Study On Serum-free Feeder-free Culture Of Rabbit Primordial Germ Cells

Attracted much attention because of their infinite proliferation and multi-directional differentiation ability.Traditional culture systems often use serum and feeder layers to provide support for embryonic finessing.However,the heterologous proteins and the inhibitory factors that are unfavorable to cell growth,which contain a large number of components in the serum and feeder layers,have potential cytotoxicity.At the same time,it also brings great difficulties to the study of the regulation of cellular products such as isolation and cell specialization.In this study,fetal rabbit primordial germ cells from 14-18d gestational age were selected as the research object,and a serum-free and feeder-free method for culturing rabbit primordial germ cells was established.1.In this study,Japanese white rabbits were randomly selected 14-18d fetal age rabbits fetal sterilized aseptically exfoliated genital warts,PGCs were isolated in vitro,KSR serum substitutes were used instead of serum,and serum-free medium was added to make rabbit serum-free medium to culture PGCs.The PGCs were identified by enzymatic histochemical detection of alkaline phosphatase activity and the expression of the transcription factor Oct-4 by RT-PCR.The relationship between the cell passage number and the recovery rate after 30 days of cryopreservation was investigated.The results showed that:①AKP staining and RT-PCR test results are positive,indicating that the use of LIF added culture medium can successfully cultivate rabbit PGCs.② The recovery rate of serum-free culture method after 30 days of cryopreservation had no difference with conventional culture(P>0.05)2.In this experiment,rabbit PGCs in a serum-free culture system were used for the transition culture of conditioned medium,and then cultured under serum-free feeder-free culture conditions supplemented with LIF,bFGF,and TGF-β1.RT-PCR was used to detect the expression of PGCs transcription factor Oct-4 in different generations and culture days.In vitro embryoid body formation experiments were conducted.After 30 days of cryopreservation,the method of serum-free,feeder-free culture of rabbit PGCs was investigated.:① Serum-free and feeder-free culture can be successfully performed through the transition culture of conditioned medium.① Addition of LIF,bFGF and TGF-β 1 cytokines in feeder-free serum-free medium can sustain rabbit primordial germ cells Proliferation and maintenance of totipotency.③ The recovery rate of serum-free feeder-free culture after 30 days of cryopreservation was not different from that of conventional culture(P>0.05).