Serum-free medium cultivation to improve efficacy in establishment of human embryonic stem cell lines.

Background: Serum-containing and serum-free media were used to derive human embryonic stem (HES) cells from donated oocytes and embryos.

Methods And Results: Inner cell masses (ICM) were isolated by immunosurgery. The HES cells were found to be easily obtained and expanded in a serum-free medium. The efficacy in establishing human embryonic stem cell lines improved in a serum-free medium compared with that in serum-containing media. Four HES cell lines were derived from 13 isolated ICM on mouse embryonic fibroblast feeder layers. All four cell lines possess the same characteristics and differentiating potency: normal 46, XX or 46, XY karyotype; and expressing a series of surface markers such as APase, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, but not SSEA-1. They can form embryoid bodies in suspension culture and develop teratomas comprising derivatives of three embryonic germ layers when injected into severe combined immunodeficient mice.

Conclusion: These preliminary results suggest that serum-free cultivation may be superior to serum-containing cultivation for deriving human embryonic stem cells.