This technology is related to ideal culture system for hES cells. Difficulties in culture hinders mass culture of hES cells. Although there have been many efforts to establish feeder-free culture for hES cells, they are expensive and less effective. Feeder cells provide various factors dispensable for hES cells to self-renew which we do not fully understand. Co-culture with feeders is the most efficient and economical way to control self-renewal of hES cells. Nevertheless, co-culture system with feeders has many disadvantages such as possibility of feeder cell contamination, culture fluctuation depending on condition of feeder cells, difficulty in pre-treatment of cytostatic agents or irradiation of feeder cells. However, porous membrane-based co-culture system allows co-culture of hES cells eliminating these difficulties in that:
1) No feeder cell contamination since this system allows only soluble factor exchanges between hES cells and feeder cells
2) No need to pre-treat cytostatic agents to arrest growth of feeder cells
3) No need to coat culture surfaces with extracellular matrix, the main source of xenogene contamination.
4) Less culture fluctuation due to feeder cell condition
5) No time limit for sub-culturing depending on feeder cell deaths
6) Maintaining optimal composition of culture medium for each type of cell
Although membrane-based co-culture system has long been used, it has never been applied for hES cells. Furthermore we found that there is an optimal range of several physical factors and materials of polymer membrane to support undifferentiated state of hES cells maximally. When optimized, we found that membrane-based co-culture system enhanced self-renewal and growth of hES cells far more than conventional culture system as follows:
1) Reduced spontaneous differentiation
2) Increased cell production rate due to ~1% of apoptosis comparing to ~20-30% of apoptosis in conventional culture
We further confirmed that genetic and epigenetic stability and differentiation capacity are well maintained when hES cells are cultured on membrane. In this regard this technology can be applied for mass culture of hES cells for drug discovery and therapeutic purposes and for derivation of pluripotent stem cells for human being and animals.