The Application And Mechanism Of Preparing Feeder Cells Fixed With Methanol

Feeder layer cells play an important role on culturing pluripotent stem cells in vitro.Mitomycin C or irradiation inactivating mouse embryonic fibroblasts are two traditional methods for preparation feeder layer cells.The inactivated feeder cells can secrete growth factors to inhibiting differentiation and enhancing self-renewal of pluripotent stem cells.The traditional methods to making feeder layer cells are limited because the complex proceeds,the necessary of isolation mouse embryonic fibroblasts,the resudial of mitomycin C and the high demands of irradiation mechines.Preparation of feeder layer cells fixed with methanol is the first reported in this study and this method is simple and high efficiency.The results showed below:1.Establishment method of preparing feeder layer cells fixed with methanolThe results of optimizing the proportion of methanol and acetone showed that the feeder layer cells were not good when the proportion of acetone was over 70 percent.We compared the effect of different concentration of methanol on feeder cells and the results demonstrated that the fixation was loose when the methanol concentration was low.Two results showed that the 100 percent of methanol was the best fixer for preparing feeder cells.By comparing the effect of methanol temperature and fixation time on feeder cells and pluripotent stem cells,we confirmed that the methanol temperature was 4°C and fixation time was 5 min based on the simple principle.The results of optimization of the feeder cells concentration showed that the concentration of feeder cells should be above 2×10~4/cm~2.Not only the primary mouse fibroblasts but also the immortalized cell lines and other species cells could be fixed with methanol.2.The application of methanol fixed feeder cells in culturing pluripotent stem cellsMouse ES and iPS cells could be maintained on methanol fixed feeder cells.Comparing with mitomycin C method,mouse pluripotent stem cells cultured on methanol fixed feeder cells had the faster growth rate,the higher expression level of pluripotent genes Oct4?Nanog?Sox2,more SSEA-1 positive colonies and the same ability to differentiate into three germ layers.Human and pig i PS cells cultured on methanol fixed feeder cells also had normal morphology and stable pluripotent genes expression.The above results demonstrated that methanol fixed feeder cells could replace mitomycin C method for culturing pluripotent stem cells derived from mouse,human and pig.3.Other applications of feeder cells fixed by methanolFeeder cells could be fixed firmly on culture dish surface after be treated with methanol,based on this feature,we suspected that the cells fixed with methanol could be resistance of antibiotic drugs and low concentration trypsin.The results showed that we could obtain stable pluripotent stem cell lines quickly and the SSEA-1 positive rate of mouse pluripotent stem cells was above 90 percent after be cultured on fixed feeder cells reused for three times.By detecting the alkaline phosphatase activity and pluripotent genes in pluripotent stem cells cultured on feeder cells fixed with methanol stored at different temperature and time conditons,we confirmed that feeder cells fixed with methanol could be stored at room temperature for long term.4.The mechanism of feeder cells fixed with methanol in culturing pluripotent stem cellsCollagen-IV and fibronectin were still existed in feeder cells fixed with methanol,but they were degraded after methanol-fixed feeder cells treated with collagenase-IV.The adhesion ability of pluripotent stem cells on feeder cells treated with collagenase-IV was declined and some colonies were suspension in medium.The results of qRT-PCR showed that the suspension pluripotent stem cells had quit from na?ve state and converted into primed state.The above results showed that the adhesion ability and pluripotency state of pluripotent stem cells cultured on methanol-fixed feeder cells treated with collagenase-IV had changed,namely that the extracellular matrix proteins collagen-IV and fibronectin were crucial for adhesion and pluripotency of pluripotent stem cells.