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Research Containing: Cell Culture Techniques/*methods

Simulated Microgravity Maintains the Undifferentiated State and Enhances the Neural Repair Potential of Bone Marrow Stromal Cells

by on 9 June 2015in Biology & Biotechnology No comment

Recently, regenerative medicine with bone marrow stromal cells (BMSCs) has gained significant attention for the treatment of central nervous system diseases. Here, we investigated the activity of BMSCs under simulated microgravity conditions. Mouse BMSCs (mBMSCs) were isolated from C57BL/6 mice and harvested in 1G condition. Subjects were divided into 4 groups: cultured under simulated microgravity and 1G condition in growth medium and neural differentiation medium. After 7 days of culture, the mBMSCs were used for morphological analysis, reverse transcription (RT)-polymerase chain reaction, immunostaining analysis, and grafting. Neural-induced mBMSCs cultured under 1G conditions exhibited neural differentiation, whereas those cultured under simulated microgravity did not. Moreover, under simulated microgravity conditions, mBMSCs could be cultured in an undifferentiated state. Next, we intravenously injected cells into a mouse model of cerebral contusion. Graft mBMSCs cultured under simulated microgravity exhibited greater survival in the damaged region, and the motor function of the grafted mice improved significantly. mBMSCs cultured under simulated microgravity expressed CXCR4 on their cell membrane. Our study indicates that culturing cells under simulated microgravity enhances their survival rate by maintaining an undifferentiated state of cells, making this a potentially attractive method for culturing donor cells to be used in grafting.

Related URLs:
<Go to ISI>://WOS:000290255300013

Human Hematopoietic Progenitor Cells Grow Faster Under Rotational Laminar Flows

by on 9 June 2015in Biology & Biotechnology No comment

We report significant and reproducible growth acceleration of human progenitor cells when exposed to rotational flow when compared with stationary conditions. Nonenriched CD34+ umbilical cord derived human hematopoietic progenitor cells were cultured in Petri dishes located at different radial distances with respect to the central axis of a rotating platform. Growth dynamics under 3 or 5 rpm agitation was compared against that observed under typical stationary conditions. Cells cultured at 3 or 5 rpm exhibited (a) the absence of a latency phase, (h) an increase in final cell concentrations by 54-58.5%, and (c) reduced doubling time in their exponential phase by 12-16% in comparison with stationary culture. Cells grown under rotational agitation were confirmed to remain CD34+ by PCR. These results document a significant positive effect of exposure to laminar flow fields on the growth of human hematopoietic progenitor cells. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog. 26: 1465-1473, 2010

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<Go to ISI>://WOS:000283482100031

Production of stem cells with embryonic characteristics from human umbilical cord blood

by on 9 June 2015in Biology & Biotechnology No comment

When will embryonic stem cells reach the clinic? The answer is simple not soon! To produce large quantities of homogeneous tissue for transplantation, without feeder layers, and with the appropriate recipient's immunological phenotype, is a significant scientific hindrance, although adult stem (ADS) cells provide an alternative, more ethically acceptable, source. The annual global 100 million human birth rate proposes umbilical cord blood (UCB) as the largest untouched stem cell source, with advantages of naive immune status and relatively unshortened telomere length. Here, we report the world's first reproducible production of cells expressing embryonic stem cell markers, – cord-blood-derived embryonic-like stem cells (CBEs). UCB, after elective birth by Caesarean section, has been separated by sequential immunomagnetic removal of nucleate granulocytes, erythrocytes and haemopoietic myeloid/lymphoid progenitors. After 7 days of high density culture in microflasks, (10(5) cells/ml, IMDM, FCS 10%, thrombopoietin 10 ng/ml, flt3-ligand 50 ng/ml, c-kit ligand 20 ng/ml). CBE colonies formed adherent to the substrata; these were maintained for 6 weeks, then were subcultured and continued for a minimum 13 weeks. CBEs were positive for TRA-1-60, TRA-1-81, SSEA-4, SSEA-3 and Oct-4, but not SSEA-I, indicative of restriction in the human stem cell compartment. The CBEs were also microgravity-bioreactor cultured with hepatocyte growth medium (IMDM, FCS 10%, HGF 20 ng/ml, bFGF 10 ng/ml, EGF 10 ng/ml, c-kit ligand 10 ng/ml). After 4 weeks the cells were found to express characteristic hepatic markers, cytokeratin-18, (alpha-foetoprotein and albumin. Thus, such CBEs are a viable human alternative from embryonic stem cells for stem cell research, without ethical constraint and with potential for clinical applications.

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<Go to ISI>://WOS:000231540000006

Differentiation of Mesenchymal Stem Cells towards a Nucleus Pulposus-like Phenotype Utilizing Simulated Microgravity In Vitro

by on 9 June 2015in Biology & Biotechnology No comment

Mesenchymal stem cells (MSCs) were induced into a nucleus pulposus-like phenotype utilizing simulated microgravity in vitro in order to establish a new cell-based tissue engineering treatment for intervertebral disc degeneration. For induction of a nucleus pulposus-like phenotype, MSCs were cultured in simulated microgravity in a chemically defined medium supplemented with 0 (experimental group) and 10 ng/mL (positive control group) of transforming growth factor beta 1 (TGF-beta 1). MSCs cultured under conventional condition without TGF-beta 1 served as blank control group. On the day 3 of culture, cellular proliferation was determined by WST-8 assay. Differentiation markers were evaluated by histology and reverse transcriptase-polymerase chain reaction (RT-PCR). TGF-beta 1 slightly promoted the proliferation of MSCs. The collagen and proteoglycans were detected in both groups after culture for 7 days. The accumulation of proteoglycans was markedly increased. The RT-PCR revealed that the gene expression of Sox-9, aggrecan and type. collagen, which were chondrocyte specific, was increased in MSCs cultured under simulated microgravity for 3 days. The ratio of proteoglycans/collagen in blank control group was 3.4-fold higher than positive control group, which denoted a nucleus pulposus-like phenotype differentiation. Independent, spontaneous differentiation of MSCs towards a nucleus pulposus-like phenotype in simulated microgravity occurred without addition of any external bioactive stimulators, namely factors from TGF-beta family, which were previously considered necessary.

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<Go to ISI>://WOS:000291785900012

Establishment of three-dimensional tissue-engineered bone constructs under microgravity-simulated conditions

by on 9 June 2015in Biology & Biotechnology No comment

Bone constructs have been grown in vitro with use of isolated cells, biodegradable polymer scaffolds, and bioreactors. In our work, the relationships between the composition and mechanical properties of engineered bone constructs were studied by culturing bone marrow mesenchymal stem cells (BMSCs) on ceramic bovine bone scaffolds in different environments: static flasks and dynamic culture system in rotating vessels-which was a National Aeronautics and Space Administration-recommended, ground-based, microgravity-simulating system. After 15 days of cultivation, osteogenicity was determined according to DNA and alkaline phosphatase (ALP) analysis. DNA content and ALP were higher for cells grown on dynamic culture. Subsequently, the two kinds of engineered bone constructs were selected for transplantation into Sprague-Dawley rat cranial bone defects. After 24 weeks of in vivo implantation, the engineered bone constructs under dynamic culture were found to repair the defects better, with the engineered constructs showing histologically better bone connection. Thus, this dynamic system provides a useful in vitro model to construct the functional role and effects of osteogenesis in the proliferation, differentiation, and maturation of BMSCs. These findings suggest that the hydrodynamic microgravity conditions in tissue-culture bioreactors can modulate the composition, morphology, and function of the engineered bone.

Related URLs:
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=emed9&AN=2010123942
http://sfxhosted.exlibrisgroup.com/mayo?sid=OVID:embase&id=pmid:&id=doi:10.1111%2Fj.1525-1594.2009.00761.x&issn=0160-564X&isbn=&volume=34&issue=2&spage=118&pages=118-125&date=2010&title=Artificial+Organs&atitle=Establishment+of+three-dimensional+tissue-engineered+bone+constructs+under+microgravity-simulated+conditions&aulast=Jin&pid=%3Cauthor%3EJin+F.%3C%2Fauthor%3E&%3CAN%3E2010123942%3C%2FAN%3E

Floating culture promotes the maintenance of hematopoietic stem cells

by on 9 June 2015in Biology & Biotechnology No comment

In this study we examined the effect of the specific gravity of culture medium on the frequency of hematopoietic stem cell (HSC) maintenance. We used a newly developed high-specific-gravity media. Bone marrow cells were isolated and cultured, and HSC activity was evaluated. The number of hematopoietic progenitor/stem cells was markedly higher in the medium with high specific gravity. In high-specific-gravity media, cells did not precipitate, maintenance of HSCs was increased, and there was a concomitant accumulation of beta-catenin. This novel technique for maintaining HSC populations provides an important new tool for studies in regenerative medicine. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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<Go to ISI>://WOS:000250102900017

Reconstitution of hepatic tissue architectures from fetal liver cells obtained from a three-dimensional culture with a rotating wall vessel bioreactor

by on 9 June 2015in Biology & Biotechnology No comment

Reconstitution of tissue architecture in vitro is important because it enables researchers to investigate the interactions and mutual relationships between cells and cellular signals involved in the three-dimensional (3D) construction of tissues. To date, in vitro methods for producing tissues with highly ordered structure and high levels of function have met with limited success although a variety of 3D culture systems have been investigated. In this study, we reconstituted functional hepatic tissue including mature hepatocyte and blood vessel-like structures accompanied with bile duct-like structures from E15.5 fetal liver cells, which contained more hepatic stem/progenitor cells comparing with neonatal liver cells. The culture was performed in a simulated microgravity environment produced by a rotating wall vessel (RWV) bioreactor. The hepatocytes in the reconstituted 3D tissue were found to be capable of producing albumin and storing glycogen. Additionally, bile canaliculi between hepatocytes, characteristics of adult hepatocyte in vivo were also formed. Apart from this, bile duct structure secreting mucin was shown to form complicated tubular branches. Furthermore, gene expression analysis by semi-quantitative RT-PCR revealed the elevated levels of mature hepatocyte markers as well as genes with the hepatic function. With RWV culture system, we could produce functionally reconstituted liver tissue and this might be useful in pharmaceutical industry including drug screening and testing and other applications such as an alternative approach to experimental animals.

Related URLs:
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=emed10&AN=2011295052
http://sfxhosted.exlibrisgroup.com/mayo?sid=OVID:embase&id=pmid:&id=doi:10.1016%2Fj.jbiosc.2011.01.019&issn=1389-1723&isbn=&volume=111&issue=6&spage=711&pages=711-718&date=2011&title=Journal+of+Bioscience+and+Bioengineering&atitle=Reconstitution+of+hepatic+tissue+architectures+from+fetal+liver+cells+obtained+from+a+three-dimensional+culture+with+a+rotating+wall+vessel+bioreactor&aulast=Ishikawa&pid=%3Cauthor%3EIshikawa+M.%3C%2Fauthor%3E&%3CAN%3E2011295052%3C%2FAN%3E

Gravity, a regulation factor in the differentiation of rat bone marrow mesenchymal stem cells

by on 9 June 2015in Biology & Biotechnology No comment

BACKGROUND: Stem cell therapy has emerged as a potential therapeutic option for tissue engineering and regenerative medicine, but many issues remain to be resolved, such as the amount of seed cells, committed differentiation and the efficiency. Several previous studies have focused on the study of chemical inducement microenvironments. In the present study, we investigated the effects of gravity on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into force-sensitive or force-insensitive cells. METHODS AND RESULTS: Rat BMSCs (rBMSCs) were cultured under hypergravity or simulated microgravity (SMG) conditions with or without inducement medium. The expression levels of the characteristic proteins were measured and analyzed using immunocytochemical, RT-PCR and Western-blot analyses. After treatment with 5-azacytidine and hypergravity, rBMSCs expressed more characteristic proteins of cardiomyocytes such as cTnT, GATA4 and beta-MHC; however, fewer such proteins were seen with SMG. After treating rBMSCs with osteogenic inducer and hypergravity, there were marked increases in the expression levels of ColIA1, Cbfa1 and ALP. Reverse results were obtained with SMG. rBMSCs treated with adipogenic inducer and SMG expressed greater levels of PPARgamma. Greater levels of Cbfa1- or cTnT-positive cells were observed under hypergravity without inducer, as shown by FACS analysis. These results indicate that hypergravity induces differentiation of rBMSCs into force-sensitive cells (cardiomyocytes and osteoblasts), whereas SMG induces force-insensitive cells (adipocytes). CONCLUSION: Taken together, we conclude that gravity is an important factor affecting the differentiation of rBMSCs; this provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated or undifferentiated cells.

Related URLs:
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=emed9&AN=19772591
http://sfxhosted.exlibrisgroup.com/mayo?sid=OVID:embase&id=pmid:19772591&id=doi:&issn=1423-0127&isbn=&volume=16&issue=&spage=87&pages=87&date=2009&title=Journal+of+biomedical+science&atitle=Gravity%2C+a+regulation+factor+in+the+differentiation+of+rat+bone+marrow+mesenchymal+stem+cells&aulast=Huang&pid=%3Cauthor%3EHuang+Y.%3C%2Fauthor%3E&%3CAN%3E19772591%3C%2FAN%3E

Dynamic Three-Dimensional Culture Methods Enhance Mesenchymal Stem Cell Properties and Increase Therapeutic Potential

by on 9 June 2015in Biology & Biotechnology No comment

Mesenchymal stemcells (MSCs) are capable of self-renewal and differentiation along the osteogenic, chondrogenic, and adipogenic lineages and have potential applications in a range of therapies. MSCs can be cultured as monolayers on tissue culture plastic, but there are indications that they lose cell-specific properties with time in vitro and so poorly reflect in vivo MSC behavior. We developed dynamic three-dimensional (3D) techniques for in vitro MSC culture using spinner flasks and a rotating wall vessel bioreactor. We characterized the two methods for dynamic 3D MSC culture and compared the properties of these cultures with monolayer MSCs. Our results showed that under optimal conditions, MSCs form compact cellular spheroids and remain viable in dynamic 3D culture. We demonstrated altered cell size and surface antigen expression together with enhanced osteogenic and adipogenic differentiation potential in MSCs from dynamic 3D conditions. By microarray analysis of monolayer and spinner flask MSCs, we identified many differences in gene expression, including those confirming widespread changes to the cellular architecture and extracellular matrix. The upregulation of interleukin 24 in dynamic 3D cultures was shown to selectively impair the viability of prostate cancer cells cultured in medium conditioned by dynamic 3D MSCs. Overall, this work suggests a novel therapeutic application for dynamic 3D MSCs and demonstrates that these methods are a viable alternative to monolayer techniques and may prove beneficial for retaining MSC properties in vitro.

Related URLs:
<Go to ISI>://WOS:000280228300018

Shear-controlled single-step mouse embryonic stem cell expansion and embryoid body-based differentiation

by on 9 June 2015in Biology & Biotechnology No comment

To facilitate the exploitation of embryonic stem cells (ESCs) and ESC-derived cells, scale-up of cell production and optimization of culture conditions are necessary. Conventional ESC culture methods are impractical for large-scale cell production and lack robust microenvironmental control. We developed two stirred-suspension culture systems for the propagation of undifferentiated ESCs – microcarrier and aggregate cultures-and compared them with tissue-culture flask and Petri dish controls. ESCs cultured on glass microcarriers had population doubling times (similar to 14 – 17 hours) comparable to tissue-culture flask controls. ESC growth could be elicited in shear-controlled stirred-suspension culture, with population doubling times ranging between 24 and 39 hours at 100 rpm impeller speed. Upon removal of leukemia inhibitory factor, the size-controlled ESC aggregates developed into embryoid bodies (EBs) capable of multilineage differentiation. A comprehensive analysis of ESC developmental potential, including flow cytometry for Oct-4, SSEA-1, and E-cadherin protein expression, reverse transcription-polymerase chain reaction for Flk-1, HNF3-beta, MHC, and Sox-1 gene expression, and EB differentiation analysis, demonstrated that the suspension-cultured ESCs retained the developmental potential of the starting cell population. Analysis of E-cadherin(-/-) and E-cadherin(+/-) cells using both systems provided insight into the mechanisms behind the role of cell aggregation control, which is fundamental to these observations. These cell-culture tools should prove useful for both the production of ESCs and ESC-derived cells and for investigations into adhesion, survival, and differentiation phenomena during ESC propagation and differentiation.

Related URLs:
<Go to ISI>://WOS:000232672700013