Large‐scale propagation of four undifferentiated human embryonic stem cell lines in a feeder‐free culture system

We describe an improved and more robust protocol for transfer and subsequent propagation of human embryonic stem cells under feeder‐free conditions. The results show that mechanical dissociation for transfer of the human embryonic stem cells to Matrigel™ resulted in highest survival rates. For passage of the cultures on the other hand, enzymatic dissociation was found to be most efficient. In addition, this method reduces the time, work, and skills needed for propagation of the human embryonic stem cells. With the present protocol, the human embryonic stem cells have been cultured under feeder‐free conditions for up to 35 passages while maintaining a normal karyotype, stable proliferation rate, and high telomerase activity. Furthermore, the feeder‐free human embryonic stem cell cultures express the transcription factor Oct‐4, alkaline phosphatase, and cell surface markers SSEA‐3, SSEA‐4, Tra 1‐60, Tra 1‐81, and formed teratomas in severe combined immunodeficient mice. This method provides distinct advantages compared with previous protocols and make propagation of human embryonic stem cells less laborious and more efficient. Developmental Dynamics 233:1304–1314, 2005. © 2005 Wiley‐Liss, Inc.