Cryopreservation of isolated blastomeres and embryonic stem-like cells of Leopard danio, Brachydanio frankei.

This study aimed at developing a suitable cryopreservation protocol for embryonic stem (ES)-like cells of a tiny freshwater ¢sh Leopard danio (Brachydanio frankei). Embryonic stem (ES)-like cells derived from blastomeres of the early blastulae stage of the developing embryo were cultured in vitro in a medium containing Leibowitz-15 supplemented with 10% foetal bovine serum, leopard danio embryo extract, sodium bicarbonate, sodium selenite, basic ¢broblast growth factor, epidermal growth factor and leukaemia inhibitory factor. The ES-like cells showed properties similar to ES cells in other species. They were morphologically small, round to polygonal and present in patches and extensively expressed alkaline phosphatase and stage-speci¢c embryonic antigen. The toxicity and chilling sensitivity of these cells were determined using ethylene glycol (EG), propylene glycol (PG) and glycerol as cryoprotective agents at molar concentrations of 0.6, 1.0, 1.4, 1.8 and 2.0. Among them, 1.8 M EG showed 70% signi¢cant viable ES-like cells (Po0.05). The post-thawed cells retained similar properties of non-cryopreserved ES-like cells with a viability rate of 65%. Similarly, blastomeres cryopreserved following the slow cooling rate with EG and PG yielded a viability of more than 70%.