The invention provides a serum-free separating and culturing method for a sheep
embryo stem cell, comprising the following steps of: removing a
zona pellucida of sheep
blastula cultured
in vitro with a Tyrode's Solution and then inoculating the sheep
blastula to a serum-free culture solution of the sheep
embryo stem cell and fixing by using a needle head; placing under the conditions that the temperature is 38.6DEG C and the saturated
humidity is 5 percent CO2 for culturing; during the culturing, replacing the culture solution in a half quantity every 48 hours with the pH value of 6.8-7.2 and primarily culturing for 7-9 days; and carrying out the transfer culture once according to the ratio of 1:2-1:4 by adopting a conventional mechanical method, that is to say, transferring the sheep
embryo stem cell cultured
in vitro to the 16th generation. The serum-free culture solution of the sheep embryo stem
cell is prepared from D-MEM / F-12+80ng / mL bFGF+3muMCHIR 99021+10mu L / mL N2+20mu L / mL B27+10mu L / mL NEAA+2mM L-
Glutamine+0.2mM beta-Mercaptoethanol. Bared embryos are fixed at the bottom of a culture dish by using a No.32 needle head so as to avoid the damage to the cells in the
blastula, and a baked embryo trophocyte is stripped off. The preferable inoculating amount of the baked embryos is 45-60 / groups. The method can ensure that the
formation rate of primary colony of sheep embryo stem cells is improved to 33 percent (14 / 42).