Isolation And Serum-Free Culture Of Mouse And Human Embryonic Germ Cells

Embryonic germ (EG) cells are pluripotent cells derived from primordial germ cells of fetal gonadal ridges. At present, although the isolation and culture of the mouse and human embryonic stem cells have achieved great progress, so far, the EG cell line of Kun-ming strain mouse has not be established, and serum-free culture system has not been perfect. As a result, it is very important to reseach serum-free culture system for establishing mouse EG cells line and applying human EG cells in clinical practice, which posseses enormous actual meaning.For perfecting serum-free culture system of Kun-ming mouse and human EG cells, the following experiments were carried out in the paper: 1. From concentration of digestant and digestion time, isolation and culture of Kum-ming mouse PGCs were reseached.2. Reseach on effect of different medium on culture of mouse EG cells in vitro.3. Comparing the results of culture human EG cells when MEF and different fibrolast cells dirivation from human fetus as feeder layer. 4. Comparing the results of the different passaging method using in culture human EG cells , and identified the EG cells obtained in our experiment.1. Serum-free culture system of Kun-ming mouse EG cellsWhen digesting mouse gonadal ridge, 0.125% trypsin +0.02% EDTA was better than 0.25% trypsin +0.04% EDTA和0.05% trypsin +0.008% EDTA, and it could be digested at room temperature for 20 min or at 37℃for 10 min. Supplement of 0.1mmol/L RA and conditioned medium derived from MEF and gonadal ridge in serum-free medium can obtain more positive colonies by AP staining. Compared MEF-CM,GR-CM and cytokine (LIF and bFGF) group ,the difference between their result is not significant,but the cost of medium is cut down. Mouse EG cells were passaged for 6 times in culture medium of GR-CM and cytokine group.More colonies of primary culture and subculture can be isolated from 10.5～12.5dpc mouse fetus. After identification of Kunming mouse EG cells, staining for AP and SSEA-1,Oct-4 is positive.2. Serum-free culture system of human EG cellsAs feeder layer of culturing human EG cells in vitro, MEF and hEF1 derived from fetus gonadal ridge are better than hEF2 derived from fetus skin, it can get more primary positive PGCs colonies and average passages. When human EG cells are subcultured, Higher ratio of forming colonies and more passages can be obtained by dividing after separating colonies manual. Good results were got by digesting after separating colonies manual and digesting colonies after feeder layer, but the result of digesting colonies and feeder layer together was bad. 8～10 weeks old human fetuses are fit for isolated and culture EG cells. After identification of Human mouse EG cells, staining for AP,SSEA-1,SSEA-3,SSEA-4,TRA-1-60,TRA-1-81and Oct-4 is positive.