A novel culture technique for human embryonic stem cells using porous membranes.

We have developed a novel culture technique for human embryonic stem cells (hESCs) using a porous membrane with feeder cells. The feeder cells were seeded and attached to the bottom of a porous membrane and, subsequently, hESCs were cultured on the top of the membrane. This porous membrane technique (PMT) allowed hESCs to be successfully cultured and to be effectively and efficiently separated from the feeder cell layer without enzyme treatment. hESCs being cultured by PMT were observed to interact with feeder cells through pores of membrane, where the interaction was dependent on the pore size of the membrane used. It was also revealed that the number of attached hESC colonies depended on the concentration of feeder cells on the bottom of the membrane. On the other hand, hESC colonies did not attach to porous membrane, as feeder cells were in the presence of culture dish, not the porous membrane. The hESCs cultured on porous membranes not only exhibited expression of several undifferentiated markers and a normal karyotype, but they also formed teratomas consisting of three germ layers in in vivo study. Compared with the mechanical isolation technique conventionally used, PMT significantly decreased mouse vimentin gene expression in cultured hESCs. Thus, a PMT for hESC culture would be a useful tool to exclude enzyme treatment and to reduce contamination from feeder cells simultaneously. Disclosure of potential conflicts of interest is found at the end of this article.