Isolation And Culture Of Kunming Mouse Embryonic Germ Cells

Embryonic germ (EG) cells is pluripotent stem cells derived from primordial germ cells (PGCs). The major object of this paper is to isolate and culture Kunming mouse EG cells as well as to identify the EG cells from various aspects. Some factors influencing the efficiency of the isolation and culture of mouse EG cells have been discussed. Our work may be useful to further study the biology of mouse EG cell and establish the EG cell lines. The results obtained were as follows:1). Effects of different isolation methods on the isolation and culture of MEF cells were compared, the results showed that the effect of direct digestion method was better than that of tissue culture method and the tissue culture plus digestion method. The proliferation of MEF cells was inhibited after treated with 10μg/mL mytomycin for 90 min, and the 90 min was the appropriate duration for mytomycin treatment.2). Effects of different gestational age on isolation and passage of EG cells were studied, when the 8.5dpc～10.5dpc fetus were used, the number of ES-like clusters and the number of passages of EG cells were lesser than that of 11.5dpc～12.5dpc groups.3). Effects of disperse methods on mouse EG cell isolation and culture were studied, when severial times digest methods was used, the colony rate of primary EG cell and the average passages were significantly higher than that of one time digest group.4). Effects of different lysis buffer on mouse EG isolation and culture were studied, 0.125% trypsin+0.02% EDTA resulted in higher primary EG cell colony rate and average passages than that of 0.25% trypsin+0.04% EDTA group and 0.1% collagenaseⅣgroup, however, no significant differences was found between groups.5). Effects of different cell culture medium on the culture of mouse EG cells were studied, EG cell colony rates and maximum passage numbers of RH-CM group and EG culture medium+MEF group were higher than that of EG culture medium group. RH-CM was the best culture system for mouse EG cells culture, it could maintain the undifferentation state of EG cells for a longer time.6). Effects of different passage methods on isolation and passages of mouse EG cells were studied, the results showed that the direct digestion method and digestion with fibroblast method both resulted in higer EG cell colony rates and maximum passage numbers than that of passage by hand method.7). Effects of different cryopreservation duration and different passages on viability of EG cells after cryopreservation were compared, the results showed that there were no significant differences between groups, the viability of EG cells was not influenced by cryopreservation.