Current methods for the extended culture of human embryonic stem cells (hESCs) are far from optimal. Therefore, we proposed to use nanoporous membrane for the co-culture of feeder cells and hESCs. In this proof-of-concept study, we first studied the stability, biocompatibility and permeability of an alginate-poly-L-lysine-alginate membrane. This membrane was then used to encapsulate feeder cells to examine the cell viability and TGF-ß secretion. Finally, the encapsulated feeder-conditioned media were used to culture hESCs. The data indicate that nanoporous membrane is a potential tool for the co-culture of feeder cells and hESCs in a safe, efficient, and continuous manner. © 2009 Inderscience Enterprises Ltd.