The application discloses a method for separating and culturing
ovary granulosa cells of Chinese soft-shelled turtles, which comprises the steps of selecting female Chinese soft-shelled turtles in the positive oviposition period, executing cervical
dislocation after
anesthesia, killing the turtles, separating
ovary tissues, removing redundant parts after washing, separating II-III stage follicles in a basic culture medium, keeping
follicle integrity, performing
enzyme digestion treatment, removing supernatant through
centrifugation, obtaining a precipitate containing ovules with a
diameter of 0.1-0.5 cm, resuspending the precipitate by using a
mixed culture medium, and inoculating and culturing. The
ovary granulosa cells obtained by the
separation method have a high yield, the ovary granulosa cells are cultured in the specific
mixed culture medium, the method can replace many factors, the amount of added factors is small, the NaHCO3-free culture medium is used, the use of CO2 is reduced, and the ovary granulosa cells obtained are consistent with the in-vivo environment, so that the cost is reduced and the in-vitro
survival period of the ovary granulosa cells is prolonged.