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Research Containing: Cell Culture Techniques/instrumentation/*methods

A biaxial rotating bioreactor for the culture of fetal mesenchymal stem cells for bone tissue engineering

by cfynanon 9 June 2015in Biology & Biotechnology No comment

The generation of effective tissue engineered bone grafts requires efficient exchange of nutrients and mechanical stimulus. Bioreactors provide a manner in which this can be achieved. We have recently developed a biaxial rotating bioreactor with efficient fluidics through in-silico modeling. Here we investigated its performance for generation of highly osteogenic bone graft using polycaprolactone-tricalcium phosphate (PCL-TCP) scaffolds seeded with human fetal mesenchymal stem cell (hfMSC). hfMSC scaffolds were cultured in either bioreactor or static cultures, with assessment of cellular viability, proliferation and osteogenic differentiation it) vitro and also after transplantation into immunodeficient mice. Compared to static culture, bioreactor-cultured hfMSC scaffolds reached cellular confluence earlier (day 7 vs. day 28), with greater cellularity (2x, p < 0.01), and maintained high cellular viability in the core, which was 2000 Inn from the surface. In addition, bioreactor culture was associated with greater osteogenic induction, ALP expression (1.5x P < 0.01), calcium deposition (5.5x, p < 0.001) and bony nodule formation on SEM, and in-vivo ectopic bone formation in immunodeficient mice (3.2x, p < 0.001) compared with static-cultured scaffolds. The use of biaxial bioreactor here allowed the maintenance of cellular viability beyond the limits of conventional diffusion, with increased proliferation and osteogenic differentiation both in vitro and in vivo, suggesting its utility for bone tissue engineering applications. (C) 2009 Elsevier Ltd. All rights reserved.

Related URLs:
<Go to ISI>://WOS:000264953900005

Development of a Novel Three-Dimensional, Automatable and Integrated Bioprocess for the Differentiation of Embryonic Stem Cells into Pulmonary Alveolar Cells in a Rotating Vessel Bioreactor System

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Application of stem cells for cell therapy of respiratory diseases is a developing field. We have previously established several protocols for the differentiation of embryonic stem cells (ESC) into alveolar epithelial cells, which require a high degree of operator interference and result in a low yield of target cells. Herein, we have shown that, by provision of a medium conditioned using A549 cells and by integration of classic steps of ESC differentiation into a single step through encapsulation in hydrogels (three-dimensional) and culture in a rotary bioreactor, murine ESC (mESC) could be directed to differentiate into distal respiratory epithelial cells. Type I and II pneumocytes (with a yield of 50% for type II) and Clara cells were demonstrated by the expression of aquaporin 5, surfactant protein C, and Clara cell secretory protein, respectively. We identified target cells as early as day 5 of culture and stably maintained our differentiated cells in vitro for 100 days. Electron microscopy demonstrated microvilli and intracellular lamellar bodies (LB), and fluorescent staining confirmed the active process of exocytosis of these LB in differentiated type II cells. When these cells were decapsulated and cultured in static conditions in flask cultures (two-dimensional), they retained their characteristic type II phenotype and morphology. In conclusion, our protocol offers integrated bioprocessing, shorter time of differentiation, lower cost, no use of growth factors, high reproducibility, and high phenotypic and functional stability, as well as being amenable to automation and being scalable, which would move this field closer to future clinical applications.

Related URLs:
<Go to ISI>://WOS:000302136300003

Enhanced cardiac differentiation of mouse embryonic stem cells by use of the slow-turning, lateral vessel (STLV) bioreactor

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Embryoid body (EB) formation is a common intermediate during in vitro differentiation of pluripotent stem cells into specialized cell types. We have optimized the slow-turning, lateral vessel (STLV) for large scale and homogenous EB production from mouse embryonic stem cells. The effects of inoculating different cell numbers, time of EB adherence to gelatin-coated dishes, and rotation speed for optimal EB formation and cardiac differentiation were investigated. Using 3 x 10(5) cells/ml, 10 rpm rotary speed and plating of EBs onto gelatin-coated surfaces three days after culture, were the best parameters for optimal size and EB quality on consequent cardiac differentiation. These optimized parameters enrich cardiac differentiation in ES cells when using the STLV method.

Related URLs:
<Go to ISI>://WOS:000293752000009

Formation and differentiation of three-dimensional rat marrow stromal cell culture on microcarriers in a rotating-wall vessel

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Using a high aspect ratio vessel (HARV), this study investigated the formation of 3-D rat marrow stromal cell culture on microcarriers and the expression of bone-related biochemical markers under conditions of simulated microgravity. In addition, it calculated the shear stresses imparted on the surface of microcarriers of different densities by the medium fluid in an HARV. Secondary rat marrow stromal cells were cultured on two types of microcarriers, Cytodex-3 beads and modified bioactive glass particles. Examination of cellular morphology by scanning electron microscopy revealed the presence of three-dimensional multicellular aggregates consisting of multiple cell-covered Cytodex-3 microcarriers bridged together. Mineralization was observed in the aggregates. Spherical cell-bead aggregates were observed in an HARV, while cell-bead assemblies were mostly loosely packed in a chainlike or branched structure in a cell bag. The expressions of alkaline phosphatase activity, collagen type I, and osteopontin were shown via the use of histochemical staining, immunolabeling, and confocal scanning electron microscopy. Using a numerical approach, it was found that at a given rotational speed and for a given culture medium, a larger density difference between the microcarrier and the culture medium (e.g., a modified bioactive glass particle) imparted a higher maximum shear stress on the microcarrier.

Related URLs:
<Go to ISI>://WOS:000072925300005

Ex vivo expansion of hematopoietic stem cells derived from umbilical cord blood in rotating wall vessel

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Expansion of umbilical cord blood mononuclear cells (UCB MNCs) was carried out in a rotating wall vessel (RWV) bioreactor and tissue culture flasks (T-flasks) in serum-containing medium supplemented with relatively low doses of purified recombinant human cytokines (5.33 ng/ml IL-3, 16 ng/ml SCF, 3.33 ng/ml G-CSF, 2.13 ng/ml GM-CSF, 7.47 ng/ml FL and 7.47 ng/ml TPO) for 8 days. The cell density, pH and osmolality of the culture medium in the two culture systems were measured every 24 It. Flow cytometric assay for CD34(+) cells was carried out at 0, 144 and 197 h and methylcellulose colony assays were performed at 0, 72, 144 and 197 It. The pH and osmolality of the medium in the two culture systems were maintained in the proper ranges for hematopoietic stem cells (HSCs) and progenitors culture. The RWV bioreactor, combined with a cell-dilution feeding protocol, was efficient to expand UCB MNCs. At the end of 200 h culture, the total cell number was multiplied by 435.5 +/- 87.6 times, and CD34(+) cells 32.7 +/- 15.6 times, and colony-forming units of granulocyte-macrophage (CFU-GM) 21.7 +/- 4.9 times. While in T-flasks, however, total cells density changed mildly, CD34(+) cells and CFU-GM decreased in number. It is demonstrated that the RWV bioreactor can provide a better environment for UCB MNCs expansion, enhance the contact between HSCs and accessory cells and make the utilization of cytokines more effective than T-flask. (c) 2006 Elsevier B.V. All rights reserved.

Related URLs:
<Go to ISI>://WOS:000239156700012

NASA-approved rotary bioreactor enhances proliferation and osteogenesis of human periodontal ligament stem cells

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Previous studies have suggested that periodontal ligament stem cells (PDLSCs) play crucial role in regeneration of periodontal defects, and recently tissue engineering based on PDLSCs to enhance periodontal regeneration has been the focus of periodontal research. A theoretical way to achieve this goal would be to provide a "stimulatory'' environment to rapidly expand PDLSCs in vitro to expedite tissue engineering of periodontium. We hypothesize that three-dimensional (3D) dynamic simulated microgravity (SMG) culture system have effect on periodontal stem cells, and would benefit periodontal stem cells proliferation and differentiation, but up to now, there are no related reports on this aspect. In this study, we investigated the biological effect of three-dimensional dynamic SMG induced by rotary cell culture system (RCCS) on human periodontal ligament stem cells (hPDLSCs) in vitro. hPDLSCs were isolated from surgically extracted human teeth and enriched by collecting multiple colonies. hPDLSCs were inoculated on Cytodex 3 microcarriers and cultured in RCCS. The results showed that SMG affected the biology of hPDLSCs as indicated by promotion of proliferation and viability, alterations of morphology, and disorganization of microfilament system. Besides, SMG-treated hPDLSCs presented increased matrix mineralization and up-regulated expression of mineralization associated genes after incubation in osteogenic medium. For it is the first time to investigate effects of SMG on PDLSCs, the research may lend insight into variations of cell response in 3D environment, and contribute to achievement of desirable periodontal regeneration utilizing PDLSCs-based tissue engineering approaches.

Related URLs:
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=emed9&AN=2009606938
http://sfxhosted.exlibrisgroup.com/mayo?sid=OVID:embase&id=pmid:&id=doi:10.1089%2Fscd.2008.0371&issn=1547-3287&isbn=&volume=18&issue=9&spage=1273&pages=1273-1282&date=2009&title=Stem+Cells+and+Development&atitle=NASA-approved+rotary+bioreactor+enhances+proliferation+and+osteogenesis+of+human+periodontal+ligament+stem+cells&aulast=Li&pid=%3Cauthor%3ELi+S.%3C%2Fauthor%3E&%3CAN%3E2009606938%3C%2FAN%3E

Dynamic Cultivation of Human Mesenchymal Stem Cells in a Rotating Bed Bioreactor System Based on the Z (R) RP Platform

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Because the regeneration of large bone defects is limited by quantitative restrictions and risks of infections, the development of bioartificial bone substitutes is of great importance. To obtain a three-dimensional functional tissue-like graft, static cultivation is inexpedient due to limitations in cell density, nutrition and oxygen support. Dynamic cultivation in a bioreactor system can overcome these restrictions and furthermore provide the possibility to control the environment with regard to pH, oxygen content, and temperature. In this study, a three-dimensional bone construct was engineered by the use of dynamic bioreactor technology. Human adipose tissue derived mesenchymal stein cells were cultivated on a macroporous zirconium dioxide based ceramic disc called Sponceram (R). Furthermore, hydroxyapatite coated Sponceram (R) was used. The cells were cultivated under dynamic conditions and compared with statically cultivated cells. The differentiation into osteoblasts was initiated by osteogenic supplements. Cellular proliferation during static and dynamic cultivation was compared measuring glucose and lactate concentration. The differentiation process was analysed determining AP-expression and using different specific staining methods. Our results demonstrate much higher proliferation rates during dynamic conditions in the bioreactor-system compared to static cultivation measured by glucose consumption and lactate production. Cell densities on the scaffolds indicated higher proliferation on native Sponceram (R) compared to hydroxyapatite coated Sponceram (R). With this study, we Present an excellent method to enhance cellular proliferation and bone lineage specific growth of tissue like structures comprising fibrous (collagen) and globular (mineral) extracellular components. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 1762-1771 2009

Related URLs:
<Go to ISI>://WOS:000273152000025

Rhythmicity of engraftment and altered cell cycle kinetics of cytokine-cultured murine marrow in simulated microgravity compared with static cultures

by cfynanon 9 June 2015in Biology & Biotechnology No comment

Space flight with associated microgravity is complicated by "astronaut's anemia" and other hematologic abnormalities. Altered erythroid differentiation, red cell survival, plasma volume, and progenitor numbers have been reported. We studied the impact of microgravity on engraftable stem cells, culturing marrow cells in rotary wall vessel (RWV) culture chambers mimicking microgravity and in normal gravity nonadherent Teflon bottles. A quantitative competitive engraftment technique was assessed under both conditions in lethally irradiated hosts. We assessed 8-wk engraftable stem cells over a period spanning at least one cell cycle for cytokine (FLT-3 ligand, thrombopoietin [TPO], steel factor)-activated marrow stem cells. Engraftable stem cells were supported out to 56 h under microgravity conditions, and this support was superior to that seen in normal-gravity Teflon bottle cultures out to 40 h, with Teflon bottle culture support superior to RWV from 40 to 56 h. A nadir of stem cell number was seen at 40 h in Teflon and 48 h in RWV, suggesting altered marrow stem cell cycle kinetics under microgravity. This is the first study of engraftable stem cells under microgravity conditions, and the differences between microgravity and normal gravity cultures may present opportunities for unique future stem cell expansion strategies.

Related URLs:
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=emed5&AN=2003031697
http://sfxhosted.exlibrisgroup.com/mayo?sid=OVID:embase&id=pmid:&id=doi:10.1290%2F1071-2690%25282002%2529038%253C0343%3AROEAAC%253E2.0.CO%3B2&issn=1071-2690&isbn=&volume=38&issue=6&spage=343&pages=343-351&date=2002&title=In+Vitro+Cellular+and+Developmental+Biology+-+Animal&atitle=Rhythmicity+of+engraftment+and+altered+cell+cycle+kinetics+of+cytokine-cultured+murine+marrow+in+simulated+microgravity+compared+with+static+cultures&aulast=Colvin&pid=%3Cauthor%3EColvin+G.A.%3C%2Fauthor%3E&%3CAN%3E2003031697%3C%2FAN%3E

Improvement of culture conditions of human embryoid bodies using a controlled perfused and dialyzed bioreactor system

by cfynanon 9 June 2015in Biology & Biotechnology No comment

In parallel to the active search for therapeutic and industrial applications of human embryonic stem cells (hESCs), designing automated means of producing those cells is a timely goal. Slow-turning lateral vessels (STLVs) with low shear stress have shown promise for expanding the cells at the embryoid body stage. We have improved this technology by developing two complementary systems, allowing continuous optimization of the culture conditions. First, perfused STLV bioreactors were set up, to provide continuous delivery of culture medium to the cells growing in the rotating chamber. This allowed the external control of the culture medium, and consequently optimized oxygenation, pH, nutrient supply, and waste elimination. Second, a dialysis chamber was adapted. This led to a further enhanced controlled environment and a decrease in the quantity of adjunct products (e.g., growth factors) necessary to the cells inside the bioreactor chamber. hESC aggregation and initial differentiation-taking neural induction as an example-were compared between the perfused and dialyzed STLV system and static cultures. Perfused and dialyzed STLV bioreactors promoted formation of embryoid bodies that were differentiated more rapidly and were homogeneously synchronized in a statistically significant manner.

Related URLs:
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=emed8&AN=2008584854
http://sfxhosted.exlibrisgroup.com/mayo?sid=OVID:embase&id=pmid:&id=doi:10.1089%2Ften.tec.2008.0029&issn=1937-3384&isbn=&volume=14&issue=4&spage=289&pages=289-298&date=2008&title=Tissue+Engineering+-+Part+C%3A+Methods&atitle=Improvement+of+culture+conditions+of+human+embryoid+bodies+using+a+controlled+perfused+and+dialyzed+bioreactor+system&aulast=Come&pid=%3Cauthor%3ECome+J.%3C%2Fauthor%3E&%3CAN%3E2008584854%3C%2FAN%3E

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