Micropatterned Co-cultures of Human Embryonic Stem Cell with Murine Embryonic Fibroblasts on Microwell Patterned Substrates

Human embryonic stem (hES) cells are generally cultured on top of a feeder layer formed by mitotically inactivated murine embryonic fibroblasts (MEFs) to maintain their undifferentiated state. The culture of hES cells in this co-culture system presents several challenges since it is difficult to control cell cluster size. Large cell clusters tend to differentiate at the borders, and clusters with different sizes may lead to heterogenous differentiation patterns within embryoid bodies. In this work, we develop a new approach to culture hES cells with controlled cluster size and number through merging microfabrication, and biomaterials technologies. Polymeric microwells were fabricated and used to control the size and uniformity of hES cell clusters in coculture with MEFs. The results show that it is possible to culture hES cells homogeneously while keeping their undifferentiated state as confirmed by the expression of stem cell markers Oct-4 and alkaline phosphatase. In addition, these clusters can be recovered from the microwells to result in the generation of uniform cell clusters for differentiation experiments.