(516ag) Feeder Cell Immobilization For Stem Cell Culture

Human embryonic stem (ES) cells can proliferate extensively in vitro and differentiate to multiple cell types. As such, human ES cells hold great potential as an invaluable source of cells for various research purposes and cell transplantation therapies. The initial human ES cell lines were derived by the culture of the inner cell mass on feeder cells, mitotically inactivated mouse embryonic fibroblasts (MEFs). Tremendous efforts have since been made to improve human ES cell culture conditions. Current methods can be classified into three categories: co-culture with feeder cells, culture with feeder cells-conditioned medium, and defined culture. Among them, feeder cells-based co-culture system is the most mature and reliable method for supporting the growth of hES cells. However, this method is limited by the problem of separating and passaging hES cells due to the direct contact with feeder cells. It is not only unfavorable for the large-scale production, but also can result in the misinterpretation of experimental data. We are motivated to immobilize MEFs in the hydrogel and co-culture the immobilized MEFs with human ES cells. We found that the diffusion of proteins into the hydrogel was very fast. In less than 5 minutes, 50-70% proteins could diffuse into the hydrogel. With this co-culture system, it was easy to separate human ES cells from feeder cells. Moreover, the immobilized feeder cells were able to maintain their normal metabolisms in the hydrogel and support the proliferation of human ES cells. The immunofluorescence staining was used to characterize the expression of stem cell markers including Oct4, SSEA4, Tra1-60 and Tra1-81.