Phenotypic and functional comparison of optimum culture conditions for upscaling of bone marrow-derived mesenchymal stem cells.

Human adult bone marrow-derived mesenchymal stem cells (MSCs) are a promising tool in the newly emerging avenue of regenerative medicine. MSCs have already been translated from basic research to clinical transplantation research. However, there is still a lack of consensus on the ideal method of culturing MSCs. Here we have compared different culture conditions of human MSCs with an attempt to preserve their characteristics and multi-lineage differentiation potential. We compare the different basal culture media DMEM-F12, DMEM-high glucose (DMEM-HG), DMEM-low glucose (DMEM-LG), knock-out DMEM (DMEM-KO) and Mesencult on the proliferation rate, surface markers and differentiation potentials of MSCs. At every fifth passage until the 25th passage, the differentiation potential and the presence of a panel of surface markers was observed, using flow cytometry. We also compared the characteristics of human MSCs when cultured in reduced concentrations of fetal bovine serum (FBS), knockout serum replacement (KO-SR) and human plasma. Data indicate that the presence of serum is essential to sustain and propagate MSCs cultures. The choice of basal medium is equally important so as to preserve their characteristics and multipotent properties even after prolonged culture in vitro. With MSCs emerging as a popular tool for regenerative therapies in incurable diseases, it is essential to be able to obtain a large number of MSCs that continue to preserve their characteristics following passaging. The data reveal the optimum basal medium for prolonged culture of MSCs while retaining their ability to differentiate and hence this may be used for up-scaling to provide sufficient numbers for transplantation.

Effect of holding time, temperature and different parenteral solutions on viability and functionality of adult bone marrow-derived mesenchymal stem cells before transplantation.

Mesenchymal stem cells (MSCs) have the ability to proliferate and differentiate into various lineages, given the appropriate microenvironment, thus making MSCs promising candidates for cell transplantation. For clinical applications, MSCs need to be stored in optimal conditions so that they may be transported and made available as an off-the-shelf product for companies to market. Freshly harvested and cultured or frozen-thawed bone marrow-derived MSCs were prepared for cell transplantation. Both freshly cultured or frozen-thawed MSCs were washed and resuspended in parenteral solutions, either 0.9% saline, Dulbecco's phosphate-buffered saline (DPBS), plasmalyte A or 5%dextrose and held for 2, 4, 6 and 8 h at 4 degrees C, 37 degrees C and RT (22 degrees C). The viability of the cells, differentiation capability and expression of cell surface markers were analysed. MSCs harvested from fresh cultures, resuspended in the parenteral solutions and maintained at 4 degrees C for 6 h showed more than 90% viability, and the viability was appreciably better when suspended in 5% dextrose at 4 degrees C for 8 h. In contrast, frozen-thawed cells can be held for a maximum of 2 h after thawing before losing their viability significantly below permissible limits for transplantation. We are reporting for the first time the effect of various parenteral solutions, holding times and temperatures on the viability and functionality of bone marrow-derived freshly cultured or frozen-thawed MSCs for transplantation. Our results suggested that freshly harvested MSCs can be held for 8 h at 4 degrees C in 5% dextrose or for up to 6 h at 4 degrees C in saline, DPBS or plasmalyte A. Freeze-thawed MSCs can be held for a maximum of 2 h in plasmalyte A before transplantation without affecting their viability and ability to differentiate.

FGF2 secreting human fibroblast feeder cells: a novel culture system for human embryonic stem cells.

Human embryonic stem cell (hESC) lines are traditionally derived and maintained on mouse embryonic fibroblasts (MEF) which are xenogeneic and enter senescence rapidly. In view of the clinical implications of hESCs, the use of human fibroblast as feeders has been suggested as a plausible alternative. However, use of fibroblast cells from varying sources leads to culture variations along with the need to add FGF2 in cultures to sustain ES cell pluripotency. In this study we report the derivation of FGF2 expressing germ layer derived fibroblast cells (GLDF) from hESC lines. These feeders could support the pluripotency, karyotypes and proliferation of hESCs with or without FGF2 in prolonged cultures as efficiently as that on MEF. GLDF cells were derived from embryoid bodies and characterized for expression of fibroblast markers by RT-PCR, Immunofluorescence and by flow cytometry for CD marker expression. The expression and secretion of FGF2 was confirmed by RT-PCR, Western blot, and ELISA. The hESC lines cultured on MEF and GLDF were analyzed for various stemness markers. These feeder cells with fibroblast cells like properties maintained the properties of hESCs in prolonged culture over 30 passages. Proliferation and pluripotency of hESCs on GLDF was comparable to that on mouse feeders. Further we discovered that these GLDF cells could secrete FGF2 and maintained pluripotency of hESC cultures even in the absence of supplemental FGF2. To our knowledge, this is the first study reporting a novel hESC culture system which does not warrant FGF2 supplementation, thereby reducing the cost of hESC cultures.