfluidics. For proof of feasibility, we have performed cell-cell interaction assays between prostate cancer (PC3) cells and myoblast (C2C12) cells. The preliminary results demonstrated the potential of using electrolysis for micro-environmental control during cell culture. Also, the ratio controlled cell-cell interaction assays were successfully performed which showed that the cell pairing ratios of PC3 to C2C12 affected the proliferation rate of myoblast cells due to increased secretion of growth factors from prostate cancer cells.

  17. Enrichment of skin-derived neural precursor cells from dermal cell populations by altering culture conditions.

    Science.gov (United States)

    Bayati, Vahid; Gazor, Rohoullah; Nejatbakhsh, Reza; Negad Dehbashi, Fereshteh

    2016-01-01

    As stem cells play a critical role in tissue repair, their manipulation for being applied in regenerative medicine is of great importance. Skin-derived precursors (SKPs) may be good candidates for use in cell-based therapy as the only neural stem cells which can be isolated from an accessible tissue, skin. Herein, we presented a simple protocol to enrich neural SKPs by monolayer adherent cultivation to prove the efficacy of this method. To enrich neural SKPs from dermal cell populations, we have found that a monolayer adherent cultivation helps to increase the numbers of neural precursor cells. Indeed, we have cultured dermal cells as monolayer under serum-supplemented (control) and serum-supplemented culture, followed by serum free cultivation (test) and compared. Finally, protein markers of SKPs were assessed and compared in both experimental groups and differentiation potential was evaluated in enriched culture. The cells of enriched culture concurrently expressed fibronectin, vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors as compared to control culture. In addition, they possessed a multipotential capacity to differentiate into neurogenic, glial, adipogenic, osteogenic and skeletal myogenic cell lineages. It was concluded that serum-free adherent culture reinforced by growth factors have been shown to be effective on proliferation of skin-derived neural precursor cells (skin-NPCs) and drive their selective and rapid expansion.

  18. Vitamin A metabolism in cultured somatic cells from rat testis.

    Science.gov (United States)

    Cavazzini, Davide; Catizone, Angela; Galdieri, Michela; Ottonello, Simone

    2003-10-01

    Sertoli and peritubular myoid cells, the somatic cells of the seminiferous tubule, support growth and differentiation of developing germ cells. This action strictly depends on the availability of in situ synthesized retinoic acid and we have previously documented the ability of Sertoli, but not peritubular cell extracts, to support the oxidation of retinol to retinoic acid. Using primary cultures of somatic cells treated with a physiological concentration of free retinol, we show here that the same is essentially true also for whole cultured cells. Sertoli cells are capable of producing not only retinoic acid, but are also the major site of retinyl ester (mainly, retinyl palmitate) formation. Compared with retinyl palmitate accumulation, retinoic acid synthesis was both faster and positively influenced by prior exposure to retinol. This increase in retinoic acid synthesis was further augmented by treatment with the retinoic acid catabolic inhibitor liarozole, thus indicating that enhanced synthesis, rather than reduced catabolism, is responsible for such an effect. Myoid cells had a higher capacity to incorporate exogenously supplied retinol, yet retinoic acid synthesis, and even more so retinyl palmitate formation, were considerably lower than in Sertoli cells. Retinoic acid synthesis in myoid cells was not only depressed, but also very little influenced by prior retinol exposure and totally insensitive to liarozole. These data further support the view that myoid cells are involved in retinol uptake from the blood and its transfer to other cells, rather than in metabolic interconversion or long-term storage of vitamin A, two processes that mainly take place in Sertoli cells.

  19. Studying cell cycle checkpoints using Drosophila cultured cells

    NARCIS (Netherlands)

    Siudeja, Katarzyna; de Jong, Jannie; Sibon, Ody

    2011-01-01

    Drosophila cell lines are valuable tools to study a number of cellular processes, including DNA damage responses and cell cycle checkpoint control. Using an in vitro system instead of a whole organism has two main advantages: it saves time and simple and effective molecular techniques are available.

  20. Ultrastructure of cells of Ulmus americana cultured in vitro and exposed to the culture filtrate of Ceratocystis ulmi

    Science.gov (United States)

    Paula M. Pijut; R. Daniel Lineberger; Subhash C. Domir; Jann M. Ichida; Charles R. Krause

    1990-01-01

    Calli of American elm susceptible and resistant to Dutch elm disease were exposed to a culture filtrate of a pathogenic isolate of Ceratocystis ulmi. Cells from untreated tissue exhibited typical internal composition associated with healthy, actively growing cells. All cells exposed to culture filtrate showed appreciable ultrastructural changes....

  1. [Influence of glycosaminoglycan synthesis of cultured cornea stroma cells by variation of culture condition].

    Science.gov (United States)

    Bleckmann, H; Kresse, H

    1979-06-15

    Cultured cells derived from bovine corneal stroma synthesize all types of sulfated glycosaminoglycans and distribute these macromolecules into topographically different compartments in a reproducible manner. Each compartment is characterized by a typical glycosaminoglycan distribution pattern. Corneal fibroblasts synthesize in vitro only small amounts of keratan sulfate in contrast to the in vivo conditions. We have, therefore, investigated the synthesis and topographical distribution of sulfated glycosaminoglycans as influenced by different culture conditions. The following results were obtained: 1) Cocultivation of epithelial and stromal fibroblasts from bovine cornea led to an increased incorporation of radiosulfate into sulfated glycosaminoglycans by about 50% as compared to the theoretical value. Glycosaminoglycan distribution of mixed cultures into different compartments showed no similarity compared with pure epithelial or stromal fibroblasts. 2) Addition of native or heat inactivated anterior chamber fluid to the culture medium was followed by a twofold increase of [35S]-sulfate incorporation and by an augmented intracellular and pericellular accumulation of labeled macromolecules. 3) Reduction of the incubation temperature led to a reduced synthesis of glycosaminoglycans without influencing their topographical distribution. Growth of stromal cells on type I collagen was accompanied by a reduced glycosaminoglycan synthesis of about 25%. Extracellular macromolecules reached only half of the normal value, while intracellularly their concentration was slightly increased. 4) None of the variations of the culture condition led to a significant change of the distribution pattern of sulfated glycosaminoglycans. Especially, no significant increase of keratan sulfate biosynthesis could be detected.

  2. Lethal impacts of cigarette smoke in cultured tobacco cells

    Directory of Open Access Journals (Sweden)

    Kawano Tomonori

    2011-07-01

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

  3. Development of the medial hypothalamus: forming a functional hypothalamic-neurohypophyseal interface.

    Science.gov (United States)

    Pearson, Caroline Alayne; Placzek, Marysia

    2013-01-01

    The medial hypothalamus is composed of nuclei of the tuberal hypothalamus, the paraventricular nucleus of the anterior hypothalamus, and the neurohypophysis. Its arrangement, around the third ventricle of the brain, above the adenohypophysis, and in direct contact with the vasculature, means that it serves as an interface with circulating systems, providing a key conduit through which the brain can sample, and control, peripheral body systems. Through these interfaces, and interactions with other parts of the brain, the medial hypothalamus centrally governs diverse homeostatic processes, including energy and fluid balance, stress responses, growth, and reproductive behaviors. Here, we summarize recent studies that reveal how the diverse cell types within the medial hypothalamus are assembled in an integrated manner to enable its later function. In particular, we discuss how the temporally protracted operation of signaling pathways and transcription factors governs the appearance and regionalization of the hypothalamic primordium from the prosencephalic territory, the specification and differentiation of progenitors into neurons in organized nuclei, and the establishment of interfaces. Through analyses of mouse, chick, and zebrafish, a picture emerges of an evolutionarily conserved and highly coordinated developmental program. Early indications suggest that deregulation of this program may underlie complex human pathological conditions and dysfunctional behaviors, including stress and eating disorders. © 2013 Elsevier Inc. All rights reserved.

  4. Wave characterization for mammalian cell culture: residence time distribution.

    Science.gov (United States)

    Rodrigues, Maria Elisa; Costa, Ana Rita; Henriques, Mariana; Azeredo, Joana; Oliveira, Rosário

    2012-02-15

    The high dose requirements of biopharmaceutical products led to the development of mammalian cell culture technologies that increase biomanufacturing capacity. The disposable Wave bioreactor is one of the most promising technologies, providing ease of operation and no cross-contamination, and using an innovative undulation movement that ensures good mixing and oxygen transfer without cell damage. However, its recentness demands further characterization. This study evaluated the residence time distribution (RTD) in Wave, allowing the characterization of mixing and flow and the comparison with ideal models and a Stirred tank reactor (STR) used for mammalian cell culture. RTD was determined using methylene blue with pulse input methodology, at three flow rates common in mammalian cell culture (3.3×10(-5)m(3)/h, 7.9×10(-5)m(3)/h, and 1.25×10(-4)m(3)/h) and one typical of microbial culture (5×10(-3)m(3)/h). Samples were taken periodically and the absorbance read at 660nm. It was observed that Wave behavior diverted from ideal models, but was similar to STR. Therefore, the deviations are not related to the particular Wave rocking mechanism, but could be associated with the inadequacy of these reactors to operate in continuous mode or to a possible inability of the theoretical models to properly describe the behavior of reactors designed for mammalian cell culture. Thus, the development of new theoretical models could better characterize the performance of these reactors. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Milk stimulates growth of prostate cancer cells in culture.

    Science.gov (United States)

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

    2011-11-01

    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.

  6. An In Vitro Nematic Model for Proliferating Cell Cultures

    CERN Document Server

    Pai, Sunil; Green, Morgaine; Cordeiro, Christine; Cabral, Elise; Chen, Bertha; Baer, Thomas

    2016-01-01

    Confluent populations of elongated cells give rise to ordered patterns seen in nematic phase liquid crystals. We correlate cell elongation and intercellular distance with intercellular alignment using an amorphous spin glass model. We compare in vitro time-lapse imaging with Monte Carlo simulation results by framing a novel hard ellipses model in terms of Boltzmann statistics. Furthermore, we find a statistically distinct alignment energy at quasi-steady state among fibroblasts, smooth muscle cells, and pluripotent cell populations when cultured in vitro. These findings have important implications in both non-invasive clinical screening of the stem cell differentiation process and in relating shape parameters to coupling in active crystal systems such as nematic cell monolayers.

  7. Effects of epiplakin-knockdown in cultured corneal epithelial cells.

    Science.gov (United States)

    Kokado, Masahide; Okada, Yuka; Miyamoto, Takeshi; Yamanaka, Osamu; Saika, Shizuya

    2016-05-20

    To investigate effects of knockdown of epiplakin gene expression on the homeostasis of cultured corneal epithelial cell line. We previously reported acceleration of corneal epithelial wound healing in an epiplakin-null mouse. Gene expression of epiplakin was knockdowned by employing siRNA transfection in SV40-immortalized human corneal epithelial cell line. Protein expression of E-cadherin, keratin 6 and vimentin was examined by western blotting. Cell migration and proliferation were examined by using scratch assay and Alamar blue assay, respectively. Scratch assay and Alamar blue assay showed migration and proliferation of the cells was accelerated by epiplakin knockdown. siRNA-knockdown of epiplakin suppressed protein expression of E-cadherin, keratin 6 and vimentin. Decreased expression of E-cadherin, keratin 6 and vimentin might be included in the mechanisms of cell migration acceleration in the absence of epiplakin. The mechanism of cell proliferation stimulation by epiplakin knockdown is to be investigated.

  8. Induced Pluripotent Stem (iPS) Cell Culture Methods and Induction of Differentiation into Endothelial Cells.

    Science.gov (United States)

    Chatterjee, Ishita; Li, Fei; Kohler, Erin E; Rehman, Jalees; Malik, Asrar B; Wary, Kishore K

    2016-01-01

    The study of stem cell behavior and differentiation in a developmental context is complex, time-consuming, and expensive, and for this reason, cell culture remains a method of choice for developmental and regenerative biology and mechanistic studies. Similar to ES cells, iPS cells have the ability to differentiate into endothelial cells (ECs), and the route for differentiation appears to mimic the developmental process that occurs during the formation of an embryo. Traditional EC induction methods from embryonic stem (ES) cells rely mostly on the formation of embryoid body (EB), which employs feeder or feeder-free conditions in the presence or absence of supporting cells. Similar to ES cells, iPS cells can be cultured in feeder layer or feeder-free conditions. Here, we describe the iPS cell culture methods and induction differentiation of these cells into ECs. We use anti-mouse Flk1 and anti-mouse VE-cadherin to isolate and characterize mouse ECs, because these antibodies are commercially available and their use has been described in the literature, including by our group. The ECs produced by this method have been used by our laboratory, and we have demonstrated their in vivo potential. We also discuss how iPS cells differ in their ability to differentiate into endothelial cells in culture.

  9. Long term maintenance of myeloid leukemic stem cells cultured with unrelated human mesenchymal stromal cells.

    Science.gov (United States)

    Ito, Sawa; Barrett, A John; Dutra, Amalia; Pak, Evgenia; Miner, Samantha; Keyvanfar, Keyvan; Hensel, Nancy F; Rezvani, Katayoun; Muranski, Pawel; Liu, Paul; Larochelle, Andre; Melenhorst, J Joseph

    2015-01-01

    Mesenchymal stromal cells (MSCs) support the growth and differentiation of normal hematopoietic stem cells (HSCs). Here we studied the ability of MSCs to support the growth and survival of leukemic stem cells (LSCs) in vitro. Primary leukemic blasts isolated from the peripheral blood of 8 patients with acute myeloid leukemia (AML) were co-cultured with equal numbers of irradiated MSCs derived from unrelated donor bone marrow, with or without cytokines for up to 6weeks. Four samples showed CD34(+)CD38(-) predominance, and four were predominantly CD34(+)CD38(+). CD34(+) CD38(-) predominant leukemia cells maintained the CD34(+) CD38(-) phenotype and were viable for 6weeks when co-cultured with MSCs compared to co-cultures with cytokines or medium only, which showed rapid differentiation and loss of the LSC phenotype. In contrast, CD34(+) CD38(+) predominant leukemic cells maintained the CD34(+)CD38(+) phenotype when co-cultured with MSCs alone, but no culture conditions supported survival beyond 4weeks. Cell cycle analysis showed that MSCs maintained a higher proportion of CD34(+) blasts in G0 than leukemic cells cultured with cytokines. AML blasts maintained in culture with MSCs for up to 6weeks engrafted NSG mice with the same efficiency as their non-cultured counterparts, and the original karyotype persisted after co-culture. Chemosensitivity and transwell assays suggest that MSCs provide pro-survival benefits to leukemic blasts through cell-cell contact. We conclude that MSCs support long-term maintenance of LSCs in vitro. This simple and inexpensive approach will facilitate basic investigation of LSCs and enable screening of novel therapeutic agents targeting LSCs. Published by Elsevier B.V.

  10. Responsiveness of fetal rat brain cells to glia maturation factor during neoplastic transformation in cell culture

    DEFF Research Database (Denmark)

    Haugen, A; Laerum, O D; Bock, E

    1981-01-01

    of gestation. The brains of the treated fetuses were transferred to cell culture and underwent neoplastic transformation with a characteristic sequence of phenotypic alterations which could be divided into five different stages. During the first 40 days after explantation (stage I & II) BE induced...... morphological differentiation of epitheloid neural cells into astrocytes. This occurred in carcinogen treated cells as well as in untreated control cultures. At the same time cells with astrocyte morphology showed accumulation of glial fibrillary acidic protein (GFA) as tested by indirect immunofluorescence...... with monospecific antibodies against GFA. Thereafter, in the EtNU pre-treated cultures an increased number of cells with astrocyte morphology was seen, and BE further increased the number of cells with long cytoplasmic processes. Control cells were GFA negative, while some few strongly, as well as many weakly...

  11. Simplified microenvironments and reduced cell culture size influence the cell differentiation outcome in cellular microarrays.

    Science.gov (United States)

    Rodríguez-Seguí, Santiago A; Ortuño, María José; Ventura, Francesc; Martínez, Elena; Samitier, Josep

    2013-01-01

    Cellular microarrays present a promising tool for multiplex evaluation of the signalling effect of substrate-immobilized factors on cellular differentiation. In this paper, we compare the early myoblast-to-osteoblast cell commitment steps in response to a growth factor stimulus using standard well plate differentiation assays or cellular microarrays. Our results show that restraints on the cell culture size, inherent to cellular microarrays, impair the differentiation outcome. Also, while cells growing on spots with immobilised BMP-2 are early biased towards the osteoblast fate, longer periods of cell culturing in the microarrays result in cell proliferation and blockage of osteoblast differentiation. The results presented here raise concerns about the efficiency of cell differentiation when the cell culture dimensions are reduced to a simplified microspot environment. Also, these results suggest that further efforts should be devoted to increasing the complexity of the microspots composition, aiming to replace signalling cues missing in this system.

  12. Myxoma virus induces apoptosis in cultured feline carcinoma cells.

    Science.gov (United States)

    MacNeill, A L; Moldenhauer, T; Doty, R; Mann, T

    2012-10-01

    There is growing interest in utilizing replicating oncolytic viruses as cancer therapeutics agents. The effectiveness of myxoma virus-induced oncolysis was evaluated in two feline cancer cell cultures. Although myxoma virus is a rabbit-specific pathogen, protein expression driven by myxoma virus and production of infectious viral particles were detected. Cell death occurred in primary feline cancer cells within 48 h of inoculation with myxoma virus. Future studies to determine if other feline neoplasms are susceptible to myxoma virus infection are warranted. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. Isolation and culture of pig epiblast stem cells.

    Science.gov (United States)

    Rodriguez, Aida; Contreras, David A; Alberio, Ramiro

    2013-01-01

    Regulation of early development can be directly interrogated in the embryo or can be studied in cultured cells isolated in the laboratory. New understanding of the developmental stages and the signalling requirements of the pig embryo have enabled the development of improved protocols for the derivation of pluripotent cells from early epiblasts. Here, we provide a detailed step-by-step description of the critical parameters required for isolation and establishment of these cells and how they can be used to study their developmental properties.

  14. PDMS/glass microfluidic cell culture system for cytotoxicity tests and cells passage

    DEFF Research Database (Denmark)

    Ziolkowska, K.; Jedrych, E.; Kwapiszewski, R.

    2010-01-01

    In this paper, hybrid (PDMS/glass) microfluidic cell culture system (MCCS) integrated with the concentration gradient generator (CGG) is presented. PDMS gas permeability enabled cells' respiration in the fabricated microdevices and excellent glass hydrophilicity allowed successful cells' seeding....... The series of cytotoxicity tests in the microdevice as well as in classic way using 96-well cell culture plates were performed to compare results obtained in micro- and macroscale. Fluorescein dibutyrate (FDB) and iodide propidine (PI) were used as viable and dead cells' markers, respectively. Fabricated...

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

    Science.gov (United States)

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

    2015-07-01

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

  16. Maintenance of mesenchymal stem cells culture due to the cells with reduced attachment rate

    Directory of Open Access Journals (Sweden)

    Shuvalova N. S.

    2013-01-01

    Full Text Available Aim. The classic detachment techniques lead to changes in cells properties. We offer a simple method of cultivating the population of cells that avoided an influence on the surface structures. Methods. Mesenchymal stem cells (MSC from human umbilical cord matrix were obtained and cultivated in standard conditions. While substituting the culture media by a fresh portion, the conditioned culture medium, where the cells were maintained for three days, was transferred to other culture flacks with addition of serum and growth factors. Results. In the flacks, one day after medium transfer, we observed attached cells with typical MSC morphology. The cultures originated from these cells had the same rate of surface markers expression and clonogenic potential as those replated by standard methods. Conclusions. MSC culture, derived by preserving the cells with reduced attachment ability, actually has the properties of «parent» passage. Using this method with accepted techniques of cells reseeding would allow maintaining the cells that avoided an impact on the cell surface proteins.

  17. Plant cell tissue culture: A potential source of chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Scott, C.D.; Dougall, D.K.

    1987-08-01

    Higher plants produce many industrially important products. Among these are drugs and medicinal chemicals, essential oils and flavors, vegetable oils and fats, fine and specialty chemicals, and even some commodity chemicals. Although, currently, whole-plant extraction is the primary means of harvesting these materials, the advent of plant cell tissue culture could be a much more effective method of producing many types of phytochemicals. The use of immobilized plant cells in an advanced bioreactor configuration with excretion of the product into the reactor medium may represent the most straightforward way of commercializing such techniques for lower-value chemicals. Important research and development opportunities in this area include screening for plant cultures for nonmedical, lower-value chemicals; understanding and controlling plant cell physiology and biochemistry; optimizing effective immobilization methods; developing more efficient bioreactor concepts; and perfecting product extraction and purification techniques. 62 refs., 2 figs.

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

    Science.gov (United States)

    Marquette, Michele L.; Sognier, Marguerite A.

    2013-01-01

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

  19. Benzaldehyde dehydrogenase from chitosan-treated Sorbus aucuparia cell cultures.

    Science.gov (United States)

    Gaid, Mariam M; Sircar, Debabrata; Beuerle, Till; Mitra, Adinpunya; Beerhues, Ludger

    2009-09-01

    Cell cultures of Sorbus aucuparia respond to the addition of chitosan with the accumulation of the biphenyl phytoalexin aucuparin. The carbon skeleton of this inducible defense compound is formed by biphenyl synthase (BIS) from benzoyl-CoA and three molecules of malonyl-CoA. The formation of benzoyl-CoA proceeds via benzaldehyde as an intermediate. Benzaldehyde dehydrogenase (BD), which converts benzaldehyde into benzoic acid, was detected in cell-free extracts from S. aucuparia cell cultures. BD and BIS were induced by chitosan treatment. The preferred substrate for BD was benzaldehyde (K(m)=49 microM). Cinnamaldehyde and various hydroxybenzaldehydes were relatively poor substrates. BD activity was strictly dependent on the presence of NAD(+) as a cofactor (K(m)=67 microM).

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

    Science.gov (United States)

    Noto, Daisuke; Sakuma, Hiroshi; Takahashi, Kazuya; Saika, Reiko; Saga, Ryoko; Yamada, Masahito; Yamamura, Takashi; Miyake, Sachiko

    2014-10-01

    Microglia are the resident immune cells in the central nervous system, originating from haematopoietic-derived myeloid cells. A microglial cell is a double-edged sword, which has both pro-inflammatory and anti-inflammatory functions. Although understanding the role of microglia in pathological conditions has become increasingly important, histopathology has been the only way to investigate microglia in human diseases. To enable the study of microglial cells in vitro, we here establish a culture system to induce microglia-like cells from haematopoietic cells by coculture with astrocytes. The characteristics of microglia-like cells were analysed by flow cytometry and functional assay. We show that triggering receptor expressing on myeloid cells-2-expressing microglia-like cells could be induced from lineage negative cells or monocytes by coculture with astrocytes. Microglia-like cells exhibited lower expression of CD45 and MHC class II than macrophages, a characteristic similar to brain microglia. When introduced into brain slice cultures, these microglia-like cells changed their morphology to a ramified shape on the first day of the culture. Moreover, we demonstrated that microglia-like cells could be induced from human monocytes by coculture with astrocytes. Finally, we showed that interleukin 34 was an important factor in the induction of microglia-like cells from haematopoietic cells in addition to cell-cell contact with astrocytes. Purified microglia-like cells were suitable for further culture and functional analyses. Development of in vitro induction system for microglia will further promote the study of human microglial cells under pathological conditions as well as aid in the screening of drugs to target microglial cells. © 2013 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of the British Neuropathological Society.

  1. Trophic Effects of Mesenchymal Stem Cells in Chondrocyte Co-Cultures are Independent of Culture Conditions and Cell Sources

    NARCIS (Netherlands)

    Wu, Ling; Prins, H.J.; Helder, M.; van Blitterswijk, Clemens; Karperien, Hermanus Bernardus Johannes

    2012-01-01

    Earlier, we have shown that the increased cartilage production in pellet co-cultures of chondrocytes and bone marrow-derived mesenchymal stem cells (BM-MSCs) is due to a trophic role of the MSC in stimulating chondrocyte proliferation and matrix production rather than MSCs actively undergoing

  2. Trophic effects of mesenchymal stem cells in chondrocyte co-cultures are independent of culture conditions and cell sources

    NARCIS (Netherlands)

    Wu, L.; Prins, H.J.; Helder, M.N.; van Blitterswijk, C.A.; Karperien, M.

    2012-01-01

    Earlier, we have shown that the increased cartilage production in pellet co-cultures of chondrocytes and bone marrow-derived mesenchymal stem cells (BM-MSCs) is due to a trophic role of the MSC in stimulating chondrocyte proliferation and matrix production rather than MSCs actively undergoing

  3. Activation of intervertebral disc cells by co-culture with notochordal cells, conditioned medium and hypoxia.

    Science.gov (United States)

    Gantenbein, Benjamin; Calandriello, Elena; Wuertz-Kozak, Karin; Benneker, Lorin M; Keel, Marius J B; Chan, Samantha C W

    2014-12-11

    Notochordal cells (NC) remain in the focus of research for regenerative therapy for the degenerated intervertebral disc (IVD) due to their progenitor status. Recent findings suggested their regenerative action on more mature disc cells, presumably by the secretion of specific factors, which has been described as notochordal cell conditioned medium (NCCM). The aim of this study was to determine NC culture conditions (2D/3D, fetal calf serum, oxygen level) that lead to significant IVD cell activation in an indirect co-culture system under normoxia and hypoxia (2% oxygen). Porcine NC was kept in 2D monolayer and in 3D alginate bead culture to identify a suitable culture system for these cells. To test stimulating effects of NC, co-cultures of NC and bovine derived coccygeal IVD cells were conducted in a 1:1 ratio with no direct cell contact between NC and bovine nucleus pulposus cell (NPC) or annulus fibrosus cells (AFC) in 3D alginate beads under normoxia and hypoxia (2%) for 7 and 14 days. As a positive control, NPC and AFC were stimulated with NC-derived conditioned medium (NCCM). Cell activity, glycosaminoglycan (GAG) content, DNA content and relative gene expression was measured. Mass spectrometry analysis of the NCCM was conducted. We provide evidence by flow cytometry that monolayer culture is not favorable for NC culture with respect to maintaining NC phenotype. In 3D alginate culture, NC activated NPC either in indirect co-culture or by addition of NCCM as indicated by the gene expression ratio of aggrecan/collagen type 2. This effect was strongest with 10% fetal calf serum and under hypoxia. Conversely, AFC seemed unresponsive to co-culture with pNC or to the NCCM. Further, the results showed that hypoxia led to decelerated metabolic activity, but did not lead to a significant change in the GAG/DNA ratio. Mass spectrometry identified connective tissue growth factor (CTGF, syn. CCN2) in the NCCM. Our results confirm the requirement to culture NC in 3D to best

  4. Imprint lithography provides topographical nanocues to guide cell growth in primary cortical cell culture

    NARCIS (Netherlands)

    Xie, Sijia; Lüttge, Regina

    2014-01-01

    In this paper, we describe a technology platform to study the effect of nanocues on the cell growth direction in primary cortical cell culture. Topographical cues to cells are provided using nanoscale features created by Jet and Flash Imprint Lithography, coated with polyethylenimine. We

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

    NARCIS (Netherlands)

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

    2001-01-01

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

  6. Cell culture tracking by multivariate analysis of raw LCMS data.

    Science.gov (United States)

    Michaud, François-Thomas; Havugimana, Pierre Claver; Duchesne, Carl; Sanschagrin, François; Bernier, Alice; Lévesque, Roger C; Garnier, Alain

    2012-06-01

    Liquid chromatography mass spectrometry (LCMS) is a powerful technique that could serve to rapidly characterize cell culture protein expression profile and be used as a process monitoring and control tool. However, this application is often hampered by both the sample proteome and the LCMS signal complexities as well as the variability of this signal. To alleviate this problem, culture samples are usually extensively fractionated and pretreated before being analyzed by top-end instruments. Such an approach precludes LCMS usage for routine on-line or at-line application. In this work, by applying multivariate analysis (MA) directly on raw LCMS signals, we were able to extract relevant information from cell culture samples that were simply lyzed. By using the recombinant adenovirus production process as a model, we were able to follow the accumulation of the three major proteins produced, identified their accumulation dynamics, and draw useful conclusions from these results. The combination of LCMS and MA provides a simple, rapid, and precise means to monitor cell culture.

  7. Cell culture density affects the proliferation activity of human adipose tissue stem cells.

    Science.gov (United States)

    Kim, Dae Seong; Lee, Myoung Woo; Ko, Young Jong; Chun, Yong Hoon; Kim, Hyung Joon; Sung, Ki Woong; Koo, Hong Hoe; Yoo, Keon Hee

    2016-01-01

    In this study, we investigated the effect of cell density on the proliferation activity of human mesenchymal stem cells (MSCs) derived from adipose tissue (AT-MSCs) over time in culture. Passage #4 (P4) and #12 (P12) AT-MSCs from two donors were plated at a density of 200 (culture condition 1, CC1) or 5000 (culture condition 2, CC2) cells cm(-2) . After 7 days of incubation, P4 and P12 AT-MSCs cultured in CC1 were thin and spindle-shaped, whereas those cultured in CC2 had extensive cell-to-cell contacts and an expanded cell volume. In addition, P4 and P12 AT-MSCs in CC1 divided more than three times, while those in CC2 divided less than once on average. Flow cytometric analysis using 5(6)-carboxyfluorescein diacetate N-succinimidyl ester dye showed that the fluorescence intensity of AT-MSCs was lower in CC1 than in CC2. Furthermore, expression of proliferation-associated genes, such as CDC45L, CDC20A and KIF20A, in P4 AT-MSCs was higher in CC1 than in CC2, and this difference was also observed in P12 AT-MSCs. These data demonstrated that cell culture density affects the proliferation activity of MSCs, suggesting that it is feasible to design a strategy to prepare suitable MSCs using specific culture conditions. Copyright © 2016 John Wiley & Sons, Ltd.

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

    Science.gov (United States)

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

    2007-11-07

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

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

    Science.gov (United States)

    Vendrame, Wagner A.; Pinares, Ania

    2013-06-01

    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

  10. Characteristics of Human Endometrial Stem Cells in Tissue and Isolated Cultured Cells: An Immunohistochemical Aspect.

    Science.gov (United States)

    Fayazi, Mehri; Salehnia, Mojdeh; Ziaei, Saeideh

    2016-01-01

    The aim of this study was to investigate the percentage of the stem cells population in human endometrial tissue sections and cultured cells at fourth passage. Human endometrial specimens were divided into two parts, one part for morphological studies and the other part for in vitro culture. Full thickness of human normal endometrial sections and cultured endometrial cells at fourth passage were analyzed via immunohistochemistry for CD146 and some stemness markers such as Oct4, Nanog, Sox2, and Klf4 and the expression of typical mesenchymal stem cell markers CD90, CD105. 11.88 ± 1.29% of human endometrial cells whitin tissue sections expressed CD146 marker vs. 28±2.3% of cultured cells, CD90 and CD105 were expressed by functionalis stroma (85±2.4 and 89±3.2%) than basalis stroma (16±1.4 and 17±1.9%), respectively (Pendometrial stromal cells in endometrial sections vs. 12±3.1% and 8±2.9% of cultured cells, respectively. They reside near the glands in the basal layer of endometrium. Sox2 and Klf4 were not commonly expressed in tissue samples and cultured cells. CD9 and EpCAM were expressed by epithelial cells of the endometrium, rather than by stroma or perivascular cells. The human endometrial stem cells and pluripotency markers may be localized more in basalis layer of endometrium. The immunostaining observations of endometrial cells at fourth passage were correlated with the immunohistochemistry data.

  11. Aging and senescence of skin cells in culture

    DEFF Research Database (Denmark)

    Rattan, Suresh

    2015-01-01

    aging in vitro are dermal fibroblasts, epidermal keratinocytes, and melanocytes. Serial subcultivation of normal diploid skin cells can be performed only a limited number of times, and the emerging senescent phenotype can be categorized into structural, physiological, biochemical, and molecular......Studying age-related changes in the physiology, biochemistry, and molecular biology of isolated skin cell populations in culture has greatly expanded the understanding of the fundamental aspects of skin aging. The three main cell types that have been studied extensively with respect to cellular...... phenotypes, which can be used as biomarkers of cellular aging in vitro. The rate and phenotype of aging are different in different cell types. There are both common features and specific features of aging of skin fibroblasts, keratinocytes, melanocytes, and other cell types. A progressive accumulation...

  12. A Place to Call Home: Bioengineering Pluripotential Stem Cell Cultures

    Directory of Open Access Journals (Sweden)

    Mark Weingarten

    2015-03-01

    Full Text Available Pluripotent stem cells (PSCs have the power to revolutionize the future of cell-based therapies and regenerative medicine. However, stem/progenitor cell use in the clinical arsenal has been hampered by discrepancies resulting from stem cell engineering and expansion, as well as in their (mass differentiation in culture. Moreover, the manner in which external conditions affect PSC and induced-pluripotent stem cell lineage establishment as well as maturation remains controversial. In this review, we examine novel methods of cell engineering and the role of reprogramming transcription factors in PSC development. In addition, we explore the effect of external environmental signals on PSC cultivation and differentiation by elucidating key components of the primordial stem cell microenvironment, the blastocyst. Furthermore, we assess the effects of hypoxic conditions on DNA editing, gene expression, and protein function in PSC self-renewal and growth. Finally, we speculate on the principal use of gap junction subunit expression as relevant biomarkers of PSC fate. Improving bioreactor design and pertinent cell biomarker classification could vastly enhance manufactured stem cell yield and quality, thereby increasing the potency and safety of therapeutic cells to be used in regenerative medicine.

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

    Science.gov (United States)

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

    2015-03-01

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

  14. Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

    Directory of Open Access Journals (Sweden)

    Magnusson Magnus K

    2010-07-01

    Full Text Available Abstract Background Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D co-culture assay. Methods Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. Results In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. Conclusion Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

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

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Fenxi, E-mail: fxzhang0824@gmail.com [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); Ren, Tongming [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); Lin, Juntang [Stem Cell Center, Xinxiang Medical University, Henan 453003, People' s Republic of China (China)

    2012-10-12

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

  16. Effects of Feeder Cell Types on Culture of Mouse Embryonic Stem Cell In Vitro.

    Science.gov (United States)

    Park, Yun-Gwi; Lee, Seung-Eun; Kim, Eun-Young; Hyun, Hyuk; Shin, Min-Young; Son, Yeo-Jin; Kim, Su-Young; Park, Se-Pill

    2015-09-01

    The suitable feeder cell layer is important for culture of embryonic stem (ES) cells. In this study, we investigated the effect of two kinds of the feeder cell, MEF cells and STO cells, layer to mouse ES (mES) cell culture for maintenance of stemness. We compare the colony formations, alkaline phosphatase (AP) activities, expression of pluripotency marker genes and proteins of D3 cell colonies cultured on MEF feeder cell layer (D3/MEF) or STO cell layers (D3/STO) compared to feeder free condition (D3/-) as a control group. Although there were no differences to colony formations and AP activities, interestingly, the transcripts level of pluripotency marker genes, Pou5f1 and Nanog were highly expressed in D3/MEF (79 and 93) than D3/STO (61and 77) or D3/- (65 and 81). Also, pluripotency marker proteins, NANOG and SOX-2, were more synthesized in D3/MEF (72.8±7.69 and 81.2±3.56) than D3/STO (32.0±4.30 and 56.0±4.90) or D3/- (55.0±4.64 and 62.0±6.20). These results suggest that MEF feeder cell layer is more suitable to mES cell culture.

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

    Science.gov (United States)

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

    2014-04-01

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

  18. Isolation and culture of protoplasts of Ma-phut (Garcinia dulcis derived from cell suspension culture

    Directory of Open Access Journals (Sweden)

    Sompong Te-chato

    2008-09-01

    Full Text Available Friable callus induced from young leaves of Ma-phut on Murashige and Skoog (MS medium containing 3% sucrose,1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D, 0.5 mg/l benzyladenine (BA and 500 mg/l polyvinylpyrrolidone (PVP, was cultured in liquid medium with the same components. Various ages of cell suspension at weekly intervals were then incubated in various kinds and concentrations of cell wall digestion enzymes combined with 1% macerozyme R-10 on a rotary shaker at 100 rpm under 1500 lux illumination at 26±4oC. Purified protoplasts were cultured at various densities in MS medium (adjusted osmoticum to 0.4 M by mannitol supplemented with 3% sucrose and two types of auxin, 2,4-D and NAA at four concentrations (1, 2, 3 and 4 mg/l together with 1 mg/l BA. The results revealed that a four-day old cell suspension culture incubated in 2% cellulase Onozuka R-10 (CR10 in combination with 1% macerozyme R-10 gave an optimum result in both yield and viability of protoplasts at 5.7x106/1 ml PCV and 80%, respectively. Embedding protoplasts at a density of 2.5x105/ml in 0.2% phytagel containing MS medium supplemented with 3 mg/l NAA and 1 mg/l BA promoted the most effective division of the protoplasts (20%. The first division of the protoplasts was obtained after 2 days of culture and further divisions to form micro- and macro-colonies could be observed after 7-10 days of culture. However, callusformation and plantlet regeneration was not obtained.

  19. Enrichment of cancer stem cell-like cells by culture in alginate gel beads.

    Science.gov (United States)

    Xu, Xiao-xi; Liu, Chang; Liu, Yang; Yang, Li; Li, Nan; Guo, Xin; Sun, Guang-wei; Ma, Xiao-jun

    2014-05-10

    Cancer stem cells (CSCs) are most likely the reason of cancer reoccurrence and metastasis. For further elucidation of the mechanism underlying the characteristics of CSCs, it is necessary to develop efficient culture systems to culture and expand CSCs. In this study, a three-dimensional (3D) culture system based on alginate gel (ALG) beads was reported to enrich CSCs. Two cell lines derived from different histologic origins were encapsulated in ALG beads respectively and the expansion of CSCs was investigated. Compared with two-dimensional (2D) culture, the proportion of cells with CSC-like phenotypes was significantly increased in ALG beads. Expression levels of CSC-related genes were greater in ALG beads than in 2D culture. The increase of CSC proportion after being cultured within ALG beads was further confirmed by enhanced tumorigenicity in vivo. Moreover, increased metastasis ability and higher anti-cancer drug resistance were also observed in 3D-cultured cells. Furthermore, we found that it was hypoxia, through the upregulation of hypoxia-inducible factors (HIFs) that occurred in ALG beads to induce the increasing of CSC proportion. Therefore, ALG bead was an efficient culture system for CSC enrichment, which might provide a useful platform for CSC research and promote the development of new anti-cancer therapies targeting CSCs. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Biofunctionalized Plants as Diverse Biomaterials for Human Cell Culture.

    Science.gov (United States)

    Fontana, Gianluca; Gershlak, Joshua; Adamski, Michal; Lee, Jae-Sung; Matsumoto, Shion; Le, Hau D; Binder, Bernard; Wirth, John; Gaudette, Glenn; Murphy, William L

    2017-04-01

    The commercial success of tissue engineering products requires efficacy, cost effectiveness, and the possibility of scaleup. Advances in tissue engineering require increased sophistication in the design of biomaterials, often challenging the current manufacturing techniques. Interestingly, several of the properties that are desirable for biomaterial design are embodied in the structure and function of plants. This study demonstrates that decellularized plant tissues can be used as adaptable scaffolds for culture of human cells. With simple biofunctionalization technique, it is possible to enable adhesion of human cells on a diverse set of plant tissues. The elevated hydrophilicity and excellent water transport abilities of plant tissues allow cell expansion over prolonged periods of culture. Moreover, cells are able to conform to the microstructure of the plant frameworks, resulting in cell alignment and pattern registration. In conclusion, the current study shows that it is feasible to use plant tissues as an alternative feedstock of scaffolds for mammalian cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Effects of pulsatile flow on cultured vascular endothelial cell morphology.

    Science.gov (United States)

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

    1991-05-01

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

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

    Science.gov (United States)

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

    2014-12-15

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

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

    Science.gov (United States)

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

    2017-09-01

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

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

    Science.gov (United States)

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

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

  5. Ultra-thin Polyethylene glycol Coatings for Stem Cell Culture

    Science.gov (United States)

    Schmitt, Samantha K.

    Human mesenchymal stem cells (hMSCs) are a widely accessible and a clinically relevant cell type that are having a transformative impact on regenerative medicine. However, current clinical expansion methods can lead to selective changes in hMSC phenotype resulting from relatively undefined cell culture surfaces. Chemically defined synthetic surfaces can aid in understanding stem cell behavior. In particular we have developed chemically defined ultra-thin coatings that are stable over timeframes relevant to differentiation of hMSCs (several weeks). The approach employs synthesis of a copolymer with distinct chemistry in solution before application to a substrate. This provides wide compositional flexibility and allows for characterization of the orthogonal crosslinking and peptide binding groups. Characterization is done in solution by proton NMR and after crosslinking by X-ray photoelectron spectroscopy (XPS). The solubility of the copolymer in ethanol and low temperature crosslinking, expands its applicability to plastic substrates, in addition to silicon, glass, and gold. Cell adhesive peptides, namely Arg-Gly-Asp (RGD) fragments, are coupled to coating via different chemistries resulting in the urethane, amide or the thioester polymer-peptide bonds. Development of azlactone-based chemistry allowed for coupling in water at low peptide concentrations and resulted in either an amide or thioester bonds, depending on reactants. Characterization of the peptide functionalized coating by XPS, infrared spectroscopy and cell culture assays, showed that the amide linkages can present peptides for multiple weeks, while shorter-term presentation of a few days is possible using the more labile thioester bond. Regardless, coatings promoted initial adhesion and spreading of hMSCs in a peptide density dependent manner. These coatings address the following challenges in chemically defined cell culture simultaneously: (i) substrate adaptability, (ii) scalability over large areas

  6. Expression of proliferating cell nuclear antigen in cultured middle ear epithelial cells of the guinea pig.

    Science.gov (United States)

    Takeno, S; Hamamura, N; Hirakawa, K; Yajin, K

    1996-01-01

    Primary cultures of middle ear epithelium from the guinea pig were successfully established on type I collagen coated dishes. To characterize cellular outgrowth, antibodies to the proliferating cell nuclear antigen were used as a marker for spreading cells in the S phase of the cell cycle. A number of migrating epithelial cells positively stained for proliferating cell nuclear antigen after 7 and 14 days in culture. Confocal laser scanning microscopy was used to evaluate the localization pattern of this antigen, and the fluorescence intensity was quantified in different areas of the migrating epithelial sheet after various times in culture. Two distinct areas proved to be major sites of proliferating cell nuclear antigen expression. One was at the edge of the tissue explants from which multilayered epithelial cells had begun to migrate. The other was along the margin of the outgrowth, where the cells often had elongated shapes and were aligned in rows. The cells in both areas were identified as nonciliated cells; ciliated cells in the outgrowth showed little staining. We hypothesized that the outgrowth cells in this experiment might be identical to the migrating cells usually observed in renewing epithelia after injury. This model may provide a simple and reproducible method of evaluating the regenerative ability of the middle ear epithelium.

  7. Encapsulation and culture of mammalian cells including corneal cells in alginate hydrogels.

    Science.gov (United States)

    Hunt, Nicola C; Grover, Liam M

    2013-01-01

    The potential of cell therapy for the regeneration of diseased and damaged tissues is now widely -recognized. As a consequence there is a demand for the development of novel systems that can deliver cells to a particular location, maintaining viability, and then degrade at a predictable rate to release the cells into the surrounding tissues. Hydrogels have attracted much attention in this area, as the hydrogel structure provides an environment that is akin to that of the extracellular matrix. One widely investigated hydrogel is alginate, which has been used for cell encapsulation for more than 30 years. Alginate gels have the potential to be used as 3D cell culture systems and as prosthetic materials, both are applied to regeneration of the cornea. Here, we describe an alginate-based process that has been used for encapsulation of mammalian cells including corneal cells, with high levels of viability, and which allows subsequent retrieval of cell cultures for further characterization.

  8. An introduction to plant cell culture: the future ahead.

    Science.gov (United States)

    Loyola-Vargas, Víctor M; Ochoa-Alejo, Neftalí

    2012-01-01

    Plant cell, tissue, and organ culture (PTC) techniques were developed and established as an experimental necessity for solving important fundamental questions in plant biology, but they currently represent very useful biotechnological tools for a series of important applications such as commercial micropropagation of different plant species, generation of disease-free plant materials, production of haploid and doublehaploid plants, induction of epigenetic or genetic variation for the isolation of variant plants, obtention of novel hybrid plants through the rescue of hybrid embryos or somatic cell fusion from intra- or intergeneric sources, conservation of valuable plant germplasm, and is the keystone for genetic engineering of plants to produce disease and pest resistant varieties, to engineer metabolic pathways with the aim of producing specific secondary metabolites or as an alternative for biopharming. Some other miscellaneous applications involve the utilization of in vitro cultures to test toxic compounds and the possibilities of removing them (bioremediation), interaction of root cultures with nematodes or mycorrhiza, or the use of shoot cultures to maintain plant viruses. With the increased worldwide demand for biofuels, it seems that PTC will certainly be fundamental for engineering different plants species in order to increase the diversity of biofuel options, lower the price marketing, and enhance the production efficiency. Several aspects and applications of PTC such as those mentioned above are the focus of this edition.

  9. Isolation and culture of Celosia cristata L cell suspension protoplasts

    Directory of Open Access Journals (Sweden)

    Retno Mastuti

    2003-06-01

    Full Text Available Developmental competence of Celosia cristata L. cell suspension-derived protoplasts was investigated. The protoplasts were isolatedfrom 3- to 9-d old cultures in enzyme solution containing 2% (w/v Cellulase YC and 0.5% (w/v Macerozyme R-10 which was dissolvedin washing solution (0.4 M mannitol and 10 mM CaCl2 at pH 5.6 for 3 hours. The highest number of viable protoplasts was releasedfrom 5-d old culture of a homogenous cell suspension. Subsequently, three kinds of protoplast culture media were simultaneously examinedwith four kinds of concentration of gelling agent. Culturing the protoplasts on KM8p medium solidified with 1.2% agarose significantlyenhanced plating efficiency as well as microcolony formation. Afterwards, the microcalli actively proliferated into friable watery calluswhen they were subcultured on MS medium supplemented with 0.3 mg/l 2,4-D and 1.0 mg/l kinetin. Although the plant regenerationfrom the protoplasts-derived calli has not yet been obtained, the reproducible developmental step from protoplasts to callus in thisstudy may facilitate the establishment of somatic hybridization using C. cristata as one parent.

  10. Genotoxic activity of caramel on Salmonella and cultured mammalian cells.

    Science.gov (United States)

    Yu, Y N; Chen, X R; Ding, C; Cai, Z N; Li, Q G

    1984-04-01

    The genetic activity of 2 commercial caramel preparations, manufactured either by heating the malt sugar solution directly (non-ammoniated caramel) or by heating it with ammonia (ammoniated caramel) was studied in the Salmonella mutagenicity test and UDS assay in cultured mammalian cells. The non-ammoniated caramel was found to be mutagenic to S. typhimurium TA100, while the ammoniated one was genetically active in all the tester strains used, namely TA100, TA97 and TA98. It was also demonstrated that non-ammoniated caramel was capable of inducing UDS in cultured human amnion FL cells, but for the ammoniated one, no such activity was observed. Furthermore, based on the results obtained in the DNA synthesis inhibition assay, it was suggested that the DNA synthesis inhibition seen in our experiments with the ammoniated caramel was probably not of DNA damage in origin. These data indicate that the mutagenic fractions formed during ammoniated and non-ammoniated caramelization were quite different.

  11. Gill cell culture systems as models for aquatic environmental monitoring.

    Science.gov (United States)

    Bury, Nic R; Schnell, Sabine; Hogstrand, Christer

    2014-03-01

    A vast number of chemicals require environmental safety assessments for market authorisation. To ensure acceptable water quality, effluents and natural waters are monitored for their potential harmful effects. Tests for market authorisation and environmental monitoring usually involve the use of large numbers of organisms and, for ethical, cost and logistic reasons, there is a drive to develop alternative methods that can predict toxicity to fish without the need to expose any animals. There is therefore a great interest in the potential to use cultured fish cells in chemical toxicity testing. This review summarises the advances made in the area and focuses in particular on a system of cultured fish gill cells grown into an epithelium that permits direct treatment with water samples.

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

    Science.gov (United States)

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

    2013-01-01

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

  13. Radiation-induced bystander effects in cultured human stem cells.

    Directory of Open Access Journals (Sweden)

    Mykyta V Sokolov

    2010-12-01

    Full Text Available The radiation-induced "bystander effect" (RIBE was shown to occur in a number of experimental systems both in vitro and in vivo as a result of exposure to ionizing radiation (IR. RIBE manifests itself by intercellular communication from irradiated cells to non-irradiated cells which may cause DNA damage and eventual death in these bystander cells. It is known that human stem cells (hSC are ultimately involved in numerous crucial biological processes such as embryologic development; maintenance of normal homeostasis; aging; and aging-related pathologies such as cancerogenesis and other diseases. However, very little is known about radiation-induced bystander effect in hSC. To mechanistically interrogate RIBE responses and to gain novel insights into RIBE specifically in hSC compartment, both medium transfer and cell co-culture bystander protocols were employed.Human bone-marrow mesenchymal stem cells (hMSC and embryonic stem cells (hESC were irradiated with doses 0.2 Gy, 2 Gy and 10 Gy of X-rays, allowed to recover either for 1 hr or 24 hr. Then conditioned medium was collected and transferred to non-irradiated hSC for time course studies. In addition, irradiated hMSC were labeled with a vital CMRA dye and co-cultured with non-irradiated bystander hMSC. The medium transfer data showed no evidence for RIBE either in hMSC and hESC by the criteria of induction of DNA damage and for apoptotic cell death compared to non-irradiated cells (p>0.05. A lack of robust RIBE was also demonstrated in hMSC co-cultured with irradiated cells (p>0.05.These data indicate that hSC might not be susceptible to damaging effects of RIBE signaling compared to differentiated adult human somatic cells as shown previously. This finding could have profound implications in a field of radiation biology/oncology, in evaluating radiation risk of IR exposures, and for the safety and efficacy of hSC regenerative-based therapies.

  14. Radiation-Induced Bystander Effects in Cultured Human Stem Cells

    Science.gov (United States)

    Sokolov, Mykyta V.; Neumann, Ronald D.

    2010-01-01

    Background The radiation-induced “bystander effect” (RIBE) was shown to occur in a number of experimental systems both in vitro and in vivo as a result of exposure to ionizing radiation (IR). RIBE manifests itself by intercellular communication from irradiated cells to non-irradiated cells which may cause DNA damage and eventual death in these bystander cells. It is known that human stem cells (hSC) are ultimately involved in numerous crucial biological processes such as embryologic development; maintenance of normal homeostasis; aging; and aging-related pathologies such as cancerogenesis and other diseases. However, very little is known about radiation-induced bystander effect in hSC. To mechanistically interrogate RIBE responses and to gain novel insights into RIBE specifically in hSC compartment, both medium transfer and cell co-culture bystander protocols were employed. Methodology/Principal Findings Human bone-marrow mesenchymal stem cells (hMSC) and embryonic stem cells (hESC) were irradiated with doses 0.2 Gy, 2 Gy and 10 Gy of X-rays, allowed to recover either for 1 hr or 24 hr. Then conditioned medium was collected and transferred to non-irradiated hSC for time course studies. In addition, irradiated hMSC were labeled with a vital CMRA dye and co-cultured with non-irradiated bystander hMSC. The medium transfer data showed no evidence for RIBE either in hMSC and hESC by the criteria of induction of DNA damage and for apoptotic cell death compared to non-irradiated cells (p>0.05). A lack of robust RIBE was also demonstrated in hMSC co-cultured with irradiated cells (p>0.05). Conclusions/Significance These data indicate that hSC might not be susceptible to damaging effects of RIBE signaling compared to differentiated adult human somatic cells as shown previously. This finding could have profound implications in a field of radiation biology/oncology, in evaluating radiation risk of IR exposures, and for the safety and efficacy of hSC regenerative

  15. Biotransformation of bavachinin by three fungal cell cultures.

    Science.gov (United States)

    Luo, Jianmei; Liang, Qikun; Shen, Yanbing; Chen, Xi; Yin, Zhinan; Wang, Min

    2014-02-01

    Biotransformation of bavachinin (1) was investigated using three fungal cell cultures of Aspergillus flavus ATCC 30899, Cunninghamella elegans CICC 40250 and Penicillium raistrickii ATCC 10490, respectively. Two major converted products were identified by LC/MS, (1)H NMR and (13)C NMR and X-ray diffraction. Two biocatalyst systems, A. flavus ATCC 30899 and C. elegans CICC 40250 cell cultures, showed a great capacity of hydroxylation and two hydroxyl groups were attached at C-2″ and C-3″ positions in the side chain of the bavachinin A-ring, resulting in the formation of the same compound with a name, (S)-6-((R)-2,3-dihydroxy-3-methylbutyl)-2-(4-hydroxyphenyl)-7-methoxychromen-4-one (2). On the other hand, P. raistrickii ATCC 10490 cell cultures possessed the ability to reduction at C-4 of the substrate C-ring, resulting in the production of (2S,4R)-2-(4-hydroxyphenyl)-7-methoxy-6-(3-methylbut-2-en-1-yl)chromen-4-ol (3). Furthermore, the in vitro anti-tumor activities of the above compounds were evaluated by MTT assay. Compared with the substrate (1), product 3 possessed stronger inhibition activity on the human breast cancer cell line (MCF-7) and slightly lower inhibition activities against Hep G2, HeLa, Hep-2 and A549 cells lines; while the hydroxyl product 2 possessed much lower inhibition activity on tumor cells lines, which might be related to the insertion of two hydroxyl groups. Compounds 2 and 3 were considered to be novel. It was also the first time to biotransform bavachinin (1) by these three fungi, which suggested the potential role of microbial enzymes to synthesize novel compounds from plant secondary metabolites. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  16. Assessment of Stem Cell Markers During Long-Term Culture of Mouse Embryonic Stem Cells

    OpenAIRE

    Berrill, A.; Tan, H L; Wuang, S.C.; Fong, W.J.; Choo, Andre B. H.; Oh, Steve K. W.

    2004-01-01

    Embryonic stem (ES) cells have been in the fore front of scientific literature lately as having the potential for regeneration of many tissue types. Two important issues that need to be addressed are the culture conditions for maintaining ES cells and the accuracy of ES cell markers in monitoring the undifferentiated state. Leukaemia inhibitory factor (LIF) is routinely used to sustain mouse ES cells (mES) in a pluripotent fashion. In this paper, we assessed three markers during long-term mai...

  17. Pro-B cells propagated in stromal cell-free cultures reconstitute functional B-cell compartments in immunodeficient mice.

    Science.gov (United States)

    von Muenchow, Lilly; Tsapogas, Panagiotis; Albertí-Servera, Llucia; Capoferri, Giuseppina; Doelz, Marianne; Rolink, Hannie; Bosco, Nabil; Ceredig, Rhodri; Rolink, Antonius G

    2017-02-01

    Up to now long-term in vitro growth of pro-B cells was thought to require stromal cells. However, here we show that fetal liver (FL) and bone marrow (BM) derived pro-B cells can be propagated long-term in stromal cell-free cultures supplemented with IL-7, stem cell factor and FLT3 ligand. Within a week, most cells expressed surface CD19, CD79A, λ5, and VpreB antigens and had rearranged immunoglobulin D-J heavy chain genes. Both FL and BM pro-B cells reconstituted the B-cell compartments of immuno-incompetent Rag2-deficient mice, with FL pro-B cells generating follicular, marginal zone (MZB) and B1a B cells, and BM pro-B cells giving rise mainly to MZB cells. Reconstituted Rag2-deficient mice generated significant levels of IgM and IgG antibodies to a type II T-independent antigen; mice reconstituted with FL pro-B cells generated surprisingly high IgG1 titers. Finally, we show for the first time that mice reconstituted with mixtures of pro-B and pro-T cells propagated in stromal cell-free in vitro cultures mounted a T-cell-dependent antibody response. This novel stromal cell-free culture system facilitates our understanding of B-cell development and might be applied clinically. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. A Three-Dimensional Cell Culture Model To Study Enterovirus Infection of Polarized Intestinal Epithelial Cells.

    Science.gov (United States)

    Drummond, Coyne G; Nickerson, Cheryl A; Coyne, Carolyn B

    2016-01-01

    Despite serving as the primary entry portal for coxsackievirus B (CVB), little is known about CVB infection of the intestinal epithelium, owing at least in part to the lack of suitable in vivo models and the inability of cultured cells to recapitulate the complexity and structure associated with the gastrointestinal (GI) tract. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of Caco-2 cells to model CVB infection of the gastrointestinal epithelium. We show that Caco-2 cells grown in 3-D using the rotating wall vessel (RWV) bioreactor recapitulate many of the properties of the intestinal epithelium, including the formation of well-developed tight junctions, apical-basolateral polarity, brush borders, and multicellular complexity. In addition, transcriptome analyses using transcriptome sequencing (RNA-Seq) revealed the induction of a number of genes associated with intestinal epithelial differentiation and/or intestinal processes in vivo when Caco-2 cells were cultured in 3-D. Applying this model to CVB infection, we found that although the levels of intracellular virus production were similar in two-dimensional (2-D) and 3-D Caco-2 cell cultures, the release of infectious CVB was enhanced in 3-D cultures at early stages of infection. Unlike CVB, the replication of poliovirus (PV) was significantly reduced in 3-D Caco-2 cell cultures. Collectively, our studies show that Caco-2 cells grown in 3-D using the RWV bioreactor provide a cell culture model that structurally and transcriptionally represents key aspects of cells in the human GI tract and can thus be used to expand our understanding of enterovirus-host interactions in intestinal epithelial cells. IMPORTANCE Coxsackievirus B (CVB), a member of the enterovirus family of RNA viruses, is associated with meningitis, pericarditis, diabetes, dilated cardiomyopathy, and myocarditis, among other pathologies. CVB is transmitted via the fecal-oral route and encounters the

  19. Lactate Detection in Tumor Cell Cultures Using Organic Transistor Circuits.

    Science.gov (United States)

    Braendlein, Marcel; Pappa, Anna-Maria; Ferro, Marc; Lopresti, Alexia; Acquaviva, Claire; Mamessier, Emilie; Malliaras, George G; Owens, Róisín M

    2017-04-01

    A biosensing platform based on an organic transistor circuit for metabolite detection in highly complex biological media is introduced. The sensor circuit provides inherent background subtraction allowing for highly specific, sensitive lactate detection in tumor cell cultures. The proposed sensing platform paves the way toward rapid, label-free, and cost-effective clinically relevant in vitro diagnostic tools. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Production of volatile Sulphides in Allium Porrum cell cultures

    OpenAIRE

    Asghari Gh.R; Lockwood GB; Houshfar Gh.A "

    2002-01-01

    Production of volatile sulphides in cell cultures of Allium porrum is described. Allium porrum calluses were initiated from whole seedlings. The high growth rate of Allium porrum callus was achived in Murashige and Skoog media containing only 1 ppm 2, 4-Dichlorophenoxy acetic acid. The routine method of solvent extraction of volatile sulphides was used for Allium porrum and the concentrated extract was subjected to capillary GC and GC-MS. Dipropyl disulphide and 4-methyl thiazolethanol were i...

  1. Production of volatile Sulphides in Allium Porrum cell cultures

    Directory of Open Access Journals (Sweden)

    "Asghari Gh.R

    2002-09-01

    Full Text Available Production of volatile sulphides in cell cultures of Allium porrum is described. Allium porrum calluses were initiated from whole seedlings. The high growth rate of Allium porrum callus was achived in Murashige and Skoog media containing only 1 ppm 2, 4-Dichlorophenoxy acetic acid. The routine method of solvent extraction of volatile sulphides was used for Allium porrum and the concentrated extract was subjected to capillary GC and GC-MS. Dipropyl disulphide and 4-methyl thiazolethanol were identified in A. porrum aggregated suspension cells.

  2. Propagation and isolation of ranaviruses in cell culture

    DEFF Research Database (Denmark)

    Ariel, Ellen; Nicolajsen, Nicole; Christophersen, Maj-Britt

    2009-01-01

    The optimal in vitro propagation procedure for a panel of ranavirus isolates and the best method for isolation of Epizootic haematopoietic necrosis virus (EHNV) from organ material in cell-culture were investigated. The panel of ranavirus isolates included: Frog virus 3 (FV3), Bohle iridovirus (BIV...... consistently produced lower titers than the other cell lines at all temperatures. The optimal temperature for propagating the isolates collectively to high titers in vivo was 24 °C. Additionally, three established methods for re-isolation of virus from EHNV-infected organ material were compared. Challenged...

  3. Mesenchymal stem cells cultured on magnetic nanowire substrates

    KAUST Repository

    Perez, Jose E.

    2016-12-28

    Stem cells have been shown to respond to extracellular mechanical stimuli by regulating their fate through the activation of specific signaling pathways. In this work, an array of iron nanowires (NWs) aligned perpendicularly to the surface was fabricated by pulsed electrodepositon in porous alumina templates followed by a partial removal of the alumina to reveal 2-3 μm of the NWs. This resulted in alumina substrates with densely arranged NWs of 33 nm in diameter separated by 100 nm. The substrates were characterized by scanning electron microscopy (SEM) energy dispersive x-ray analysis and vibrating sample magnetometer. The NW array was then used as a platform for the culture of human mesenchymal stem cells (hMSCs). The cells were stained for the cell nucleus and actin filaments, as well as immuno-stained for the focal adhesion protein vinculin, and then observed by fluorescence microscopy in order to characterize their spreading behavior. Calcein AM/ethidium homodimer-1 staining allowed the determination of cell viability. The interface between the cells and the NWs was studied using SEM. Results showed that hMSCs underwent a re-organization of actin filaments that translated into a change from an elongated to a spherical cell shape. Actin filaments and vinculin accumulated in bundles, suggesting the attachment and formation of focal adhesion points of the cells on the NWs. Though the overall number of cells attached on the NWs was lower compared to the control, the attached cells maintained a high viability (>90%) for up to 6 d. Analysis of the interface between the NWs and the cells confirmed the re-organization of F-actin and revealed the adhesion points of the cells on the NWs. Additionally, a net of filopodia surrounded each cell, suggesting the probing of the array to find additional adhesion points. The cells maintained their round shape for up to 6 d of culture. Overall, the NW array is a promising nanostructured platform for studying and influencing h

  4. Mouse ES cell culture system as a model of development.

    Science.gov (United States)

    Niwa, Hitoshi

    2010-04-01

    Mouse embryonic stem (mES) cells are pluripotent stem cells derived from pre-implantation embryos. They are regarded as an essential tool for studying mouse development, as they provide a means for generating knock-out mouse lines. This, however, is not the sole utility of the mES cell system. They undergo differentiation in culture, mimicking the morphological differentiation of peri-implantation embryos from epiblast to egg-cylinder stage. Moreover, they retain the capacity to respond to triggers of differentiation toward trophectoderm and primitive endoderm by forced activation. For these reasons, mES cells can be regarded as a useful tool for analyzing molecular mechanisms underlying early mouse development.

  5. Replication of hepatitis C virus in cell culture

    Directory of Open Access Journals (Sweden)

    Fedorchenko D. B.

    2009-02-01

    Full Text Available The technique for replicating hepatitis C virus (HCV in cell culture has been modified and the susceptibility of the cells of various origin to HCV upon their infection with HCV-containing sera has been compared. The viral load on the fifth day post-infection has been assessed by reverse transcriptase polymerase chain reaction technique. The highest infection and replication efficacy have been found in cells of rat Gasser’s ganglion neurinoma. The peculiar features of the mitotic index and the anomalous forms of the mitosis have been studied in HCV-infected cells. The data presented may be used as a basis for the experimental model of HCV infection in vitro suitable for studying the effects of antiviral drugs on the infection caused by the cytopathogenic variant of HCV.

  6. In Vitro Cell Culture Infectivity Assay for Human Noroviruses

    Energy Technology Data Exchange (ETDEWEB)

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

    2007-01-30

    Human noroviruses (NoV) cause severe, self-limiting gastroenteritis that typically lasts 24 - 48 hours. The true nature of NoV pathogenesis remains unknown due to the lack of suitable tissue culture or animal models. Here we show, for the first time, that NoV can infect and replicate in an organoid, three-dimensional (3-D) model of human small intestinal epithelium (INT-407). Cellular differentiation for this model was achieved by growing the cells in 3-D on porous collagen I-coated microcarrier beads under conditions of physiological fluid shear in rotating wall vessel bioreactors. Microscopy, PCR, and fluorescent in-situ hybridization were employed to provide evidence of NoV infection. CPE and norovirus RNA was detected at each of the five cell passages for both genogroup I and II viruses. Our results demonstrate that the highly differentiated 3-D cell culture model can support the natural growth of human noroviruses, whereas previous attempts using differentiated monolayer cultures failed.

  7. Rotary orbital suspension culture of embryonic stem cell-derived neural stem/progenitor cells: impact of hydrodynamic culture on aggregate yield, morphology and cell phenotype.

    Science.gov (United States)

    Laundos, Tiago L; Silva, Joana; Assunção, Marisa; Quelhas, Pedro; Monteiro, Cátia; Oliveira, Carla; Oliveira, Maria J; Pêgo, Ana P; Amaral, Isabel F

    2017-08-01

    Embryonic stem (ES)-derived neural stem/progenitor cells (ES-NSPCs) constitute a promising cell source for application in cell therapies for the treatment of central nervous system disorders. In this study, a rotary orbital hydrodynamic culture system was applied to single-cell suspensions of ES-NSPCs, to obtain homogeneously-sized ES-NSPC cellular aggregates (neurospheres). Hydrodynamic culture allowed the formation of ES-NSPC neurospheres with a narrower size distribution than statically cultured neurospheres, increasing orbital speeds leading to smaller-sized neurospheres and higher neurosphere yield. Neurospheres formed under hydrodynamic conditions (72 h at 55 rpm) showed higher cell compaction and comparable percentages of viable, dead, apoptotic and proliferative cells. Further characterization of cellular aggregates provided new insights into the effect of hydrodynamic shear on ES-NSPC behaviour. Rotary neurospheres exhibited reduced protein levels of N-cadherin and β-catenin, and higher deposition of laminin (without impacting fibronectin deposition), matrix metalloproteinase-2 (MMP-2) activity and percentage of neuronal cells. In line with the increased MMP-2 activity levels found, hydrodynamically-cultured neurospheres showed higher outward migration on laminin. Moreover, when cultured in a 3D fibrin hydrogel, rotary neurospheres generated an increased percentage of neuronal cells. In conclusion, the application of a constant orbital speed to single-cell suspensions of ES-NSPCs, besides allowing the formation of homogeneously-sized neurospheres, promoted ES-NSPC differentiation and outward migration, possibly by influencing the expression of cell-cell adhesion molecules and the secretion of proteases/extracellular matrix proteins. These findings are important when establishing the culture conditions needed to obtain uniformly-sized ES-NSPC aggregates, either for use in regenerative therapies or in in vitro platforms for biomaterial development or

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

    Science.gov (United States)

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

  9. Light transfer in agar immobilized microalgae cell cultures

    Science.gov (United States)

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

    2017-09-01

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

  10. Transport Mechanism of Nicotine in Primary Cultured Alveolar Epithelial Cells.

    Science.gov (United States)

    Takano, Mikihisa; Nagahiro, Machi; Yumoto, Ryoko

    2016-02-01

    Nicotine is absorbed from the lungs into the systemic circulation during cigarette smoking. However, there is little information concerning the transport mechanism of nicotine in alveolar epithelial cells. In this study, we characterized the uptake of nicotine in rat primary cultured type II (TII) and transdifferentiated type I-like (TIL) epithelial cells. In both TIL and TII cells, [(3)H]nicotine uptake was time and temperature-dependent, and showed saturation kinetics. [(3)H]Nicotine uptake in these cells was not affected by Na(+), but was sensitive to extracellular and intracellular pH, suggesting the involvement of a nicotine/proton antiport system. The uptake of [(3)H]nicotine in these cells was potently inhibited by organic cations such as clonidine, diphenhydramine, and pyrilamine, but was not affected by substrates and/or inhibitors of known organic cation transporters such as carnitine, 1-methyl-4-phenylpyridinium, and tetraethylammonium. In addition, the uptake of [(3)H]nicotine in TIL cells was stimulated by preloading the cells with unlabeled nicotine, pyrilamine, and diphenhydramine, but not with tetraethylammonium. These results suggest that a novel proton-coupled antiporter is involved in the uptake of nicotine in alveolar epithelial cells and its absorption from the lungs into the systemic circulation. Copyright © 2016. Published by Elsevier Inc.

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

    Science.gov (United States)

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

    2017-03-01

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

  12. In vitro co-culture of epithelial cells and smooth muscle cells on aligned nanofibrous scaffolds.

    Science.gov (United States)

    Kuppan, Purushothaman; Sethuraman, Swaminathan; Krishnan, Uma Maheswari

    2017-12-01

    Esophagus is a complex, hollow organ consisting of epithelial cells in the inner mucosal layer and smooth muscle cells in the outer muscle layer. In the present study, we have evaluated the in vitro co-culture of epithelial cells and smooth muscle cells on the aligned nanofibrous scaffold made of PHBV, PHBV-gelatin, PCL and PCL-gelatin developed through electrospinning using rotating drum collector. Epithelial cells were labeled with cell tracker green while the smooth muscle cells were labeled with cell tracker red. Labeled cells were seeded on the aligned nanofibers matrices and tracked using laser scanning confocal microscopy. The results demonstrate that both epithelial and smooth muscle cells attach, extend, and proliferate over these nanofibrous matrices. Confocal z-sectioning shows that epithelial and smooth muscle cells tend to separate into two distinct layers on a single nanofiber system mimicking the in vivo anatomy. Cell viability assay showed that both types of cells are viable and also interact with each other. The functional gene expression of respective cell types demonstrates that both epithelial and smooth muscle cells are phenotypically as well as functionally active when they were co-cultured. Thus the study highlighted that aligned nanofibrous scaffolds could be potential alternative graft for esophageal tissue regeneration. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Smooth muscle myosin regulation by serum and cell density in cultured rat lung connective tissue cells.

    Science.gov (United States)

    Babij, P; Zhao, J; White, S; Woodcock-Mitchell, J; Mitchell, J; Absher, M; Baldor, L; Periasamy, M; Low, R B

    1993-08-01

    RNA and protein analyses were used to detect expression of SM1 and SM2 smooth muscle myosin heavy chain (MHC) in cultured adult rat lung connective tissue cells (RL-90). Smooth muscle MHC mRNA expression in confluent cells grown in 10% serum was approximately 50% of the level in adult stomach. Similar results were obtained in cells cultured at low density (25% confluency) in 1% serum. However, in low-density cultures transferred to 10% serum for 24 h, the level of MHC mRNA decreased to approximately 20% of that in adult stomach. Smooth muscle alpha-actin showed a pattern of expression similar to that for smooth muscle MHC. Expression of nonmuscle MHC-A mRNA was higher in all culture conditions compared to stomach. MHC-A mRNA expression was less in low-density cultures in low serum and increased when low-density cultures were transferred to 10% serum for 24 h. MHC-B mRNA expression was less in low- vs. high-density cultures. In contrast to MHC-A, however, MHC-B mRNA expression in low-density cultures was higher in low serum. Immunofluorescence and immunoblotting with SM1-specific antibody demonstrated the presence of the SM1 protein isoform as well as reactivity to a protein band migrating slightly faster than SM2. These results demonstrate that cultured rat lung connective tissue cells express smooth muscle MHC and that expression is modulated by culture conditions.

  14. Culturing of Mouse Mesenchymal Stem Cells on Poly-3-Hydroxybutyrate Scaffolds.

    Science.gov (United States)

    Andreeva, N V; Bonartsev, A P; Zharkova, I I; Makhina, T K; Myshkina, V L; Kharitonova, E P; Voinova, V V; Bonartseva, G A; Shaitan, K V; Belyavskii, A V

    2015-08-01

    We studied the possibility of long-term culturing of mouse mesenchymal stem cells on a porous scaffold made of biocompatible polymer poly-3-hydroxybutyrate. The cells remained viable for at least 2 months and passed more than 65 population doublings in culture. Culturing on the scaffold did not change surface phenotype of cells. 3D poly-3-hydroxybutyrate scaffolds are appropriate substrate for long-term culturing of mesenchymal stem cells.

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

    Directory of Open Access Journals (Sweden)

    Islam M. Saadeldin

    2017-01-01

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

  16. Thymic epithelial cells. I. Expression of strong suppressive (veto) activity in mouse thymic epithelial cell cultures

    DEFF Research Database (Denmark)

    Claesson, Mogens Helweg; Ropke, C

    1990-01-01

    We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level. It is conclu....... It is concluded that the epithelial stromal cells of the thymus, by acting as veto cells, may be responsible for the negative intrathymic selection of self-reactive thymocytes leading to elimination of the vast majority of immature thymic lymphocytes....

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

    Science.gov (United States)

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

    1999-01-01

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

  18. Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure.

    Science.gov (United States)

    Akahane, M; Shimizu, T; Kira, T; Onishi, T; Uchihara, Y; Imamura, T; Tanaka, Y

    2016-11-01

    To assess the structure and extracellular matrix molecule expression of osteogenic cell sheets created via culture in medium with both dexamethasone (Dex) and ascorbic acid phosphate (AscP) compared either Dex or AscP alone. Osteogenic cell sheets were prepared by culturing rat bone marrow stromal cells in a minimal essential medium (MEM), MEM with AscP, MEM with Dex, and MEM with Dex and AscP (Dex/AscP). The cell number and messenger (m)RNA expression were assessed in vitro, and the appearance of the cell sheets was observed after mechanical retrieval using a scraper. β-tricalcium phosphate (β-TCP) was then wrapped with the cell sheets from the four different groups and subcutaneously implanted into rats. After mechanical retrieval, the osteogenic cell sheets from the MEM, MEM with AscP, and MEM with Dex groups appeared to be fragmented or incomplete structures. The cell sheets cultured with Dex/AscP remained intact after mechanical retrieval, without any identifiable tears. Culture with Dex/AscP increased the mRNA and protein expression of extracellular matrix proteins and cell number compared with those of the other three groups. More bridging bone formation was observed after transplantation of the β-TCP scaffold wrapped with cell sheets cultured with Dex/AscP, than in the other groups. These results suggest that culture with Dex/AscP improves the mechanical integrity of the osteogenic cell sheets, allowing retrieval of the confluent cells in a single cell sheet structure. This method may be beneficial when applied in cases of difficult tissue reconstruction, such as nonunion, bone defects, and osteonecrosis.Cite this article: M. Akahane, T. Shimizu, T. Kira, T. Onishi, Y. Uchihara, T. Imamura, Y. Tanaka. Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure. Bone Joint Res 2016;5:569-576. DOI: 10.1302/2046-3758.511.BJR-2016-0013.R1. © 2016 Akahane et al.

  19. Is cell culture a risky business? Risk analysis based on scientist survey data.

    Science.gov (United States)

    Shannon, Mark; Capes-Davis, Amanda; Eggington, Elaine; Georghiou, Ronnie; Huschtscha, Lily I; Moy, Elsa; Power, Melinda; Reddel, Roger R; Arthur, Jonathan W

    2016-02-01

    Cell culture is a technique that requires vigilance from the researcher. Common cell culture problems, including contamination with microorganisms or cells from other cultures, can place the reliability and reproducibility of cell culture work at risk. Here we use survey data, contributed by research scientists based in Australia and New Zealand, to assess common cell culture risks and how these risks are managed in practice. Respondents show that sharing of cell lines between laboratories continues to be widespread. Arrangements for mycoplasma and authentication testing are increasingly in place, although scientists are often uncertain how to perform authentication testing. Additional risks are identified for preparation of frozen stocks, storage and shipping. © 2015 UICC.

  20. Mutation Analysis in Cultured Cells of Transgenic Rodents

    Directory of Open Access Journals (Sweden)

    Ahmad Besaratinia

    2018-01-01

    Full Text Available To comply with guiding principles for the ethical use of animals for experimental research, the field of mutation research has witnessed a shift of interest from large-scale in vivo animal experiments to small-sized in vitro studies. Mutation assays in cultured cells of transgenic rodents constitute, in many ways, viable alternatives to in vivo mutagenicity experiments in the corresponding animals. A variety of transgenic rodent cell culture models and mutation detection systems have been developed for mutagenicity testing of carcinogens. Of these, transgenic Big Blue® (Stratagene Corp., La Jolla, CA, USA, acquired by Agilent Technologies Inc., Santa Clara, CA, USA, BioReliance/Sigma-Aldrich Corp., Darmstadt, Germany mouse embryonic fibroblasts and the λ Select cII Mutation Detection System have been used by many research groups to investigate the mutagenic effects of a wide range of chemical and/or physical carcinogens. Here, we review techniques and principles involved in preparation and culturing of Big Blue® mouse embryonic fibroblasts, treatment in vitro with chemical/physical agent(s of interest, determination of the cII mutant frequency by the λ Select cII assay and establishment of the mutation spectrum by DNA sequencing. We describe various approaches for data analysis and interpretation of the results. Furthermore, we highlight representative studies in which the Big Blue® mouse cell culture model and the λ Select cII assay have been used for mutagenicity testing of diverse carcinogens. We delineate the advantages of this approach and discuss its limitations, while underscoring auxiliary methods, where applicable.

  1. 21 CFR 864.2220 - Synthetic cell and tissue culture media and components.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Synthetic cell and tissue culture media and... Products § 864.2220 Synthetic cell and tissue culture media and components. (a) Identification. Synthetic cell and tissue culture media and components are substances that are composed entirely of defined...

  2. Digital microfluidics for automated hanging drop cell spheroid culture.

    Science.gov (United States)

    Aijian, Andrew P; Garrell, Robin L

    2015-06-01

    Cell spheroids are multicellular aggregates, grown in vitro, that mimic the three-dimensional morphology of physiological tissues. Although there are numerous benefits to using spheroids in cell-based assays, the adoption of spheroids in routine biomedical research has been limited, in part, by the tedious workflow associated with spheroid formation and analysis. Here we describe a digital microfluidic platform that has been developed to automate liquid-handling protocols for the formation, maintenance, and analysis of multicellular spheroids in hanging drop culture. We show that droplets of liquid can be added to and extracted from through-holes, or "wells," and fabricated in the bottom plate of a digital microfluidic device, enabling the formation and assaying of hanging drops. Using this digital microfluidic platform, spheroids of mouse mesenchymal stem cells were formed and maintained in situ for 72 h, exhibiting good viability (>90%) and size uniformity (% coefficient of variation microfluidic platform provides a viable tool for automating cell spheroid culture and analysis. © 2014 Society for Laboratory Automation and Screening.

  3. Effect of antioxidants on PDT treatment of cultured tumor cells

    Science.gov (United States)

    Melnikova, Vladislava; Bezdetnaya, Lina N.; Belitchenko, Irina; Potapenko, Alexander Y.; Merlin, Jean-Louis; Guillemin, Francois H.

    1998-05-01

    Lipid peroxidation (LP) is involved in cell damage induced by photodynamic treatment (PDT) sensitized by some lipophylic porphyrins. We investigated an effect of lipophylic antioxidant (alpha) -tocopherol and its water-soluble analog, trolox, on meta-tetra(hydroxyphenyl)chlorin (mTHPC) sensitized PDT (413 nm) of cultured human colon adenocarcinoma cells (HT29). Cell survival was measured by the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide conversion to farmazan (MTT assay). Both antioxidants in concentrations lower than 0.1 mM did not affect photokilling of HT29 cells. These data might suggest that LP is not of crucial importance in cell damage photosensitized by mTHPC. One mM (alpha) -tocopherol or trolox decreased cell survival by ca. 15 and 13% respectively. Both antioxidants increased PDT- induced damage of HT29. Potentiation was evident as the decrease in the initial shoulder part of fluence dependence curve. We propose that antioxidants at height, pro-oxidant concentrations can potentiate PDT induced killing of tumor cells.

  4. A three-dimensional approach to in vitro culture of immune-related cells

    DEFF Research Database (Denmark)

    Hartmann, Sofie Bruun

    in culture and to measure cell activity such as IGRA, as described above. The traditional way of culturing cells are done using polystyrene (PS) plastic ware in flask-, Petri dish- or micro titer plate format. However, these artificial two dimensional (2D) surfaces on which the cells grow, has shown...... on the differentiation of porcine monocytes. Monocytes are immune cells of high plasticity, and thus we speculated that they might be sensitive to culture conditions. Indeed, monocytes differentiated into monocyte-derived DC (moDCs) when cultured conventionally (2D PS) in the presence of GM-CSF and IL-4, but adopted...... a macrophage-like gene expression profile when cultured on PDMS. Further it was found that 3D culturing resulted in increased activation of the monocyte-derived cells. The work in this thesis covers several aspects within primary cell culture, but central to the work is the investigation of 3D cell culture...

  5. Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages.

    Directory of Open Access Journals (Sweden)

    Christiane E Wobus

    2004-12-01

    Full Text Available Noroviruses are understudied because these important enteric pathogens have not been cultured to date. We found that the norovirus murine norovirus 1 (MNV-1 infects macrophage-like cells in vivo and replicates in cultured primary dendritic cells and macrophages. MNV-1 growth was inhibited by the interferon-alphabeta receptor and STAT-1, and was associated with extensive rearrangements of intracellular membranes. An amino acid substitution in the capsid protein of serially passaged MNV-1 was associated with virulence attenuation in vivo. This is the first report of replication of a norovirus in cell culture. The capacity of MNV-1 to replicate in a STAT-1-regulated fashion and the unexpected tropism of a norovirus for cells of the hematopoietic lineage provide important insights into norovirus biology.

  6. Glycosylation-mediated phenylpropanoid partitioning in Populus tremuloides cell cultures

    Directory of Open Access Journals (Sweden)

    Babst Benjamin A

    2009-12-01

    Full Text Available Abstract Background Phenylpropanoid-derived phenolic glycosides (PGs and condensed tannins (CTs comprise large, multi-purpose non-structural carbon sinks in Populus. A negative correlation between PG and CT concentrations has been observed in several studies. However, the molecular mechanism underlying the relationship is not known. Results Populus cell cultures produce CTs but not PGs under normal conditions. Feeding salicyl alcohol resulted in accumulation of salicins, the simplest PG, in the cells, but not higher-order PGs. Salicin accrual reflected the stimulation of a glycosylation response which altered a number of metabolic activities. We utilized this suspension cell feeding system as a model for analyzing the possible role of glycosylation in regulating the metabolic competition between PG formation, CT synthesis and growth. Cells accumulated salicins in a dose-dependent manner following salicyl alcohol feeding. Higher feeding levels led to a decrease in cellular CT concentrations (at 5 or 10 mM, and a negative effect on cell growth (at 10 mM. The competition between salicin and CT formation was reciprocal, and depended on the metabolic status of the cells. We analyzed gene expression changes between controls and cells fed with 5 mM salicyl alcohol for 48 hr, a time point when salicin accumulation was near maximum and CT synthesis was reduced, with no effect on growth. Several stress-responsive genes were up-regulated, suggestive of a general stress response in the fed cells. Salicyl alcohol feeding also induced expression of genes associated with sucrose catabolism, glycolysis and the Krebs cycle. Transcript levels of phenylalanine ammonia lyase and most of the flavonoid pathway genes were reduced, consistent with down-regulated CT synthesis. Conclusions Exogenous salicyl alcohol was readily glycosylated in Populus cell cultures, a process that altered sugar utilization and phenolic partitioning in the cells. Using this system, we

  7. In vitro cell culture lethal dose submitted to gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Moreno, Carolina S.; Rogero, Sizue O.; Rogero, Jose Roberto [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)], e-mail: carolina_sm@hotmail.com; Ikeda, Tamiko I.; Cruz, Aurea S. [Instituto Adolfo Lutz, Sao Paulo, SP (Brazil)

    2009-07-01

    The present study was designed to evaluate the in vitro effect of gamma radiation in cell culture of mouse connective tissue exposed to different doses of gamma radiation and under several conditions. The cell viability was analyzed by neutral red uptake methodology. This assay was developed for establish a methodology to be used in the future in the study of resveratrol radioprotection. Resveratrol (3,4',5- trihydroxystilbene), a phenolic phytoalexin that occurs naturally in some spermatophytes, such as grapevines, in response to injury as fungal infections and exposure to ultraviolet light. In the wines this compound is found at high levels and is considered one of the highest antioxidant constituents. The intense antioxidant potential of resveratrol provides many pharmacological activities including cardioprotection, chemoprevention and anti-tumor effects. Our results demonstrated that {sup 60}Co gamma radiation lethal dose (LD50) on NCTC clone 929 cells was about 340Gy. (author)

  8. Ribavirin Inhibits Parrot Bornavirus 4 Replication in Cell Culture.

    Directory of Open Access Journals (Sweden)

    Jeffrey M B Musser

    Full Text Available Parrot bornavirus 4 is an etiological agent of proventricular dilatation disease, a fatal neurologic and gastrointestinal disease of psittacines and other birds. We tested the ability of ribavirin, an antiviral nucleoside analog with antiviral activity against a range of RNA and DNA viruses, to inhibit parrot bornavirus 4 replication in duck embryonic fibroblast cells. Two analytical methods that evaluate different products of viral replication, indirect immunocytochemistry for viral specific nucleoprotein and qRT-PCR for viral specific phosphoprotein gene mRNA, were used. Ribavirin at concentrations between 2.5 and 25 μg/mL inhibited parrot bornavirus 4 replication, decreasing viral mRNA and viral protein load, in infected duck embryonic fibroblast cells. The addition of guanosine diminished the antiviral activity of ribavirin suggesting that one possible mechanism of action against parrot bornavirus 4 may likely be through inosine monophosphate dehydrogenase inhibition. This study demonstrates parrot bornavirus 4 susceptibility to ribavirin in cell culture.

  9. Cell-free DNA in a three-dimensional spheroid cell culture model: A preliminary study.

    Science.gov (United States)

    Aucamp, Janine; Calitz, Carlemi; Bronkhorst, Abel J; Wrzesinski, Krzysztof; Hamman, Sias; Gouws, Chrisna; Pretorius, Piet J

    2017-08-01

    Investigating the biological functions of cell-free DNA (cfDNA) is limited by the interference of vast numbers of putative sources and causes of DNA release into circulation. Utilization of three-dimensional (3D) spheroid cell cultures, models with characteristics closer to the in vivo state, may be of significant benefit for cfDNA research. CfDNA was isolated from the growth medium of C3A spheroid cultures in rotating bioreactors during both normal growth and treatment with acetaminophen. Spheroid growth was monitored via planimetry, lactate dehydrogenase activity and glucose consumption and was related to isolated cfDNA characteristics. Changes in spheroid growth and stability were effectively mirrored by cfDNA characteristics. CfDNA characteristics correlated with that of previous two-dimensional (2D) cell culture and human plasma research. 3D spheroid cultures can serve as effective, simplified in vivo-simulating "closed-circuit" models since putative sources of cfDNA are limited to only the targeted cells. In addition, cfDNA can also serve as an alternative or auxiliary marker for tracking spheroid growth, development and culture stability. 3D cell cultures can be used to translate "closed-circuit" in vitro model research into data that is relevant for in vivo studies and clinical applications. In turn, the utilization of cfDNA during 3D culture research can optimize sample collection without affecting the stability of the growth environment. Combining 3D culture and cfDNA research could, therefore, optimize both research fields. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Responsiveness of fetal rat brain cells to glia maturation factor during neoplastic transformation in cell culture

    DEFF Research Database (Denmark)

    Haugen, A; Laerum, O D; Bock, E

    1981-01-01

    The effect of partially purified extracts from adult pig brains containing a glia maturation protein factor (BE) has been investigated on neural cells during carcinogenesis. Pregnant BD IX-rats were given a single transplacental dose of the carcinogen ethylnitrosourea (EtNU) on the 18th day...... of gestation. The brains of the treated fetuses were transferred to cell culture and underwent neoplastic transformation with a characteristic sequence of phenotypic alterations which could be divided into five different stages. During the first 40 days after explantation (stage I & II) BE induced...... on GFA-content was seen any longer, although some few weakly GFA positive cells could be observed in all permanent cell lines. Fetal rat brain cells therefore seem to become less responsive to this differentiation inducer during neoplastic transformation in cell culture....

  11. Establishment of callus, cell suspension and shoot cultures of Leonurus cardiaca L. and diterpene analysis.

    Science.gov (United States)

    Knöss, W

    1995-10-01

    Callus cultures, cell suspension cultures and shoot cultures of Leonurus cardiaca L. (Motherwort) were established and growth conditions optimized. Shoot cultures showed constant growth whether in the dark or under continuous light, accumulating varying amounts of the furanic labdane diterpenes leosibiricin, preleosibirin, leosibirin and isoballotenol acetate, which are also present in the soil-grown plants. Only traces of leosibiricin were detected in callus cultures, while cell suspension cultures did not produce any furanic diterpenes. A small amount of furanic labdane diterpenes was found in the medium of shoot cultures. Callus and shoot culture induction of several other Lamiaceae species is also described.

  12. Optimization of Storage Temperature for Cultured ARPE-19 Cells

    Directory of Open Access Journals (Sweden)

    Lara Pasovic

    2013-01-01

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

  13. EFFECT OF MACROLIDE ANTIBIOTICS ON VARIOUS CELL CULTURES IN VITRO: 1. CELL MORPHOLOGY

    Directory of Open Access Journals (Sweden)

    Renáta Kováčová

    2012-08-01

    Full Text Available The aim of our study was to evaluate the cytotoxicity of macrolide antibiotics (tilmicosin, tylosin and spiramycin of various concentrations on different cell cultures in vitro. Cellular lines from animal tissues (VERO cells - kidney cells of Macacus rhesus, FE cells - feline embryonal cells, BHK 21 cellular line from young hamster kidneys were used. Tilmicosin effect: BHK cells are most sensitive, significant decrease in vital cells occurs already at the concentration of 50 μg.ml-1. VERO cells were most resistant, significant decrease of vital cells was observed only at the concentration of 300 μg.ml-1. Tylosin effect: BHK cells can be considered most sensitive, since at concentrations higher than 500 μg.ml-1, no vital cells were observed. At the concentration of 1000 μg.ml-1 were 3.13% of vital and 70.52% of subvital FE cells. In Vero cells, we observed a significant decrease at the concentration of 750 μg.ml-1. Spiramycin effect: Significant decrease of vital BHK cells was observed at the concentration of 150 μg.ml-1, at the concentration of 300 μg.ml-1, no vital cells and only 7.53% of subvital cells were observed. At the concentration of 500 μg.ml-1 reported 10.34% of vital FE cells. At the concentration of 500 μg.ml-1 22.48% of vital and 71.16% of subvital VERO cells were recorded.

  14. Culture and detection of primary cilia in endothelial cell models.

    Science.gov (United States)

    Lim, Yi Chung; McGlashan, Sue R; Cooling, Michael T; Long, David S

    2015-01-01

    The primary cilium is a sensor of blood-induced forces in endothelial cells (ECs). Studies that have examined EC primary cilia have reported a wide range of cilia incidence (percentage of ciliated cells). We hypothesise that this variation is due to the diversity in culture conditions in which the cells are grown. We studied two EC types: human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMEC-1s). Both cell types were grown in media containing foetal bovine serum (FBS) at high (20 % FBS and 10 % FBS for HUVECs and HMEC-1s, respectively) or low (2 % FBS) concentrations. Cells were then either fixed at confluence, serum-starved or grown post-confluence for 5 days in corresponding expansion media (cobblestone treatment). For each culture condition, we quantified cilia incidence and length. HUVEC ciliogenesis is dependent on serum concentration during the growth phase; low serum (2 % FBS) HUVECs were not ciliated, whereas high serum (20 % FBS) confluent HUVECs have a cilia incidence of 2.1 ± 2.2 % (median ± interquartile range). We report, for the first time, the presence of cilia in the HMEC-1 cell type. HMEC-1s have between 2.2 and 3.5 times greater cilia incidence than HUVECs (p cilia compared to HUVECs (3.0 ± 1.0 μm versus 5.1 ± 2.4 μm, at confluence, p = 0.003). We demonstrate that FBS plays a role in determining the prevalence of cilia in HUVECs. In doing so, we highlight the importance of considering a commonly varied parameter (% FBS), in the experimental design. We recommend that future studies examining large blood vessel EC primary cilia use confluent HUVECs grown in high serum medium, as we found these cells to have a higher cilia incidence than low serum media HUVECs. For studies interested in microvasculature EC primary cilia, we recommend using cobblestone HMEC-1s grown in high serum medium, as these cells have a 19.5 ± 6.2 % cilia incidence.

  15. Over-pressurized bioreactors: application to microbial cell cultures.

    Science.gov (United States)

    Lopes, Marlene; Belo, Isabel; Mota, Manuel

    2014-01-01

    In industrial biotechnology, microbial cultures are exposed to different local pressures inside bioreactors. Depending on the microbial species and strains, the increased pressure may have detrimental or beneficial effects on cellular growth and product formation. In this review, the effects of increased air pressure on various microbial cultures growing in bioreactors under moderate total pressure conditions (maximum, 15 bar) will be discussed. Recent data illustrating the diversity of increased air pressure effects at different levels in microbial cells cultivation will be presented, with particular attention to the effects of oxygen and carbon dioxide partial pressures on cellular growth and product formation, and the concomitant effect of oxygen pressure on antioxidant cellular defense mechanisms. © 2014 American Institute of Chemical Engineers.

  16. Biosynthesis of titin in cultured skeletal muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Isaacs, W.B.; Kim, I.S.; Struve, A.; Fulton, A.B. (Univ. of Iowa, Iowa City (USA))

    1989-11-01

    Although significant progress has been made regarding the structure and function of titin, little data exist on the biosynthesis of this large protein in developing muscle. Using pulse-labeling with ({sup 35}S)methionine and immunoprecipitation with an anti-titin mAb, we have examined the biosynthesis of titin in synchronized cultures of skeletal muscle cells derived from day 12 chicken embryos. We find that: (a) titin synthesis increases greater than 4-fold during the first week in culture and during this same time period, synthesis of muscle-specific myosin heavy chain increases greater than 12-fold; (b) newly synthesized titin has a t1/2 of approximately 70 h; (c) titin is resistant to extraction with Triton X-100 both during and immediately after its synthesis. These observations suggest that newly synthesized titin molecules are stable proteins that rapidly associate with the cytoskeleton of developing myotubes.

  17. Cytotoxicity testing of wound dressings using methylcellulose cell culture.

    Science.gov (United States)

    van Luyn, M J; van Wachem, P B; Nieuwenhuis, P; Jonkman, M F

    1992-01-01

    Wound dressings may induce cytotoxic effects. In this study, we check several, mostly commercially available, wound dressings for cytotoxicity. We used our previously described, newly developed and highly sensitive 7 d methylcellulose cell culture with fibroblasts as the test system. Cytotoxicity is assessed by monitoring cell growth inhibition, supported by cell morphological evaluation using light and transmission electron microscopy. We tested conventional wound dressings, polyurethane-based films, composites, hydrocolloids and a collagen-based dressing. It was shown that only 5 out of 16 wound dressings did not induce cytotoxic effects. All 5 hydrocolloids were found to inhibit cell growth (greater than 70%), while cells had strongly deviant morphologies. The remaining wound dressings showed medium cytotoxic effects, with cell growth inhibition, which varied from low (+/- 15%), medium-low (+/- 25%) to medium-high (+/- 50%). Measurable cytotoxic effects of dressings detected in vitro are likely to interfere with wound healing when applied in vivo. The results are discussed in view of the clinical uses with contaminated wounds, impaired epithelialization or hypergranulation.

  18. Defining cell culture conditions to improve human norovirus infectivity assays

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Hutchison, Janine R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Bartholomew, Rachel A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Valdez, Catherine O. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Valentine, Nancy B. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Dohnalkova, Alice [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Ozanich, Richard M. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Bruckner-Lea, Cindy J. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2013-01-10

    Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that leads to more reproducible hNoV infectivity in vitro requires that the cell line be 1) of human gastrointestinal origin, 2) expresses apical microvilli, and 3) be a positive secretor cell line. The C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log10 increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both reverse transcription quantitative PCR (qRT-PCR) and microscopy. Using 3-D C2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using quantitative reverse transcription PCR (qRT-PCR) that measures all RNA vs. plaque assays that measure infectious virus.

  19. Centering Single Cells in Microgels via Delayed Crosslinking Supports Long-Term 3D Culture by Preventing Cell Escape

    NARCIS (Netherlands)

    Kamperman, Tom; Henke, Sieger; Visser, Claas Willem; Karperien, Marcel; Leijten, Jeroen

    2017-01-01

    Single-cell-laden microgels support physiological 3D culture conditions while enabling straightforward handling and high-resolution readouts of individual cells. However, their widespread adoption for long-term cultures is limited by cell escape. In this work, it is demonstrated that cell escape is

  20. Induction of viral interference by IPNV-carrier cells on target cells: A cell co-culture study.

    Science.gov (United States)

    Parreño, Ricardo; Torres, Susana; Almagro, Lucía; Belló-Pérez, Melissa; Estepa, Amparo; Perez, Luis

    2016-11-01

    IPNV is a salmonid birnavirus that possesses the ability to establish asymptomatic persistent infections in a number of valuable fish species. The presence of IPNV may interfere with subsequent infection by other viruses. In the present study we show that an IPNV-carrier cell line (EPC(IPNV)) can induce an antiviral state in fresh EPC by co-cultivating both cell types in three different ways: a "droplet" culture system, a plastic chamber setup, and a transmembrane (Transwell(®)) system. All three cell co-culture methods were proven useful to study donor/target cell interaction. Naïve EPC cells grown in contact with EPC(IPNV) cells develop resistance to VHSV superinfection. The transmembrane system seems best suited to examine gene expression in donor and target cells separately. Our findings point to the conclusion that one or more soluble factors produced by the IPNV carrier culture induce the innate immune response within the target cells. This antiviral response is associated to the up-regulation of interferon (ifn) and mx gene expression in target EPC cells. To our knowledge this is the first article describing co-culture systems to study the interplay between virus-carrier cells and naive cells in fish. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  1. A biocompatible micro cell culture chamber for culturing and on-line monitoring of Eukaryotic cells

    DEFF Research Database (Denmark)

    Stangegaard, Michael

    2006-01-01

    at holde celler i live over længere tidsperioder I det foreliggende arbejde præsenteres et nyt perfusions baseret mikro celle dyrknings kultur kammer med integreret termisk overvågning og regulering. Kammeret opretholdt både dyrkning og on-line overvågning af både kræft celler såvel som stam celler over...... at dyrknings betingelserne i kammeret var sammenlignelige med dem i konventionelle celle kultur dyrknings flaske, hvis lys intensiteten på mikroskopet og omgivelserne blev minimeret mest muligt. Overflade modificeringer af den strukturelle fotoresist SU-8, der ofte bliver brugt til fabrikation af mikro kanaler...

  2. Immunological identification and isolation of phosphotyrosyl proteins in cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Martensen, T.M.; Madoff, D.H.; Lane, M.D.

    1987-05-01

    Affinity-purified sheep anti-phosphotyrosine antibodies were utilized to probe for phosphotyrosyl proteins from cultured cells. Western blots of cell extract proteins separated by SDS PAGE were incubated with anti-Tyr-P antibodies followed by peroxidase labeled anti-sheep antibodies or ( SVI)protein G to decorate the immune complexes. Normal, retrovirus transformed, and preadipocyte fibroblasts showed a variety of phosphotyrosyl proteins. Specific binding was indicated by the ability of Tyr-P but not Ser-P or Thr-P to inhibit the immunolocalization. Anti-phosphotyrosine antibodies covalently coupled to Sepharose were used to isolate phosphotyrosyl proteins from cultured cells. The binding capacity of the gel was determined by the quantity of unlabeled phosphotyrosyl protein needed to displace (TSP)labeled phosphotyrosyl protein binding. (TSP)labeled insulin receptor from 3T3-11 adipocytes could be immuno-adsorbed in high yield. Desorption was achieved in good yields under native conditions by incubation with Tyr-P. Treatment of (TSP)insulin receptor with SDS prior to incubation with immobilized antibody did not inhibit immuno-adsorption or -elution. This feature enabled antiphosphotyrosine antibodies to distinguish phosphotyrosyl proteins whose modified residues appear to be inaccessible for antibody binding in the native state.

  3. Moles of a Substance per Cell Is a Highly Informative Dosing Metric in Cell Culture.

    Directory of Open Access Journals (Sweden)

    Claire M Doskey

    Full Text Available The biological consequences upon exposure of cells in culture to a dose of xenobiotic are not only dependent on biological variables, but also the physical aspects of experiments e.g. cell number and media volume. Dependence on physical aspects is often overlooked due to the unrecognized ambiguity in the dominant metric used to express exposure, i.e. initial concentration of xenobiotic delivered to the culture medium over the cells. We hypothesize that for many xenobiotics, specifying dose as moles per cell will reduce this ambiguity. Dose as moles per cell can also provide additional information not easily obtainable with traditional dosing metrics.Here, 1,4-benzoquinone and oligomycin A are used as model compounds to investigate moles per cell as an informative dosing metric. Mechanistic insight into reactions with intracellular molecules, differences between sequential and bolus addition of xenobiotic and the influence of cell volume and protein content on toxicity are also investigated.When the dose of 1,4-benzoquinone or oligomycin A was specified as moles per cell, toxicity was independent of the physical conditions used (number of cells, volume of medium. When using moles per cell as a dose-metric, direct quantitative comparisons can be made between biochemical or biological endpoints and the dose of xenobiotic applied. For example, the toxicity of 1,4-benzoquinone correlated inversely with intracellular volume for all five cell lines exposed (C6, MDA-MB231, A549, MIA PaCa-2, and HepG2.Moles per cell is a useful and informative dosing metric in cell culture. This dosing metric is a scalable parameter that: can reduce ambiguity between experiments having different physical conditions; provides additional mechanistic information; allows direct comparison between different cells; affords a more uniform platform for experimental design; addresses the important issue of repeatability of experimental results, and could increase the

  4. Nitration of plant apoplastic proteins from cell suspension cultures.

    Science.gov (United States)

    Szuba, Agnieszka; Kasprowicz-Maluśki, Anna; Wojtaszek, Przemysław

    2015-04-29

    Nitric oxide causes numerous protein modifications including nitration of tyrosine residues. This modification, though one of the greatest biological importance, is poorly recognized in plants and is usually associated with stress conditions. In this study we analyzed nitrotyrosines from suspension cultures of Arabidopsis thaliana and Nicotiana tabacum, treated with NO modulators and exposed to osmotic stress, as well as of BY2 cells long-term adapted to osmotic stress conditions. Using confocal microscopy, we showed that the cell wall area is one of the compartments most enriched in nitrotyrosines within a plant cell. Subsequently, we analyzed nitration of ionically-bound cell-wall proteins and identified selected proteins with MALDI-TOF spectrometry. Proteomic analysis indicated that there was no significant increase in the amount of nitrated proteins under the influence of NO modulators, among them 3-morpholinosydnonimine (SIN-1), considered a donor of nitrating agent, peroxynitrite. Moreover, osmotic stress conditions did not increase the level of nitration in cell wall proteins isolated from suspension cells, and in cultures long-term adapted to stress conditions; that level was even reduced in comparison with control samples. Among identified nitrotyrosine-containing proteins dominated the ones associated with carbon circulation as well as the numerous proteins responding to stress conditions, mainly peroxidases. High concentrations of nitric oxide found in the cell wall and the ability to produce large amounts of ROS make the apoplast a site highly enriched in nitrotyrosines, as presented in this paper. Analysis of ionically bound fraction of the cell wall proteins indicating generally unchanged amounts of nitrotyrosines under influence of NO modulators and osmotic stress, is noticeably different from literature data concerning, however, the total plant proteins analysis. This observation is supplemented by further nitroproteome analysis, for cells long

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

    Science.gov (United States)

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

    2014-01-01

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

  6. Label-free hybridoma cell culture quality control by a chip-based impedance flow cytometer.

    Science.gov (United States)

    Pierzchalski, Arkadiusz; Hebeisen, Monika; Mittag, Anja; Bocsi, Jozsef; Di Berardino, Marco; Tarnok, Attila

    2012-11-07

    Impedance flow cytometry (IFC) was evaluated as a possible alternative to fluorescence-based methods for on-line quality monitoring of hybridoma cells. Hybridoma cells were cultured at different cell densities and viability was estimated by means of IFC and fluorescence-based flow cytometry (FCM). Cell death was determined by measuring the impedance phase value at high frequency in low conductivity buffer. IFC data correlate well with reference FCM measurements using AnnexinV and 7-AAD staining. Hybridoma cells growing at different densities in cell culture revealed a density-dependent subpopulation pattern. Living cells of high density cultures show reduced impedance amplitudes, indicating particular cellular changes. Dead cell subpopulations become evident in cultures with increasing cell densities. In addition, a novel intermediate subpopulation, which most probably represents apoptotic cells, was identified. These results emphasize the extraordinary sensitivity of high frequency impedance measurements and their suitability for hybridoma cell culture quality control.

  7. Phenotypic expression of human hepatoma cells in culture.

    Science.gov (United States)

    Cutroneo, Kenneth R; White, Sheryl L; Buttolph, Thomas R; Allison, Gretchen; Ehrlich, H Paul

    2007-04-01

    Hepatomas thrive in a hypoxic environment resulting in the induction of a cluster of hypoxia related genes. The protein phenotypic expression include hypoxia inducible factor-alpha, prolyl-4-hydroxylase, vascular endothelear growth factor and erythropoietin. The present study was undertaken to determine if human hepatoma cells when cultured for 72 h in the presence of serum under normoxia would maintain their cancerous phenotypic expression of certain hypoxia inducible genes. Our positive results affords an in vitro model system to test hypoxia inhibitors on the expression and the intracellular compartmentalization or the secretion of these hypoxia-inducible proteins. c 2007 Wiley-Liss, Inc.

  8. Cellular Changes of Stem Cells in 3-Dimensional Culture.

    Science.gov (United States)

    Green, Matthew P; Hou, Bo

    2017-11-01

    During various operations and procedures, such as distraction osteogenesis and orthodontics, skeletal tissues use mechanotransduction. Mechanotransduction is important for maintaining bone health and converting mechanical forces into biochemical signals. We hypothesized that cells put under mechanical stress would adapt and change morphologically and respond with a decrease in cellular proliferation to accommodate the stress differences. These differences will be measured at the molecular and genetic level. We also wanted to test the practicality of an in vitro 3-dimensional gel model system. We implemented a 3-dimensional cell culture model. The sample was composed of isolated mouse mesenchymal prefibroblast bone marrow cells from the femurs and tibias of 6- to 8-week-old wild-type C57BL6 mice. The cells were seeded on fibronectin-coated hydrogels along with fibrin and nodulin growth factors. The variables tested were a no-force model (control) and a force model. The force model required two 0.1-mm suture pins put through one 0.25-cm length of cell-gel matrix. After the experiments were run to completion, the samples were fixed with 4% paraformaldehyde and embedded in paraffin. Serial sections were cut at a thickness of 5 μm along the long axis for the force construct and encompassing the entire circular area of the control construct. Descriptive and bivariate statistics were computed, and the P value was set at 5%. There was a statistically significant difference between the 2 models. The force model had longer and straighter primary cilia, less apoptosis, and an increase in cell proliferation. In addition, the shape of the cells was markedly different after the experiment. The results of the study suggest cells put under tensile stress have the ability to mechanically sense the environment to provide improved adaptation. Our work also confirms the usefulness of the in vitro 3-dimensional gel model system to mimic in vivo applications. Published by Elsevier

  9. Cytotoxicity Profile of Endodontic Sealers Provided by 3D Cell Culture Experimental Model.

    Science.gov (United States)

    Silva, Emmanuel João Nogueira Leal; Carvalho, Nancy Kudsi de; Ronconi, Carina Taboada; De-Deus, Gustavo; Zuolo, Mario Luis; Zaia, Alexandre Augusto

    2016-01-01

    The aim of the present study was to evaluate the cytotoxic effects of five endodontic sealers (AH Plus, Endomethasone N, EndoSequence BC, MTA Fillapex and Pulp Canal Sealer EWT) using a three-dimensional (3D) cell culture model. A conventional bi-dimensional (2D) cell culture model was used as reference technique for comparison. Balb/c 3T3 fibroblasts were cultured in conventional bi-dimensional cell culture and in rat-tail collagen type I three-dimensional cell culture models. Then, both cell cultures were incubated with elutes of freshly mixed endodontic sealers for 24 h. Cell viability was measured by the methyl-thiazol-diphenyltetrazolium assay (MTT). Data were statistically analyzed using ANOVA and the Tukey test at a significance level of pculture model- and sealer-dependent. Sealers showed higher cytotoxicity in 2D than in 3D cell culture model (pculture model (p>0.05). Endomethasone N and Pulp Canal Sealer EWT showed lower cytotoxic effects than AH Plus in 2D cell culture model (p3D cell culture model. It may be concluded that cytotoxicity was higher in 2D cell culture compared to 3D cell culture. EndoSequence BC sealer exhibited the highest cytocompatibility and MTA Fillapex the lowest cytocompatibility.

  10. Serum-free media formulations are cell line-specific and require optimization for microcarrier culture.

    Science.gov (United States)

    Tan, Kah Yong; Teo, Kim Leng; Lim, Jessica F Y; Chen, Allen K L; Choolani, Mahesh; Reuveny, Shaul; Chan, Jerry; Oh, Steve Kw

    2015-08-01

    Mesenchymal stromal cells (MSCs) are being investigated as potential cell therapies for many different indications. Current methods of production rely on traditional monolayer culture on tissue-culture plastic, usually with the use of serum-supplemented growth media. However, the monolayer culturing system has scale-up limitations and may not meet the projected hundreds of billions to trillions batches of cells needed for therapy. Furthermore, serum-free medium offers several advantages over serum-supplemented medium, which may have supply and contaminant issues, leading to many serum-free medium formulations being developed. We cultured seven MSC lines in six different serum-free media and compared their growth between monolayer and microcarrier culture. We show that (i) expansion levels of MSCs in serum-free monolayer cultures may not correlate with expansion in serum-containing media; (ii) optimal culture conditions (serum-free media for monolayer or microcarrier culture) differ for each cell line; (iii) growth in static microcarrier culture does not correlate with growth in stirred spinner culture; (iv) and that early cell attachment and spreading onto microcarriers does not necessarily predict efficiency of cell expansion in agitated microcarrier culture. Current serum-free media developed for monolayer cultures of MSCs may not support MSC proliferation in microcarrier cultures. Further optimization in medium composition will be required for microcarrier suspension culture for each cell line. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  11. Cell culture-derived influenza vaccines from Vero cells: a new horizon for vaccine production.

    Science.gov (United States)

    Montomoli, Emanuele; Khadang, Baharak; Piccirella, Simona; Trombetta, Claudia; Mennitto, Elisa; Manini, Ilaria; Stanzani, Valerio; Lapini, Giulia

    2012-05-01

    In the 20th century, three influenza pandemics killed approximately 100 million people. The traditional method of influenza vaccine manufacturing is based on using chicken eggs. However, the necessity of the availability of millions of fertile eggs in the event of a pandemic has led research to focus on the development of cell culture-derived vaccines, which offer shorter lead-in times and greater flexibility of production. So far, the cell substrates being evaluated and in use include Vero, Madin-Darby canine kidney, PER.C6 and insect cells. However, Vero cells are the most widely accepted among others. This review introduces briefly the concepts of advanced cell culture-derived influenza vaccine production and highlights the advantages of these vaccines in terms of efficiency, speed and immunogenicity based on the clinical data obtained from different studies.

  12. Cabergoline protects dopaminergic neurons against rotenone-induced cell death in primary mesencephalic cell culture.

    Science.gov (United States)

    Meinel, J; Radad, K; Rausch, W-D; Reichmann, H; Gille, G

    2015-01-01

    In the present study, primary mesencephalic cell cultures prepared from embryonic mouse mesencephala were used to investigate the neuroprotective effect of cabergoline, an ergoline D2 receptor agonist, against the pesticide and neurotoxin rotenone relevant to Parkinson disease (PD). Treatment of cultures with cabergoline alone significantly increased the number of tyrosine hydroxylase immunoreactive (THir) neurons and reduced the release of lactate dehydrogenase (LDH) into the culture medium compared to untreated controls. Against rotenone toxicity, cabergoline significantly rescued degenerating THir neurons, reduced the release of LDH into the culture medium and improved the morphology of surviving THir neurons. The neuroprotective effects afforded by cabergoline were independent of dopaminergic stimulation as blocking of dopamine receptors by the dopamine receptor antagonist sulpiride did not prevent them. Furthermore, rotenone-induced formation of reactive oxygen species (ROS) was significantly reduced by cabergoline. Although cabergoline increased the glutathione (GSH) content in the culture, the protective effect for dopaminergic neurons seemed not to be predominantly mediated by increasing GSH, as depletion of GSH by L-buthionine-(S,R)-sulfoximine (BSO), a GSH biosynthesis inhibitor, did not prevent cabergoline-mediated neuroprotection of THir neurons in rotenone-treated cultures. Moreover, cabergoline significantly increased the ATP/protein ratio in primary mesencephalic cell cultures when added alone or prior to rotenone treatment. These results indicate a neuroprotective effect of cabergoline for dopaminergic neurons against rotenone toxicity. This effect was independent of dopamine receptor stimulation and was at least partially mediated by reducing ROS production and increasing the ATP/protein ratio.

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

    Science.gov (United States)

    Richmond, Robert C.

    2001-01-01

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

  14. Cell cycle effects of hypertonic stress on various human cells in culture.

    Science.gov (United States)

    Pellicciari, C; Filippini, C; De Grada, L; Fuhrman Conti, A M; Manfredi Romanini, M G

    1995-03-01

    Long-term exposure to hypertonic (HT) culture media has been found to perturb the cell cycle and change gene expression in various animal cell types. A lower growth rate, with exit of cells from the cycling compartment has been observed previously in human transformed EUE cells. The aim of this study was to investigate if the kinetic changes after long-term HT stress, were typical of transformed cells or could be also found in primary cultures of normal cells. Human transformed cells from normal and neoplastic tissues, and normal human cells of epithelial and connective origin have been studied. After the incorporation of bromodeoxyuridine (BrdUrd), the frequency of S-phase cells was estimated by dual-parameter flow cytometry of DNA content versus BrdUrd immunolabelling; the total growth fraction was also estimated, after immunolabelling with an anti-PCNA antibody. We also investigated, by polyacrylamide gel electrophoresis, changes in the amount of a 35 kDa protein band, which increased in EUE cells grown in an HT medium, and which may be directly involved in cell resistance to hypertonicity. Lower BrdUrd labelling indices and higher frequencies of cells in the G0/1 range of DNA content were common features of all the cells in HT media, irrespective of their tissue of origin; other cycle phases may also be involved, depending on the cell type considered. The mechanisms by which cells cope with the HT environment could however differ, since only some cell types showed an increase of the 35 kDa stress protein found originally in HT EUE cells.

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

    Energy Technology Data Exchange (ETDEWEB)

    Campisi, Judith

    2000-12-21

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

  16. Pros and cons of fish skin cells in culture: long-term full skin and short-term scale cell culture from rainbow trout, Oncorhynchus mykiss.

    Science.gov (United States)

    Rakers, Sebastian; Klinger, Matthias; Kruse, Charli; Gebert, Marina

    2011-12-01

    Here, we report the establishment of a permanent skin cell culture from rainbow trout (Oncorhynchus mykiss). The cells of the fish skin cell culture could be propagated over 60 passages so far. Furthermore, we show for the first time that it is possible to integrate freshly harvested rainbow trout scales into this new fish skin cell culture. We further demonstrated that epithelial cells derived from the scales survived in the artificial micro-environment of surrounding fibroblast-like cells. Also, antibody staining indicated that both cell types proliferated and started to build connections with the other cell type. It seems that it is possible to generate an 'artificial skin' with two different cell types. This could lead to the development of a three-dimensional test system, which might be a better in vitro representative of fish skin in vivo than individual skin cell lines. Copyright © 2011 Elsevier GmbH. All rights reserved.

  17. CD34+ cells cultured in stem cell factor and interleukin-2 generate CD56+ cells with antiproliferative effects on tumor cell lines

    Directory of Open Access Journals (Sweden)

    Hensel Nancy

    2005-04-01

    Full Text Available Abstract In vitro stimulation of CD34+ cells with IL-2 induces NK cell differentiation. In order to define the stages of NK cell development, which influence their generation from CD34 cells, we cultured G-CSF mobilized peripheral blood CD34+ cells in the presence of stem cell factor and IL-2. After three weeks culture we found a diversity of CD56+ subsets which possessed granzyme A, but lacked the cytotoxic apparatus required for classical NK-like cytotoxicity. However, these CD56+ cells had the unusual property of inhibiting proliferation of K562 and P815 cell lines in a cell-contact dependent fashion.

  18. Cell-cycle research with synchronous cultures: an evaluation

    Science.gov (United States)

    Helmstetter, C. E.; Thornton, M.; Grover, N. B.

    2001-01-01

    The baby-machine system, which produces new-born Escherichia coli cells from cultures immobilized on a membrane, was developed many years ago in an attempt to attain optimal synchrony with minimal disturbance of steady-state growth. In the present article, we put forward a model to describe the behaviour of cells produced by this method, and provide quantitative evaluation of the parameters involved, at each of four different growth rates. Considering the high level of selection achievable with this technique and the natural dispersion in interdivision times, we believe that the output of the baby machine is probably close to optimal in terms of both quality and persistence of synchrony. We show that considerable information on events in the cell cycle can be obtained from populations with age distributions very much broader than those achieved with the baby machine and differing only modestly from steady state. The data presented here, together with the long and fruitful history of findings employing the baby-machine technique, suggest that minimisation of stress on cells is the single most important factor for successful cell-cycle analysis.

  19. Quantitative determination of geranyl diphosphate levels in cultured human cells.

    Science.gov (United States)

    Holstein, Sarah A; Tong, Huaxiang; Kuder, Craig H; Hohl, Raymond J

    2009-11-01

    Geranyl diphosphate (GPP), a 10-carbon isoprenoid, is a key intermediate in the isoprenoid biosynthetic pathway. This pathway, in addition to leading to sterol synthesis, results in the synthesis of farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), which serve as substrates for protein isoprenylation reactions. Basal levels of GPP in mammalian cells previously have been undetectable. Here we present a novel, sensitive, nonradioactive method which allows for measurement of GPP in mammalian cells. This methodology involves extraction of isoprenoids from cultured cells followed by enzymatic conjugation of GPP to a fluorescent dansylated-peptide via farnesyl transferase and quantification with high-performance liquid chromatography (HPLC). The lower limit of detection of GPP is 5 pg, or 0.015 pmol. Basal levels of GPP were determined in three human multiple myeloma cell lines (RPMI-8226, U266, H929). Treatment of cells with inhibitors of the isoprenoid biosynthetic pathway results in marked changes in GPP levels: the HMG-CoA reductase inhibitor lovastatin decreases GPP levels by over 50%, while the FPP synthase inhibitor zoledronic acid increases GPP levels 16- to 107-fold. This method also allows for the simultaneous measurement of GPP, FPP, and GGPP, thus leading to improved understanding of the pathway in a multitude of biological systems. Furthermore, as drugs targeting this pathway are developed, their biological activity can be more directly linked to effects on isoprenoid levels.

  20. Quantitative determination of isopentenyl diphosphate in cultured mammalian cells.

    Science.gov (United States)

    Tong, Huaxiang; Kuder, Craig H; Wasko, Brian M; Hohl, Raymond J

    2013-02-01

    Isopentenyl diphosphate (IPP), an intermediate of the isoprenoid biosynthetic pathway (IBP), has several important biological functions, yet a method to determine its basal level has not been described. Here, we describe a nonradioactive and sensitive analytical method to isolate and specifically quantify IPP from cultured mammalian cells. This method applies an enzymatic coupling reaction to determine intracellular concentrations of IPP. In this reaction, geranylgeranyl diphosphate synthase catalyzes the formation of geranylgeranyl diphosphate (GGPP) from IPP and farnesyl diphosphate (FPP). Subsequently, geranylgeranyl protein transferase I conjugates GGPP with a fluorescently labeled peptide. The geranylgeranylated peptide can be quantified by high-performance liquid chromatography (HPLC) with a fluorescence detector, thereby allowing for IPP quantification. The detection lower limit of the fluorescence-labeled geranylgeranyl peptide is approximately 5 pg (~0.017 pmol). This method was used to examine the effects of IBP inhibitors such as lovastatin and zoledronate on intracellular levels of IPP. Inhibition of hydroxymethylglutaryl coenzyme A reductase (HMGCR) by lovastatin (50 nM) decreases IPP levels by 78% and 53% in K562 and MCF-7 cells, respectively. Whereas zoledronic acid (10 μM) increased IPP levels 12.6-fold when compared with untreated cells in the K562 cell line, an astonishing 960-fold increase was observed in the MCF-7 cells. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Potassium currents in cultured human pulmonary arterial smooth muscle cells.

    Science.gov (United States)

    Peng, W; Karwande, S V; Hoidal, J R; Farrukh, I S

    1996-04-01

    In this study, using whole cell and single-channel configurations of the patch-clamp technique, we characterized K+ currents (IK) in cultured human pulmonary arterial smooth muscle cells. The net whole cell outward membrane current (IKo) was activated at potentials positive to -60 mV. One component of IKo, IK(dr), was inhibited by 4-aminopyridine (4-AP) and high concentrations of tetraethylammonium (TEA) but was Ca2+ and charybdotoxin (CTX) insensitive. The other component of IKo, IK(Ca), was voltage and Ca2+ dependent and was inhibited by CTX and low concentrations of TEA. Activation of IKo in single-channel recordings was voltage dependent and demonstrated a high-conductance channel (245 +/- 2 pS) that was Ca2+ and CTX sensitive [IK(Ca)] and a low-conductance channel (109 +/- 2 pS) that was inhibited by 4-AP [IK(dr)] but was insensitive to low concentrations of TEA or to an increase in intracellular [Ca2+]. In isolated pulmonary arterial rings, TEA and 4-AP caused an additive increase in arterial tension. To our knowledge these data provide the first characterization of the IK in human pulmonary arterial smooth muscle cells and indicate that IK(Ca) and IK(dr) play an important role in maintaining pulmonary vascular tone. The data confirm previous observations in pulmonary smooth muscle cells of animal models.

  2. Cell Monitoring and Manipulation Systems (CMMSs based on Glass Cell-Culture Chips (GC3s

    Directory of Open Access Journals (Sweden)

    Sebastian M. Buehler

    2016-06-01

    Full Text Available We developed different types of glass cell-culture chips (GC3s for culturing cells for microscopic observation in open media-containing troughs or in microfluidic structures. Platinum sensor and manipulation structures were used to monitor physiological parameters and to allocate and permeabilize cells. Electro-thermal micro pumps distributed chemical compounds in the microfluidic systems. The integrated temperature sensors showed a linear, Pt1000-like behavior. Cell adhesion and proliferation were monitored using interdigitated electrode structures (IDESs. The cell-doubling times of primary murine embryonic neuronal cells (PNCs were determined based on the IDES capacitance-peak shifts. The electrical activity of PNC networks was detected using multi-electrode arrays (MEAs. During seeding, the cells were dielectrophoretically allocated to individual MEAs to improve network structures. MEA pads with diameters of 15, 20, 25, and 35 µm were tested. After 3 weeks, the magnitudes of the determined action potentials were highest for pads of 25 µm in diameter and did not differ when the inter-pad distances were 100 or 170 µm. Using 25-µm diameter circular oxygen electrodes, the signal currents in the cell-culture media were found to range from approximately −0.08 nA (0% O2 to −2.35 nA (21% O2. It was observed that 60-nm thick silicon nitride-sensor layers were stable potentiometric pH sensors under cell-culture conditions for periods of days. Their sensitivity between pH 5 and 9 was as high as 45 mV per pH step. We concluded that sensorized GC3s are potential animal replacement systems for purposes such as toxicity pre-screening. For example, the effect of mefloquine, a medication used to treat malaria, on the electrical activity of neuronal cells was determined in this study using a GC3 system.

  3. Phase-segregated model for plant cell culture: The effect of cell volume fraction

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, W. [Univ. of Adelaide, Adelaide (Australia). Dept. of Chemical Engineering]|[Tokyo Univ. (Japan)hinese Academy of Sciences, Dalian (China). Dalian Inst. of Chemical Physics; Furusaki, S. [Tokyo Univ. (Japan)] Middelberg, A. [Univ. of Adelaide, Adelaide (Australia). Dept. of Chemical Engineering

    1998-06-01

    Plant cells are characterized by low water content, so the fraction of cell volume (volume fraction) in a vessel is large compared with other cell systems, even if the cell concentrations are the same. Therefore, concentration of plant cells should preferably be expressed by the liquid volume basis rather than by the total vessel volume basis. In this paper, a new model is proposed to analyze behavior of a plant cell culture by dividing the cell suspension into the biotic- and abiotic-phases. Using this model, we analyzed the cell-growth and the alkaloid production by Catharanthus roseus. Large errors in the simulated results were observed if the phase-segregation was not considered. 12 refs., 3 figs.

  4. Fibroblasts Cultured on Nanowires Exhibit Low Motility, Impaired Cell Division, and DNA Damage

    DEFF Research Database (Denmark)

    Persson, H.; Købler, Carsten; Mølhave, Kristian

    2013-01-01

    Mouse fibroblasts cultured on 7-μm-long vertical nanowires are reported on page 4006 by C. N. Prinz and co-workers. Culturing cells on this kind of substrate interferes greatly with cell function, causing the cells to develop into widely different morphologies. The cells' division is impaired...

  5. Cell-Culture Reactor Having a Porous Organic Polymer Membrane

    Science.gov (United States)

    Koontz, Steven L. (Inventor)

    2000-01-01

    A method for making a biocompatible polymer article using a uniform atomic oxygen treatment is disclosed. The substrate may be subsequently optionally grated with a compatibilizing compound. Compatibilizing compounds may include proteins, phosphory1choline groups, platelet adhesion preventing polymers, albumin adhesion promoters, and the like. The compatibilized substrate may also have a living cell layer adhered thereto. The atomic oxygen is preferably produced by a flowing afterglow microwave discharge, wherein the substrate resides in a sidearm out of the plasma. Also, methods for culturing cells for various purposes using the various membranes are disclosed as well. Also disclosed are porous organic polymers having a distributed pore chemistry (DPC) comprising hydrophilic and hydrophobic regions, and a method for making the DPC by exposing the polymer to atomic oxygen wherein the rate of hydrophilization is greater than the rate of mass loss.

  6. SPONTANEOUS TRANSFORMATION OF CULTURED PORCINE BONE MARROW STROMAL CELLS

    DEFF Research Database (Denmark)

    Zou, Lijin; Zou, Xuenong; Li, Haisheng

    activity detection assay and analysis of the expression of p53, Fas and c- Myc genes. Multipotency was investigated by biochemical and histological assays and analysis of gene expression. RESULTS BMSC showed a change in appearance, from the initial spindle shape to a more flatted morphology then to small...... contact shape. After additional passages, BMSC gradually acquired recovery of proliferating capacity and transformation properties such as anchorage-independent growth, chromosomal abnormality, and abnormal gene expression. The expression of P53 and Fas was decreased, while the expression of c-Myc gene......-term culture are transformed into malignant cells. MATERIAL AND METHODS BMSC from 6 pigs were isolated and propagated continuously. Cell morphology was observed. Transformation properties were evaluated by means of serum dependence assay, Ki- 67 immunostaining, soft agar colony assay, karyotyping, telomerase...

  7. NAC, tiron and trolox impair survival of cell cultures containing glioblastoma tumorigenic initiating cells by inhibition of cell cycle progression.

    Science.gov (United States)

    Monticone, Massimiliano; Taherian, Razieh; Stigliani, Sara; Carra, Elisa; Monteghirfo, Stefano; Longo, Luca; Daga, Antonio; Dono, Mariella; Zupo, Simona; Giaretti, Walter; Castagnola, Patrizio

    2014-01-01

    Reactive oxygen species (ROS) are metabolism by-products that may act as signaling molecules to sustain tumor growth. Antioxidants have been used to impair cancer cell survival. Our goal was to determine the mechanisms involved in the response to antioxidants of a human cell culture (PT4) containing glioblastoma (GBM) tumorigenic initiating cells (TICs). ROS production in the absence or presence of N-acetyl-L-cysteine (NAC), tiron, and trolox was evaluated by flow cytometry (FCM). The effects of these antioxidants on cell survival and apoptosis were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) and FCM. The biological processes modulated by these drugs were determined by oligonucleotide microarray gene expression profiling. Our results showed that NAC, tiron and trolox impaired PT4 cell survival, had minor effects on ROS levels and caused wide deregulation of cell cycle genes. Furthermore, tiron and trolox caused inhibition of cell survival in two additional cell cultures containing TICs, FO-1 and MM1, established from a melanoma and a mesothelioma patient, respectively. NAC, instead, impaired survival of the MM1 cells but not of the FO-1 cells. However, when used in combination, NAC enhanced the inhibitory effect of PLX4032 (BRAF V600E inhibitor) and Gefitinib (EGFR inhibitor), on FO-1 and PT4 cell survival. Collectively, NAC, tiron and trolox modulated gene expression and impaired the growth of cultures containing TICs primarily by inhibiting cell cycle progression.

  8. Sensitivity of neoplastic cells to senescence unveiled under standard cell culture conditions.

    Science.gov (United States)

    Zieba, Jolanta; Ksiazkiewcz, Magdalena; Janik, Karolina; Banaszczyk, Mateusz; Peciak, Joanna; Piaskowski, Sylwester; Lipinski, Marek; Olczak, Michal; Stoczynska-Fidelus, Ewelina; Rieske, Piotr

    2015-05-01

    Cancer cells are typically defined as infinitely proliferating, whereas normal cells (except stem cells) are considered as being programmed to become senescent. Our data show that this characterization is misleading. Multiplex Ligation-dependent Probe Amplification, TP53 sequencing, real-time polymerase chain reaction (PCR) for MUC1 and SCGB2A2 and immunocytochemistry, together with senescence detection assay and real-time microscopic observations were used to analyze primary neoplastic cells isolated from prostate, breast and colorectal tumors, as well as stable cancer cell lines (MCF7, MDA-MB-468, SW962, SK-MEL28, NCI-H1975 and NCI-H469). In all cases of primary cancer cell cultures, in vitro conditions rapidly revealed senescence in the majority of cells. Two out of six stable cancer cell lines did not exhibit any senescence-associated-β-Galactosidase-positive cells. Interestingly, four cell lines had small sub-populations of senescent cells (single SA-β-Gal-positive cells). Primary neoplastic cells from different types of cancer (prostate, breast, colon cancer) appear to be senescent in vitro. Apparently, cancer cell lines that have been used for many years in drug-testing analyses have constantly been misleading researchers in terms of the general sensitivity of cancer cells to senescence. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  9. Comparison of biophysical properties characterized for microtissues cultured using microencapsulation and liquid crystal based 3D cell culture techniques.

    Science.gov (United States)

    Soon, Chin Fhong; Tee, Kian Sek; Wong, Soon Chuan; Nayan, Nafarizal; Sargunan Sundra; Ahmad, Mohd Khairul; Sefat, Farshid; Sultana, Naznin; Youseffi, Mansour

    2017-11-30

    Growing three dimensional (3D) cells is an emerging research in tissue engineering. Biophysical properties of the 3D cells regulate the cells growth, drug diffusion dynamics and gene expressions. Scaffold based or scaffoldless techniques for 3D cell cultures are rarely being compared in terms of the physical features of the microtissues produced. The biophysical properties of the microtissues cultured using scaffold based microencapsulation by flicking and scaffoldless liquid crystal (LC) based techniques were characterized. Flicking technique produced high yield and highly reproducible microtissues of keratinocyte cell lines in alginate microcapsules at approximately 350 ± 12 pieces per culture. However, microtissues grown on the LC substrates yielded at lower quantity of 58 ± 21 pieces per culture. The sizes of the microtissues produced using alginate microcapsules and LC substrates were 250 ± 25 μm and 141 ± 70 μm, respectively. In both techniques, cells remodeled into microtissues via different growth phases and showed good integrity of cells in field-emission scanning microscopy (FE-SEM). Microencapsulation packed the cells in alginate scaffolds of polysaccharides with limited spaces for motility. Whereas, LC substrates allowed the cells to migrate and self-stacking into multilayered structures as revealed by the nuclei stainings. The cells cultured using both techniques were found viable based on the live and dead cell stainings. Stained histological sections showed that both techniques produced cell models that closely replicate the intrinsic physiological conditions. Alginate microcapsulation and LC based techniques produced microtissues containing similar bio-macromolecules but they did not alter the main absorption bands of microtissues as revealed by the Fourier transform infrared spectroscopy. Cell growth, structural organization, morphology and surface structures for 3D microtissues cultured using both techniques appeared to be

  10. Hemopoietic cell precursor responses to erythropoietin in plasma clot cultures

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, W.L.

    1979-01-01

    The time dependence of the response of mouse bone marrow cells to erythropoietin (Ep) in vitro was studied. Experiments include studies on the Ep response of marrow cells from normal, plethoric, or bled mice. Results with normal marrow reveal: (1) Not all erythroid precursors (CFU-E) are alike in their response to Ep. A significant number of the precursors develop to a mature erythroid colony after very short Ep exposures, but they account for only approx. 13% of the total colonies generated when Ep is active for 48 hrs. If Ep is active more than 6 hrs, a second population of erythroid colonies emerges at a nearly constant rate until the end of the culture. Full erythroid colony production requires prolonged exposure to erythropoietin. (2) The longer erythropoietin is actively present, the larger the number of erythroid colonies that reach 17 cells or more. Two distinct populations of immediate erythroid precursors are also present in marrow from plethoric mice. In these mice, total colony numbers are equal to or below those obtained from normal mice. However, the population of fast-responding CFU-E is consistently decreased to 10 to 20% of that found in normal marrow. The remaining colonies are formed from plethoric marrow at a rate equal to normal marrow. With increasing Ep exposures, the number of large colonies produced increases. From the marrow of bled mice, total erythroid colony production is equal to or above that of normal marrow. Two populations of colony-forming cells are again evident, with the fast-responding CFU-E being below normal levels. The lack of colonies from this group was compensated in bled mice by rapid colony production in the second population. A real increase in numbers of precursors present in this pool increased the rate of colony production in culture to twice that of normal marrow. The number of large colonies obtained from bled mice was again increased as the Ep exposure was lengthened. (ERB)

  11. Cellulose Biosynthesis Inhibitors: Comparative Effect on Bean Cell Cultures

    Directory of Open Access Journals (Sweden)

    José L. Acebes

    2012-03-01

    Full Text Available The variety of bioassays developed to evaluate different inhibition responses for cellulose biosynthesis inhibitors makes it difficult to compare the results obtained. This work aims (i to test a single inhibitory assay for comparing active concentrations of a set of putative cellulose biosynthesis inhibitors and (ii to characterize their effect on cell wall polysaccharides biosynthesis following a short-term exposure. For the first aim, dose-response curves for inhibition of dry-weight increase following a 30 days exposure of bean callus-cultured cells to these inhibitors were obtained. The compound concentration capable of inhibiting dry weight increase by 50% compared to control (I50 ranged from subnanomolar (CGA 325′615 to nanomolar (AE F150944, flupoxam, triazofenamide and oxaziclomefone and micromolar (dichlobenil, quinclorac and compound 1 concentrations. In order to gain a better understanding of the effect of the putative inhibitors on cell wall polysaccharides biosynthesis, the [14C]glucose incorporation into cell wall fractions was determined after a 20 h exposure of cell suspensions to each inhibitor at their I50 value. All the inhibitors tested decreased glucose incorporation into cellulose with the exception of quinclorac, which increased it. In some herbicide treatments, reduction in the incorporation into cellulose was accompanied by an increase in the incorporation into other fractions. In order to appreciate the effect of the inhibitors on cell wall partitioning, a cluster and Principal Component Analysis (PCA based on the relative contribution of [14C]glucose incorporation into the different cell wall fractions were performed, and three groups of compounds were identified. The first group included quinclorac, which increased glucose incorporation into cellulose; the second group consisted of compound 1, CGA 325′615, oxaziclomefone and AE F150944, which decreased the relative glucose incorporation into cellulose but

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

    Energy Technology Data Exchange (ETDEWEB)

    Sestili, Piero, E-mail: piero.sestili@uniurb.it [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Paolillo, Marco [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Lenzi, Monia [Dipartimento di Farmacologia, Universita degli Studi di Bologna, Via Irnerio 48, 40126 Bologna (Italy); Colombo, Evelin; Vallorani, Luciana; Casadei, Lucia; Martinelli, Chiara [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Fimognari, Carmela [Dipartimento di Farmacologia, Universita degli Studi di Bologna, Via Irnerio 48, 40126 Bologna (Italy)

    2010-07-07

    Sulforaphane (SFR), an isothiocyanate from cruciferous vegetables, possesses growth-inhibiting and apoptosis-inducing activities in cancer cell lines. Recently, SFR has been shown to promote the mitochondrial formation of reactive oxygen species (ROS) in human cancer cell lines. The present study was undertaken to see whether SFR-derived ROS might cause DNA damage in cultured human cells, namely T limphoblastoid Jurkat and human umbilical vein endothelial cells (HUVEC). 1-3 h treatments with 10-30 {mu}M SFR elicited intracellular ROS formation (as assayed with dihydrorhodamine, DHR, oxidation) as well as DNA breakage (as assessed with fast halo assay, FHA). These effects lacked cell-type specificity, since could be observed in both Jurkat and HUVEC. Differential-pH FHA analysis of damaged DNA showed that SFR causes frank DNA single strand breaks (SSBs); no DNA double strand breaks (DSBs) were found within the considered treatment times (up to 3 h). SFR-derived ROS were formed at the mitochondrial respiratory chain (MRC) level: indeed rotenone or myxothiazol (MRC Complex I and III inhibitors, respectively) abrogated ROS formation. Furthermore ROS were not formed in Jurkat cells pharmacologically depleted of respiring mitochondria (MRC-/Jurkat). Formation of ROS was causally linked to the induction of SSBs: indeed all the experimental conditions capable of preventing ROS formation also prevented the damage of nuclear DNA from SFR-intoxicated cells. As to the toxicological relevance of SSBs, we found that their prevention slightly but significantly attenuated SFR cytotoxicity, suggesting that high-dose SFR toxicity is the result of a complex series of events among which GSH depletion seems to play a pivotal role. In conclusion, the present study identifies a novel mechanism contributing to SFR toxicity which - since DNA damage is a prominent mechanism underlying the cytotoxic activity of established antineoplastic agents - might help to exploit the therapeutic value

  13. Propagation of Human Embryonic Stem Cells on Human Amniotic Fluid Cells as Feeder Cells in Xeno-Free Culture Conditions.

    Science.gov (United States)

    Jung, Juwon; Baek, Jin Ah; Seol, Hye Won; Choi, Young Min

    2016-03-01

    Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feederlayers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KOSR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xenofree conditions for clinical grade hESCs culture will be useful data in future clinical studies.

  14. Micro fluidic System for Culturing and Monitoring of Neuronal Cells and Tissue

    DEFF Research Database (Denmark)

    Bakmand, Tanya; Waagepetersen, Helle S.

    The aim of this Ph.D. project was to combine experience within cell and tissue culturing, electrochemistry and microfabrication in order to develop an in vivo-like fluidic culturing platform, challenging the traditional culturing methods. The first goal was to develope a fluidic system...... for culturing of brain tissue. The second goal was to develop a sensor system with the potential for incorporation into both conventional culture systems and fluidic culturing systems. The third and final goal of this project was to develop a system for culturing of neuronal cells with the possibility...... neuronal cells on a Peptide Nano Wires (PNW) modified substrate aiming to bring conventional neuronal cultures closer to mimic the in vivo situation. The work describes both the fabrication of the culture substrates and results comparing the performance of PNWcultured neurons and conventional cultures...

  15. Metabolite profiling of microfluidic cell culture conditions for droplet based screening

    DEFF Research Database (Denmark)

    Björk, Sara M.; Sjoström, Staffan L.; Svahn, Helene Andersson

    2015-01-01

    We investigate the impact of droplet culture conditions on cell metabolic state by determining key metabolite concentrations in S. cerevisiae cultures in different microfluidic droplet culture formats. Control of culture conditions is critical for single cell/clone screening in droplets......, such as directed evolution of yeast, as cell metabolic state directly affects production yields from cell factories. Here, we analyze glucose, pyruvate, ethanol, and glycerol, central metabolites in yeast glucose dissimilation to establish culture formats for screening of respiring as well as fermenting yeast...... limited cultures, whereas the metabolite profiles of cells cultured in the alternative wide tube droplet incubation format resemble those from aerobic culture. Furthermore, we demonstrate retained droplet stability and size in the new better oxygenated droplet incubation format....

  16. Defining process design space for monoclonal antibody cell culture.

    Science.gov (United States)

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

    2010-08-15

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

  17. Culture of human mesenchymal stem cells using a candidate pharmaceutical grade xeno-free cell culture supplement derived from industrial human plasma pools.

    Science.gov (United States)

    Díez, José M; Bauman, Ewa; Gajardo, Rodrigo; Jorquera, Juan I

    2015-03-13

    Fetal bovine serum (FBS) is an animal product used as a medium supplement. The animal origin of FBS is a concern if cultured stem cells are to be utilized for human cell therapy. Therefore, a substitute for FBS is desirable. In this study, an industrial, xeno-free, pharmaceutical-grade supplement for cell culture (SCC) under development at Grifols was tested for growth of human mesenchymal stem cells (hMSCs), cell characterization, and differentiation capacity. SCC is a freeze-dried product obtained through cold-ethanol fractionation of industrial human plasma pools from healthy donors. Bone marrow-derived hMSC cell lines were obtained from two commercial suppliers. Cell growth was evaluated by culturing hMSCs with commercial media or media supplemented with SCC or FBS. Cell viability and cell yield were assessed with an automated cell counter. Cell surface markers were studied by indirect immunofluorescence assay. Cells were cultured then differentiated into adipocytes, chondrocytes, osteoblasts, and neurons, as assessed by specific staining and microscopy observation. SCC supported the growth of commercial hMSCs. Starting from the same number of seeded cells in two consecutive passages of culture with medium supplemented with SCC, hMSC yield and cell population doubling time were equivalent to the values obtained with the commercial medium and was consistent among lots. The viability of hMSCs was higher than 90%, while maintaining the characteristic phenotype of undifferentiated hMSCs (positive for CD29, CD44, CD90, CD105, CD146, CD166 and Stro-1; negative for CD14 and CD19). Cultured hMSCs maintained the potential for differentiation into adipocytes, chondrocytes, osteoblasts, and neurons. The tested human plasma-derived SCC sustains the adequate growth of hMSCs, while preserving their differentiation capacity. SCC can be a potential candidate for cell culture supplement in advanced cell therapies.

  18. Efficient Culture of Human Naive and Memory B Cells for Use as APCs.

    Science.gov (United States)

    Su, Kuei-Ying; Watanabe, Akiko; Yeh, Chen-Hao; Kelsoe, Garnett; Kuraoka, Masayuki

    2016-11-15

    The ability to culture and expand B cells in vitro has become a useful tool for studying human immunity. A limitation of current methods for human B cell culture is the capacity to support mature B cell proliferation. We developed a culture method to support the efficient activation and proliferation of naive and memory human B cells. This culture supports extensive B cell proliferation, with ∼103-fold increases following 8 d in culture and 106-fold increases when cultures are split and cultured for 8 more days. In culture, a significant fraction of naive B cells undergo isotype switching and differentiate into plasmacytes. Culture-derived (CD) B cells are readily cryopreserved and, when recovered, retain their ability to proliferate and differentiate. Significantly, proliferating CD B cells express high levels of MHC class II, CD80, and CD86. CD B cells act as APCs and present alloantigens and microbial Ags to T cells. We are able to activate and expand Ag-specific memory B cells; these cultured cells are highly effective in presenting Ag to T cells. We characterized the TCR repertoire of rare Ag-specific CD4+ T cells that proliferated in response to tetanus toxoid (TT) presented by autologous CD B cells. TCR Vβ usage by TT-activated CD4+ T cells differs from resting and unspecifically activated CD4+ T cells. Moreover, we found that TT-specific TCR Vβ usage by CD4+ T cells was substantially different between donors. This culture method provides a platform for studying the BCR and TCR repertoires within a single individual. Copyright © 2016 by The American Association of Immunologists, Inc.

  19. Studies of baby hamster kidney natural cell aggregation in suspended batch cultures.

    Science.gov (United States)

    Moreira, J L; Alves, P M; Rodrigues, J M; Cruz, P E; Aunins, J G; Carrondo, M J

    1994-11-30

    Microcarrier cultures of animal cells of industrial relevance are known to shed aggregates into the suspension phase. For a BHK cell line, which is known to be prone to aggregate naturally, microcarrier and aggregate forms of culture are compared in spinner culture. In microcarrier cultures, it is shown that increasing initial microcarrier concentration yields decreasing concentration of smaller aggregates in suspension; roughly equivalent concentrations of total cells and single cells in suspension are obtained. In the absence of Cytodex 3, aggregate final size is hydrodynamically controlled in batch and semicontinuous suspension culture. Rate of agitation is the main variable controlling aggregate size in batch cultures. The range of agitation rates studied (20 to 70 rpm in 250 mL spinner flasks) produced aggregates with maximum sizes of 200 microns. Necrotic centers were not observed; this was confirmed by Trypan blue viability measurements after mechanical dissociation of aggregates and also by the constant productivity obtained from different aggregate sizes. Comparing aggregate and microcarrier culture conditions, it is shown that at 100 rpm maximum total cell concentration is larger in the absence of microcarriers; dead cell concentrations, most of which exist in suspension, are slightly larger in microcarrier culture. Total viable cell concentrations in aggregate, hydrodynamically controlled culture, are almost one order of magnitude higher than in microcarrier cultures. These results suggest that there might be advantages in using aggregate cultures under hydrodynamic control of aggregate size in lieu of microcarrier cultures for naturally aggregating cell lines.

  20. Animal cell cultures in microsporidial research: their general roles and their specific use for fish microsporidia

    OpenAIRE

    Monaghan, S. Richelle; Michael L. Kent; Watral, Virginia G.; Kaufman, R. John; Lee, Lucy E. J.; Bols, Niels C

    2009-01-01

    The use of animal cell cultures as tools for studying the microsporidia of insect and mammals is briefly reviewed, along with an in depth review of the literature on using fish cell cultures to study the microsporidia of fish. Fish cell cultures have been used less often but have had some successes. Very short-term primary cultures have been used to show how microsporidia spores can modulate the activities of phagocytes. The most successful microsporidia/fish cell culture system has been rela...

  1. Cellular Microenvironment Dictates Androgen Production by Murine Fetal Leydig Cells in Primary Culture1

    Science.gov (United States)

    Carney, Colleen M.; Muszynski, Jessica L.; Strotman, Lindsay N.; Lewis, Samantha R.; O'Connell, Rachel L.; Beebe, David J.; Theberge, Ashleigh B.; Jorgensen, Joan S.

    2014-01-01

    ABSTRACT Despite the fact that fetal Leydig cells are recognized as the primary source of androgens in male embryos, the mechanisms by which steroidogenesis occurs within the developing testis remain unclear. A genetic approach was used to visualize and isolate fetal Leydig cells from remaining cells within developing mouse testes. Cyp11a1-Cre mice were bred to mT/mG dual reporter mice to target membrane-tagged enhanced green fluorescent protein (GFP) within steroidogenic cells, whereas other cells expressed membrane-tagged tandem-dimer tomato red. Fetal Leydig cell identity was validated using double-labeled immunohistochemistry against GFP and the steroidogenic enzyme 3beta-HSD, and cells were successfully isolated as indicated by qPCR results from sorted cell populations. Because fetal Leydig cells must collaborate with neighboring cells to synthesize testosterone, we hypothesized that the fetal Leydig cell microenvironment defined their capacity for androgen production. Microfluidic culture devices were used to measure androstenedione and testosterone production of fetal Leydig cells that were cultured in cell-cell contact within a mixed population, were isolated but remained in medium contact via compartmentalized co-culture with other testicular cells, or were isolated and cultured alone. Results showed that fetal Leydig cells maintained their identity and steroidogenic activity for 3–5 days in primary culture. Microenvironment dictated proficiency of testosterone production. As expected, fetal Leydig cells produced androstenedione but not testosterone when cultured in isolation. More testosterone accumulated in medium from mixed cultures than from compartmentalized co-cultures initially; however, co-cultures maintained testosterone synthesis for a longer time. These data suggest that a combination of cell-cell contact and soluble factors constitute the ideal microenvironment for fetal Leydig cell activity in primary culture. PMID:25143354

  2. Cellular microenvironment dictates androgen production by murine fetal Leydig cells in primary culture.

    Science.gov (United States)

    Carney, Colleen M; Muszynski, Jessica L; Strotman, Lindsay N; Lewis, Samantha R; O'Connell, Rachel L; Beebe, David J; Theberge, Ashleigh B; Jorgensen, Joan S

    2014-10-01

    Despite the fact that fetal Leydig cells are recognized as the primary source of androgens in male embryos, the mechanisms by which steroidogenesis occurs within the developing testis remain unclear. A genetic approach was used to visualize and isolate fetal Leydig cells from remaining cells within developing mouse testes. Cyp11a1-Cre mice were bred to mT/mG dual reporter mice to target membrane-tagged enhanced green fluorescent protein (GFP) within steroidogenic cells, whereas other cells expressed membrane-tagged tandem-dimer tomato red. Fetal Leydig cell identity was validated using double-labeled immunohistochemistry against GFP and the steroidogenic enzyme 3beta-HSD, and cells were successfully isolated as indicated by qPCR results from sorted cell populations. Because fetal Leydig cells must collaborate with neighboring cells to synthesize testosterone, we hypothesized that the fetal Leydig cell microenvironment defined their capacity for androgen production. Microfluidic culture devices were used to measure androstenedione and testosterone production of fetal Leydig cells that were cultured in cell-cell contact within a mixed population, were isolated but remained in medium contact via compartmentalized co-culture with other testicular cells, or were isolated and cultured alone. Results showed that fetal Leydig cells maintained their identity and steroidogenic activity for 3-5 days in primary culture. Microenvironment dictated proficiency of testosterone production. As expected, fetal Leydig cells produced androstenedione but not testosterone when cultured in isolation. More testosterone accumulated in medium from mixed cultures than from compartmentalized co-cultures initially; however, co-cultures maintained testosterone synthesis for a longer time. These data suggest that a combination of cell-cell contact and soluble factors constitute the ideal microenvironment for fetal Leydig cell activity in primary culture. © 2014 by the Society for the Study of

  3. Cellular Cytotoxicity of Antiglaucoma Drugs in Cultured Corneal Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Kwou-Yeung Wu

    2007-03-01

    Full Text Available In this study, the various antiglaucoma drugs including betaxolol, timolol, levobunolol, carteolol, brimonidine, dipivefrin, dorzolamide, brinzolamide, latanoprost, unoprostone, and pilocarpine were used to investigate the effects of cellular cytotoxicity in cultured bovine corneal endothelial cells. After exposure to the drugs in three dilutions, 1/100, 1/1,000, and 1/10,000, for 100 minutes, cells were estimated based on the release assay of lactate dehydrogenase (LDH enzyme. It was found that cellular LDH was significantly released in the medium only at 1/100th dilution of betaxolol, brimonidine, dorzolamide, dipivefrin, latanoprost and unoprostone to 130%, 123%, 145%, 157%, 128% and 237%, respectively, compared with controls upon exposure to drugs for 100 minutes. Moreover, benzalkonium chloride preservative at the concentrations ranging from 0.001 to 0.00001mg/mL did not affect cellular LDH release in bovine corneal endothelial cells. These results indicate that high concentrations of antiglaucoma drugs may induce cytotoxicity in corneal endothelial cells.

  4. Azo-polysiloxanes as new supports for cell cultures

    Energy Technology Data Exchange (ETDEWEB)

    Hurduc, Nicolae, E-mail: nhurduc@ch.tuiasi.ro [“Gheorghe Asachi” Technical University of Iasi, Department of Natural and Synthetic Polymers, Prof. Dimitrie, Mangeron Street, 73, 700050-Iasi (Romania); Macovei, Alina [Institute of Biochemistry of the Romanian Academy, Department of Viral Glycoproteins, Splaiul Independentei 296, Sector 6, 060041-Bucuresti (Romania); Paius, Cristina; Raicu, Alina [“Gheorghe Asachi” Technical University of Iasi, Department of Natural and Synthetic Polymers, Prof. Dimitrie, Mangeron Street, 73, 700050-Iasi (Romania); Moleavin, Ioana [CEA, LIST Saclay, Laboratoire Capteurs et Architectures Électroniques, F-91191 Gif-sur-Yvette, Cedex (France); Branza-Nichita, Norica, E-mail: nichita@biochim.ro [Institute of Biochemistry of the Romanian Academy, Department of Viral Glycoproteins, Splaiul Independentei 296, Sector 6, 060041-Bucuresti (Romania); Hamel, Matthieu [CEA, LIST Saclay, Laboratoire Capteurs et Architectures Électroniques, F-91191 Gif-sur-Yvette, Cedex (France); Rocha, Licinio, E-mail: Licinio.ROCHA@cea.fr [CEA, LIST Saclay, Laboratoire Capteurs et Architectures Électroniques, F-91191 Gif-sur-Yvette, Cedex (France)

    2013-05-01

    The paper introduces a new class of materials with azo-polysiloxanic structure bearing the property to generate nano-structured surfaces by laser irradiation. The ability to modulate the optical response of the film, through a modification of the polymer chemical structure, has been investigated. The azo-materials were tested for their ability to support cell adhesion and growth, with very promising results. A future use of these materials as growth support in cell cultures is of great interest, due to an easy, one step-method to generate the surface relief grating and to the possibility to introduce a large range of chemical modifications due to the presence of the chlorobenzyl groups in the polymeric side-chain. - Graphical abstract: Cell development on a nano-structured surface obtained from an azo-polysiloxanic film. Highlights: ► New azo-polysiloxanic films for biological applications were reported. ► Nanostructured surfaces with controllable geometry are obtained by laser irradiation. ► Cells are very sensitive to the chemical and physical properties of the polymeric substrate.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  7. CELL SHAPE AND HEXOSE TRANSPORT IN NORMAL AND VIRUS-TRANSFORMED CELLS IN CULTURE

    Energy Technology Data Exchange (ETDEWEB)

    Bissell, M.J.; Farson, D.; Tung, A.S.C.

    1976-07-01

    The rate of hexose transport was compared in normal and virus-transformed cells on a monolayer and in suspension. It was shown that: (1) Both trypsin-removed cells and those suspended for an additional day in methyl cellulose had decreased rates of transport and lower available water space when compared with cells on a monolayer. Thus, cell shape affects the overall rate of hexose transport, especially at higher sugar concentrations. (2) Even in suspension, the initial transport rates remained higher in transformed cells with reference to normal cells. Scanning electron micrographs of normal and transformed chick cells revealed morphological differences only in the flat state. This indicates that the increased rate of hexose transport after transformation is not due to a difference in the shape of these cells on a monolayer. The relation between the geometry of cells, transport rates, and growth regulation is undoubtedly very complex, and our knowledge of these relationships is still very elementary. In a recent review on the influence of geometry on control of cell growth, Folkman and Greenspan (1) pointed out that the permeability of cells in a flat versus a spherical state may indeed be very different. The growth properties of cells on a surface and in suspension have been compared often (1-5). However, with one exception. little is known about the changes in transport properties when cell shape is changed. Foster and Pardee (6) demonstrated that the active transport of a-aminoisobutyric acid was reduced 2.5 times in suspension cultures of Chinese hamster cells with respect to the cells grown on a coverslip. They attributed this to the smaller surface area of suspended cells. While it is not clear why active transport should be dependent on the surface area available, it is possible that once the cells assume a spherical configuration, the carrier proteins are redistributed in such a way as to make them less accessible to the substrate. What happens to

  8. Culture conditions defining glioblastoma cells behavior: what is the impact for novel discoveries?

    Science.gov (United States)

    Ledur, Pítia Flores; Onzi, Giovana Ravizzoni; Zong, Hui; Lenz, Guido

    2017-09-15

    In cancer research, the use of established cell lines has gradually been replaced by primary cell cultures due to their better representation of in vivo cancer cell behaviors. However, a major challenge with primary culture involves the finding of growth conditions that minimize alterations in the biological state of the cells. To ensure reproducibility and translational potentials for research findings, culture conditions need to be chosen so that the cell population in culture best mimics tumor cells in vivo . Glioblastoma (GBM) is one of the most aggressive and heterogeneous tumor types and the GBM research field would certainly benefit from culture conditions that could maintain the original plethora of phenotype of the cells. Here, we review culture media and supplementation options for GBM cultures, the rationale behind their use, and how much those choices affect drug-screening outcomes. We provide an overview of 120 papers that use primary GBM cultures and discuss the current predominant conditions. We also show important primary research data indicating that "mis-cultured" glioma cells can acquire unnatural drug sensitivity, which would have devastating effects for clinical translations. Finally, we propose the concurrent test of four culture conditions to minimize the loss of cell coverage in culture.

  9. Influence of co-culture on osteogenesis and angiogenesis of bone marrow mesenchymal stem cells and aortic endothelial cells.

    Science.gov (United States)

    Gurel Pekozer, Gorke; Torun Kose, Gamze; Hasirci, Vasif

    2016-11-01

    Co-culture of bone forming cells and endothelial cells to induce pre-vascularization is one of the strategies used to solve the insufficient vascularization problem in bone tissue engineering attempts. In the study, primary cells isolated from 2 different tissues of the same animal, rat bone marrow stem cells (RBMSCs) and rat aortic endothelial cells (RAECs) were co-cultured to study the effects of co-culturing on both osteogenesis and angiogenesis. The formation of tube like structure in 2D culture was observed for the first time in the literature by the co-culture of primary cells from the same animal and also osteogenesis and angiogenesis were investigated at the same time by using this co-culture system. Co-cultured cells mineralized and formed microvasculature beginning from 14days of incubation. After 28days of incubation in the osteogenic medium, expression of osteogenic genes in co-cultures was significantly upregulated compared to RBMSCs cultured alone. These results suggest that the co-culture of endothelial cells with mesenchymal stem cells induces both osteogenesis and angiogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. The usefulness of three-dimensional cell culture in induction of cancer stem cells from esophageal squamous cell carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Fujiwara, Daisuke [Department of Esophageal and Gastroenterological Surgery, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Kato, Kazunori, E-mail: kzkatou@juntendo.ac.jp [Department of Biomedical Engineering, Toyo University, 2100 Kujirai, Kawagoe, Saitama 350-8585 (Japan); Department of Atopy Research Center, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Nohara, Shigeo; Iwanuma, Yoshimi; Kajiyama, Yoshiaki [Department of Esophageal and Gastroenterological Surgery, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan)

    2013-05-17

    Highlights: •Spheroids were created from esophageal carcinoma cells using NanoCulture® Plates. •The proportion of strongly ALDH-positive cells increased in 3-D culture. •Expression of cancer stem cell-related genes was enhanced in 3-D culture. •CA-9 expression was enhanced, suggesting hypoxia had been induced in 3-D culture. •Drug resistance was increased. 3-D culture is useful for inducing cancer stem cells. -- Abstract: In recent years, research on resistance to chemotherapy and radiotherapy in cancer treatment has come under the spotlight, and researchers have also begun investigating the relationship between resistance and cancer stem cells. Cancer stem cells are assumed to be present in esophageal cancer, but experimental methods for identification and culture of these cells have not yet been established. To solve this problem, we created spheroids using a NanoCulture® Plate (NCP) for 3-dimensional (3-D) cell culture, which was designed as a means for experimentally reproducing the 3-D structures found in the body. We investigated the potential for induction of cancer stem cells from esophageal cancer cells. Using flow cytometry we analyzed the expression of surface antigen markers CD44, CD133, CD338 (ABCG2), CD318 (CDCP1), and CD326 (EpCAM), which are known cancer stem cell markers. None of these surface antigen markers showed enhanced expression in 3-D cultured cells. We then analyzed aldehyde dehydrogenase (ALDH) enzymatic activity using the ALDEFLUOR reagent, which can identify immature cells such as stem cells and precursor cells. 3-D-cultured cells were strongly positive for ALDH enzyme activity. We also analyzed the expression of the stem cell-related genes Sox-2, Nanog, Oct3/4, and Lin28 using RT-PCR. Expression of Sox-2, Nanog, and Lin28 was enhanced. Analysis of expression of the hypoxic surface antigen marker carbonic anhydrase-9 (CA-9), which is an indicator of cancer stem cell induction and maintenance, revealed that CA-9 expression

  11. Electric pulses to prepare feeder cells for sustaining and culturing of undifferentiated embryonic stem cells.

    Science.gov (United States)

    Browning, Lauren M; Huang, Tao; Xu, Xiao-Hong Nancy

    2010-06-01

    Current challenges in embryonic-stem cell (ESC) research include the inability of sustaining and culturing of undifferentiated ESCs over time. Growth-arrested feeder cells are essential to the culture and sustaining of undifferentiated ESCs, and they are currently prepared using gamma-radiation and chemical inactivation. Both techniques have severe limitations. In this study, we developed a new, simple and effective technique (pulsed electric fields, PEFs) to produce viable growth-arrested cells (RTS34st) and used them as high-quality feeder cells to culture and sustain undifferentiated zebrafish ESCs over time. The cells were exposed to 25 sequential 10-ns electric pulses (10nsEPs) of 25, 40 and 150 kV/cm with 1-s pulse interval, or 2 sequential 50-mus electric pulses (50microsEPs) of 2.83, 1.78 and 0.78 kV/cm with 5-s pulse interval, respectively. We found that the cellular effects of PEFs depended directly upon the duration, number and electric field strength of the pulses, showing the feasibility of tuning them to produce various types of growth-arrested cells for culturing undifferentiated ESCs. Both 10nsEPs of 40 kV/cm produced by a 10nsEP generator and 50microsEPs of 1.78 kV/cm provided by inexpensive and widely available conventional electroporators, generated high-quality growth-arrested feeder cells for proliferation of undifferentiated ESCs over time. PEFs can therefore be used to replace radiation and chemical inactivation methods for preparation of growth-arrested feeder cells for advancing ESC research.

  12. Standardized cell sources and recommendations for good cell culture practices in genotoxicity testing.

    Science.gov (United States)

    Lorge, E; Moore, M M; Clements, J; O'Donovan, M; Fellows, M D; Honma, M; Kohara, A; Galloway, S; Armstrong, M J; Thybaud, V; Gollapudi, B; Aardema, M J; Tanir, J Y

    2016-10-01

    Good cell culture practice and characterization of the cell lines used are of critical importance in in vitro genotoxicity testing. The objective of this initiative was to make continuously available stocks of the characterized isolates of the most frequently used mammalian cell lines in genotoxicity testing anywhere in the world ('IVGT' cell lines). This project was organized under the auspices of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing. First, cell isolates were identified that are as close as possible to the isolate described in the initial publications reporting their use in genotoxicity testing. The depositors of these cell lines managed their characterization and their expansion for preparing continuously available stocks of these cells that are stored at the European Collection of Cell Cultures (ECACC, UK) and the Japanese Collection of Research Bioresources (JCRB, Japan). This publication describes how the four 'IVGT' cell lines, i.e. L5178Y TK+/- 3.7.2C, TK6, CHO-WBL and CHL/IU, were prepared for deposit at the ECACC and JCRB cell banks. Recommendations for handling these cell lines and monitoring their characteristics are also described. The growth characteristics of these cell lines (growth rates and cell cycles), their identity (karyotypes and genetic status) and ranges of background frequencies of select endpoints are also reported to help in the routine practice of genotoxicity testing using these cell lines. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  13. Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids.

    Science.gov (United States)

    Bonazza, Camila; Andrade, Sheila Siqueira; Sumikawa, Joana Tomomi; Batista, Fabrício Pereira; Paredes-Gamero, Edgar J; Girão, Manoel J B C; Oliva, Maria Luiza V; Castro, Rodrigo Aquino

    2016-01-01

    Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity.

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

    Science.gov (United States)

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

    2015-08-01

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

  15. LLC-PK(1) cells maintained in a new perfusion cell culture system exhibit an improved oxidative metabolism

    NARCIS (Netherlands)

    Felder, Edward; Jennings, Paul; Seppi, Thomas; Pfaller, Walter

    2002-01-01

    Cultured renal proximal tubule cells dedifferentiate from an oxidative metabolism to high rates of glycolysis over time. There are many reasons why cells in culture dedifferentiate, not least being a lack of homogenous nutrient supply and poor oxygenation. To this end we have developed a new cell

  16. Development and application of computer software for cell culture laboratory management.

    Science.gov (United States)

    Selznick, S H; Thatcher, M L; Brown, K S; Haussler, C A

    2001-01-01

    Prototype computer software for a Cell Culture Laboratory Management System (CCLMS) has been developed to relieve cell culture specialists of the burden of manual recordkeeping. Conventional data archives in cell culture laboratories are prone to error and expensive to maintain. The reliance upon cell culture to provide models for biochemical and molecular biological research serves to magnify errors at great expense. The CCLMS prototype encapsulates a modular software application that manages the many aspects of cell culture laboratory recordkeeping. A transaction-based database stores detailed information on subcultures, freezes and thaws, prints waterproof labels for culture vessels, and provides for immediate historical trace-back of any cultured cell line. Linked database files store information specific to an individual culture flask while removing redundancy between similar groups of flasks. A frozen cell log maintains locations of all vials within any type of cryogenic storage unit, locates spaces for newly frozen cell lines, and generates alphabetical or numerical reports. Finally, modules for maintaining cell counts, user records, and culture vessel specifications to support a comprehensive automation process are incorporated within this software. The developed CCLMS prototype has been demonstrated to be an adaptable, reliable tool for improving training, efficiency, and historical rigor for two independent cell culture facilities.

  17. Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells

    Science.gov (United States)

    Schmelzer, Eva; Over, Patrick; Nettleship, Ian; Gerlach, Joerg C.

    2016-01-01

    Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications. PMID:27403430

  18. [Cell culture of middle-ear epithelium of the guinea pig--histochemical localization of mitochondrial enzymatic activities in cultured cells].

    Science.gov (United States)

    Takeno, S

    1990-01-01

    Primary cell culture system from middle-ear epithelium of the guinea pig was established in defined condition. Mucosal cells were dispersed with enzymatic procedure and over 90% of the cell viability was obtained. Collagen gel and fibronectin coated Thermanox plate were used as culture substrates, and cultured cells on both materials formed confluent epithelial linings. Histochemical localization of succinate dehydrogenase, cytochrome oxidase and adenosine triphosphatase in mitochondria were examined. Cultured ciliated cells and some non-ciliated cells with numerous microvilli showed strong activities of succinate dehydrogenase and cytochrome oxidase. Also in vivo, normal ciliated epithelium near the eustachian tube in the middle-ear cavity of the guinea pig revealed strong mitochondrial metabolic activities. We concluded that this system would be useful for the study of cellular multiplication and differentiation systems of the middle-ear epithelium.

  19. Identification of 13 new mutations in the vasopressin-neurophysin II gene in 17 kindreds with familial autosomal dominant neurohypophyseal diabetes insipidus

    Energy Technology Data Exchange (ETDEWEB)

    Rittig, S.; Siggaard, C.; Pedersen, E.B. [Aahus Univ. Hospital and Faculty of Health Sciences (Denmark)] [and others

    1996-01-01

    Familial neurohypophyseal diabetes insipidus (FNDI) is an autosomal dominant disorder characterized by progressive postnatal deficiency of arginine vasopressin as a result of mutation in the gene that encodes the hormone. To determine the extent of mutations in the coding region that produce the phenotype, we studied members of 17 unrelated kindreds with the disorder. We sequenced all 3 exons of the gene by using a rapid, direct dye-terminator method and found the causative mutation in each kindred. In four kindreds, the mutations were each identical to mutations described in other affected families. In the other 13 kindreds each mutation was unique. There were two missense mutations that altered the cleavage region of the signal peptide, seven missense mutations in exon 2, which codes for the conserved portion of the protein, one nonsense mutation in exon 2, and three nonsense mutations in exon 3. These findings, together with the clinical features of FNDI, suggest that each of the mutations exerts an effect by directing the production of a pre-prohormone that cannot be folded, processed, or degraded properly and eventually destroys vasopressinergic neurons. 63 refs., 5 figs., 6 tabs.

  20. Glucose Transport in Cultured Animal Cells: An Exercise for the Undergraduate Cell Biology Laboratory

    Science.gov (United States)

    Ledbetter, Mary Lee S; Lippert, Malcolm J

    2002-01-01

    Membrane transport is a fundamental concept that undergraduate students of cell biology understand better with laboratory experience. Formal teaching exercises commonly used to illustrate this concept are unbiological, qualitative, or intricate and time consuming to prepare. We have developed an exercise that uses uptake of radiolabeled nutrient analogues by attachment-dependent animal cells cultured on multiwell trays. This system can readily be manipulated within a typical 3-h laboratory period to yield reproducible, biologically relevant, quantitative data regarding key aspects of membrane transport. Each 24-well tray of cultures allows a group of two to four students to compare eight conditions in triplicate. If different groups of students test different conditions or different types of cells, data can be shared for an even broader experience. The exercise is also readily adaptable for open-ended student projects. Here we illustrate the exercise measuring uptake of the nonmetabolizable glucose analogue [3H]-2-deoxy-d-glucose. Students successfully tested the effects of competing sugars, putative inhibitors of the GLUT1 transporter, and changes in cell physiology that might be expected to affect glucose transport in epithelial cells and fibroblasts. In this exercise students find the nutritional and medical implications of glucose transport and its regulation intriguing. They also learn to handle radioisotopes and cultured cells. PMID:12459793

  1. Flow perfusion culture of human mesenchymal stem cells on coralline hydroxyapatite scaffolds with various pore sizes

    DEFF Research Database (Denmark)

    Bjerre, Lea; Bünger, Cody; Baatrup, Anette

    2011-01-01

    of this study was to obtain a clinically relevant substitute size using a direct perfusion culture system. Human bone marrowderived mesenchymal stem cells were seeded on coralline hydroxyapatite scaffolds with 200 μm or 500 μm pores, and resulting constructs were cultured in a perfusion bioreactor or in static...... μm ones. Adhesion and proliferation of the cells was seen on both scaffold sizes, but the vitality and morphology of cells changed unfavorably during perfusion culture. In contrast to previous studies using spinner flask that show increased cellularity and osteogenic properties of cells when cultured...... dynamically, the perfusion culture in our study did not enhance the osteogenic properties of cell/scaffold constructs. The statically cultured constructs showed increasing cell numbers and abundant osteogenic differentiation probably because of weak initial cell adhesion due to the surface morphology...

  2. Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

    Science.gov (United States)

    A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus th...

  3. Control of cell growth on 3D-printed cell culture platforms for tissue engineering.

    Science.gov (United States)

    Tan, Zhikai; Liu, Tong; Zhong, Juchang; Yang, Yikun; Tan, Weihong

    2017-12-01

    Biocompatible tissue growth has excellent prospects for tissue engineering. These tissues are built over scaffolds, which can influence aspects such as cell adhesion, proliferation rate, morphology, and differentiation. However, the ideal 3D biological structure has not been developed yet. Here, we applied the electro-hydrodynamic jet (E-jet) 3D printing technology using poly-(lactic-co-glycolic acid, PLGA) solution to print varied culture platforms for engineered tissue structures. The effects of different parameters (electrical voltage, plotting speed, and needle sizes) on the outcome were investigated. We compared the biological compatibility of the 3D printed culture platforms with that of random fibers. Finally, we used the 3D-printed PLGA platforms to culture fibroblasts, the main cellular components of loose connective tissue. The results show that the E-jet printed platforms could guide and improve cell growth. These highly aligned fibers were able to support cellular alignment and proliferation. Cell angle was consistent with the direction of the fibers, and cells cultured on these fibers showed a much faster migration, potentially enhancing wound healing performance. Thus, the potential of this technology for 3D biological printing is large. This process can be used to grow biological scaffolds for the engineering of tissues. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3281-3292, 2017. © 2017 Wiley Periodicals, Inc.

  4. Methods for animal satellite cell culture under a variety of conditions.

    Science.gov (United States)

    Burton, N M; Vierck, J; Krabbenhoft, L; Bryne, K; Dodson, M V

    2000-03-01

    Primary and clonal culture systems have been devised and refined for animal-derived satellite cells. Satellite cell (SC) culture development includes efficient cell isolation techniques, establishment of effective plating and growth conditions, formulation of media requirements and thorough evaluation of experimental limitations. As the field of muscle cell culture has expanded, the number of animal species from which satellite cells have been isolated has increased. The focus of this paper is to compare and contrast SC culture conditions presently used by a variety of researchers and to introduce a new source of SC from wapiti (elk).

  5. Iridovirus infection of cell cultures from the Diaprepes root weevil, Diaprepes abbreviatus

    Directory of Open Access Journals (Sweden)

    W.B. Hunter

    2003-12-01

    Full Text Available We here report the development and viral infection of a Diaprepes root weevil cell culture. Embryonic tissues of the root weevil were used to establish cell cultures for use in screening viral pathogens as potential biological control agents. Tissues were seeded into a prepared solution of insect medium and kept at a temperature of 24°C. The cell culture had primarily fibroblast-like morphology with some epithelial monolayers. Root weevil cells were successfully infected in vitro with a known insect virus, Invertebrate Iridescent Virus 6. Potential uses of insect cell cultures and insect viruses are discussed.

  6. Effect of cell shape change on the function and differentiation of rabbit mammary cells in culture.

    Science.gov (United States)

    Haeuptle, M T; Suard, Y L; Bogenmann, E; Reggio, H; Racine, L; Kraehenbuhl, J P

    1983-05-01

    We examined the role of cell shape, cytodifferentiation, and tissue topography on the induction and maintenance of functional differentiation in rabbit mammary cells grown as primary cultures on two-dimensional collagen surfaces or in three-dimensional collagen matrices. Mammary glands from mid-pregnant rabbits were dissociated into single cells, and epithelial cells were enriched by isopycnic centrifugation. Small spheroids of epithelial cells (approximately 50 cells) that formed on a rotary shaker were plated on or embedded in collagen gels. The cells were cultured for 1 d in serum-containing medium and then for up to 25 d in chemically defined medium. In some experiments, epithelial monolayers on gels were mechanically freed from the dishes on day 2 or 5. These gels retracted and formed floating collagen gels. On attached collagen gels, flat monolayers of a single cell type developed within a few days. The cells synthesized DNA until the achievement of confluence but did not accumulate milk proteins. No morphological changes were induced by prolactin (PRL). On floating gels, two cell types appeared in the absence of cell proliferation. The cells in direct contact with the medium became cuboidal and developed intracellular organelles typical of secretory cells. PRL-induced lipogenesis, resulting in large fat droplets filling the apical cytoplasm and accumulation of casein and alpha-lactalbumin in vesicles surrounding the fat droplets. We detected tranferrin in the presence or absence of PRL intracellularly in small vesicles but also in the collagen matrix in contact with the cell layer. The second cell type, rich in microfilaments and reminiscent of the myoepithelial cells, was situated between the secretory cell layer and the collagen matrix. In embedding gels, the cells formed hollow ductlike structures, which grew continuously in size. Secretory cells formed typical lumina distended by secretory products. We found few microfilament-rich cells in contact with

  7. Cell Culture Isolation of Piscine Nodavirus (Betanodavirus) in Fish-Rearing Seawater

    National Research Council Canada - National Science Library

    Nishi, Shinnosuke; Yamashita, Hirofumi; Kawato, Yasuhiko; Nakai, Toshihiro

    2016-01-01

    ...) in a variety of cultured fish species, particularly marine fish. In the present study, we developed a sensitive method for cell culture isolation of the virus from seawater and applied the method to a spontaneous fish-rearing environment...

  8. Modeling spatial distribution of oxygen in 3d culture of islet beta-cells.

    Science.gov (United States)

    McReynolds, John; Wen, Yu; Li, Xiaofei; Guan, Jianjun; Jin, Sha

    2017-01-01

    Three-dimensional (3D) scaffold culture of pancreatic β-cell has been proven to be able to better mimic physiological conditions in the body. However, one critical issue with culturing pancreatic β-cells is that β-cells consume large amounts of oxygen, and hence insufficient oxygen supply in the culture leads to loss of β-cell mass and functions. This becomes more significant when cells are cultured in a 3D scaffold. In this study, in order to understand the effect of oxygen tension inside a cell-laden collagen culture on β-cell proliferation, a culture model with encapsulation of an oxygen-generator was established. The oxygen-generator was made by embedding hydrogen peroxide into nontoxic polydimethylsiloxane to avoid the toxicity of a chemical reaction in the β-cell culture. To examine the effectiveness of the oxygenation enabled 3D culture, the spatial-temporal distribution of oxygen tension inside a scaffold was evaluated by a mathematical modeling approach. Our simulation results indicated that an oxygenation-aided 3D culture would augment the oxygen supply required for the β-cells. Furthermore, we identified that cell seeding density and the capacity of the oxygenator are two critical parameters in the optimization of the culture. Notably, cell-laden scaffold cultures with an in situ oxygen supply significantly improved the β-cells' biological function. These β-cells possess high insulin secretion capacity. The results obtained in this work would provide valuable information for optimizing and encouraging functional β-cell cultures. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:221-228, 2017. © 2016 American Institute of Chemical Engineers.

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

    Science.gov (United States)

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

    2015-09-01

    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.

  10. Transcriptional profiles of valvular interstitial cells cultured on tissue culture polystyrene, on 2D hydrogels, or within 3D hydrogels

    Directory of Open Access Journals (Sweden)

    Kelly M. Mabry

    2015-12-01

    Full Text Available Valvular interstitial cells (VICs actively maintain and repair heart valve tissue; however, persistent activation of VICs to a myofibroblast phenotype can lead to aortic stenosis (Chen and Simmons, 2011 [1]. To better understand and quantify how microenvironmental cues influence VIC phenotype, we compared expression profiles of VICs cultured on/in poly(ethylene glycol (PEG gels to those cultured on tissue culture polystyrene (TCPS, as well as fresh isolates. Here, we present both the raw and processed microarray data from these culture conditions. Interpretation of this data can be found in a research article entitled “Microarray analyses to quantify advantages of 2D and 3D hydrogel culture systems in maintaining the native valvular interstitial cell phenotype” (Mabry et al., 2015 [2].

  11. Culture medium refinement by dialysis for the expansion of human induced pluripotent stem cells in suspension culture.

    Science.gov (United States)

    Nath, Suman Chandra; Nagamori, Eiji; Horie, Masanobu; Kino-Oka, Masahiro

    2017-01-01

    Human induced pluripotent stem cells (hiPSCs) secrete essential autocrine factors that are removed along with toxic metabolites when the growth medium is exchanged daily. In this study, after determining the minimum inhibitory level of lactic acid for hiPSCs, a medium refining system was constructed by which toxic metabolites were removed from used culture medium and autocrine factors as well as other growth factors were recycled. Specifically, about 87 % of the basic fibroblast growth factor and 80 % of transforming growth factor beta 1 were retained in the refined medium after dialysis. The refined medium efficiently potentiated the proliferation of hiPS cells in adherent culture. When the refining system was used to refresh medium in suspension culture, a final cell density of (1.1 ± 0.1) × 106 cells mL-1 was obtained, with 99.5 ± 0.2 % OCT 3/4 and 78.3 ± 1.1 % TRA-1-60 expression, on day 4 of culture. These levels of expression were similar to those observed in the conventional suspension culture. With this method, culture medium refinement by dialysis was established to remove toxic metabolites, recycle autocrine factors as well as other growth factors, and reduce the use of macromolecules for the expansion of hiPSCs in suspension culture.

  12. A flexible thermoresponsive cell culture substrate for direct transfer of keratinocyte cell sheets.

    Science.gov (United States)

    Praveen, Wulligundam; Madathil, Bernadette K; Sajin Raj, R S; Kumary, T V; Anil Kumar, P R

    2017-10-25

    Most cell sheet engineering systems require a support or carrier to handle the harvested cell sheets. In this study, polyethylene terephthalate-based overhead projection transparency sheets (OHPS) were subjected to surface hydrolysis by alkali treatment to increase pliability and hydrophilicity and enable poly(N-isopropylacrylamide-co-glycidylmethacrylate) copolymer (NGMA) coating to impart thermoresponsiveness. NGMA was applied on the modified OHPS by the technique of spin coating using an indigenously designed spin coater. The spin coating had the advantage of using low volumes of the polymer and a reduced coating time. The surface chemistry and thermoresponsive coating was analyzed by Fourier transform infrared spectroscopy and water contact angle. Human keratinocyte cells were cultured on the spin coated surface and scaffold-free cell sheets were successfully harvested by simple variation of temperature. These cell sheets were found to be viable, exhibited epithelial characteristic and cell-cell contact as confirmed by positive immunostaining for ZO-1. The integrity and morphology of the cell sheet was confirmed by stereomicroscopy and E-SEM. These results highlight the potential of the NGMA spin coated modified OHPS to serve as a thermoresponsive culture surface-cum-flexible transfer tool.

  13. Pigment Cell Differentiation in Sea Urchin Blastula-Derived Primary Cell Cultures

    Science.gov (United States)

    Ageenko, Natalya V.; Kiselev, Konstantin V.; Dmitrenok, Pavel S.; Odintsova, Nelly A.

    2014-01-01

    The quinone pigments of sea urchins, specifically echinochrome and spinochromes, are known for their effective antioxidant, antibacterial, antifungal, and antitumor activities. We developed in vitro technology for inducing pigment differentiation in cell culture. The intensification of the pigment differentiation was accompanied by a simultaneous decrease in cell proliferation. The number of pigment cells was two-fold higher in the cells cultivated in the coelomic fluids of injured sea urchins than in those intact. The possible roles of the specific components of the coelomic fluids in the pigment differentiation process and the quantitative measurement of the production of naphthoquinone pigments during cultivation were examined by MALDI and electrospray ionization mass spectrometry. Echinochrome A and spinochrome E were produced by the cultivated cells of the sand dollar Scaphechinus mirabilis in all tested media, while only spinochromes were found in the cultivated cells of another sea urchin, Strongylocentrotus intermedius. The expression of genes associated with the induction of pigment differentiation was increased in cells cultivated in the presence of shikimic acid, a precursor of naphthoquinone pigments. Our results should contribute to the development of new techniques in marine biotechnology, including the generation of cell cultures producing complex bioactive compounds with therapeutic potential. PMID:24979272

  14. Polarity establishment, morphogenesis, and cultured plant cells in space

    Science.gov (United States)

    Krikorian, Abraham D.

    1989-01-01

    Plant development entails an orderly progression of cellular events both in terms of time and geometry. There is only circumstantial evidence that, in the controlled environment of the higher plant embryo sac, gravity may play a role in embryo development. It is still not known whether or not normal embryo development and differentiation in higher plants can be expected to take place reliably and efficiently in the micro g space environment. It seems essential that more attention be given to studying aspects of reproductive biology in order to be confident that plants will survive seed to seed to seed in a space environment. Until the time arrives when successive generations of plants can be grown, the best that can be done is utilize the most appropriate systems and begin, piece meal, to accumulate information on important aspects of plant reproduction. Cultured plant cells can play an important role in these activities since they can be grown so as to be morphogenetically competent, and thus can simulate those embryogenic events more usually identified with fertilized eggs in the embryo sac of the ovule in the ovary. Also, they can be manipulated with relative ease. The extreme plasticity of such demonstrably totipotent cell systems provides a means to test environmental effects such as micro g on a potentially free-running entity. The successful manipulation and management of plant cells and propagules in space also has significance for exploitation of biotechnologies in space since such systems, perforce, are an important vehicle whereby many genetic engineering manipulations are achieved.

  15. Proliferation of cultured mouse choroid plexus epithelial cells.

    Directory of Open Access Journals (Sweden)

    Basam Z Barkho

    Full Text Available The choroid plexus (ChP epithelium is a multifunctional tissue found in the ventricles of the brain. The major function of the ChP epithelium is to produce cerebrospinal fluid (CSF that bathes and nourishes the central nervous system (CNS. In addition to the CSF, ChP epithelial cells (CPECs produce and secrete numerous neurotrophic factors that support brain homeostasis, such as adult hippocampal neurogenesis. Accordingly, damage and dysfunction to CPECs are thought to accelerate and intensify multiple disease phenotypes, and CPEC regeneration would represent a potential therapeutic approach for these diseases. However, previous reports suggest that CPECs rarely divide, although this has not been extensively studied in response to extrinsic factors. Utilizing a cell-cycle reporter mouse line and live cell imaging, we identified scratch injury and the growth factors insulin-like growth factor 1 (IGF-1 and epidermal growth factor (EGF as extrinsic cues that promote increased CPEC expansion in vitro. Furthermore, we found that IGF-1 and EGF treatment enhances scratch injury-induced proliferation. Finally, we established whole tissue explant cultures and observed that IGF-1 and EGF promote CPEC division within the intact ChP epithelium. We conclude that although CPECs normally have a slow turnover rate, they expand in response to external stimuli such as injury and/or growth factors, which provides a potential avenue for enhancing ChP function after brain injury or neurodegeneration.

  16. Aromatase activity in human skin fibroblasts grown in cell culture.

    Science.gov (United States)

    Berkovitz, G D; Brown, T R; Fujimoto, M

    1987-01-01

    Recent studies in this laboratory have described an unusual kindred in which gynecomastia resulted from abnormally elevated levels of extraglandular aromatase activity. In order to better understand the molecular mechanisms responsible for the abnormal aromatase activity in these and other patients, we explored the aromatase activity of genital skin fibroblasts. Our studies demonstrate that the kinetic parameters for aromatase in skin are similar to those of other cultured cells and suggest that skin is an important site of extraglandular aromatase activity. These cells also contain 5 alpha-reductase activity and androgen receptors and are, therefore, a model for androgen action and metabolism. For example, they provided a system for the study of the potency and specificity of the aromatase inhibitors 4-OHA and MDL 18,962. Finally, the influence of DEX on aromatase in genital skin fibroblasts differs in some important respects from the pattern of control observed in adipose tissue stromal-vascular cells. These findings suggest that investigating the molecular mechanisms for the regulation of aromatase in skin may provide unique information about the control of the enzyme.

  17. Culture models of human mammary epithelial cell transformation

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R.; Yaswen, Paul

    2000-11-10

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

  18. Metabolism of strobilurins by wheat cell suspension cultures.

    Science.gov (United States)

    Myung, Kyung; Williams, Daniel A; Xiong, Quanbo; Thornburgh, Scott

    2013-01-09

    Strobilurin fungicides are a leading class of antifungal chemicals used today in agricultural applications. Although degradation of some strobilurin fungicides has been assessed in plant residues, little information has appeared in the literature concerning the rates of metabolism of these fungicides in plants. In this study, we explored plant metabolism of three strobilurin fungicides, azoxystrobin, kresoxim-methyl, and trifloxystrobin, using wheat cell suspension cultures. Trifloxystrobin and kresoxim-methyl were completely metabolized within 24 h, whereas the metabolism of azoxystrobin was relatively slow with half-lives up to 48 h depending on specific experimental conditions. Metabolic rates of these fungicides were affected by the amounts of compound and cells added to the media. Structural analysis of metabolites of trifloxystrobin and kresoxim-methyl by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance spectroscopy (NMR) indicated