Derivation of Human Embryonic Stem Cell Lines from Vitrified Human Blastocysts

Human embryonic stem cells are pluripotent cells typically derived from blastulating embryos that have become excess to clinical needs in assisted reproduction programs. They provide cellular models for embryonic development and disease, and are thought to be useful for future cell replacement therapies and regenerative medicine. Here we describe methods to derive human embryonic stem cell lines. This includes blastocyst cryopreservation using a highly efficient vitrification protocol, the production and use of fibroblast feeder cells, embryo plating and passaging of resulting cellular outgrowths, and cryopreservation of putative stem cells lines.