Optimization And Establishment Of An In Vitro Culture System For Rabbit Embryonic Stem Cell

This paper is to systematically study the effect of isolating and cloning Embryonic stem cells (ESC) and embryonic germ cells( EGC) in the different culture conditions and the passage way. It might provide datas to perfect route to establish rabbit ES,EG cell line. The results obtained were as follows:1 The research Compared the Effect of Different culture medium and Feeder Layers on isolating and cloning of ES cells. With the culture medium of DMEM, 15%FBS, 0.1mMβ-mercaptoethanol, 0.01mM non-essential amino acid, 100IU/ml penicillin, 100IU/ml streptomycin, 1000IU/ml LIF and 10mg/mL insulin, the Rabbit Embryo can Attach and proliferate very fast. ES cell-like line clone in this culture medium had maintained an undifferentiated state for 5 passages. Compare with the feeder layer of MEF and the REF, the rates of embryo attaching, ICM propagating for the embryo cultivated on free-feeding layer culture system were significant differences(P<0.05), and have no passage. With the feeder layer of MEF and the REF, The rate of attaching of embryos is 67.4% and 63.2%, and they had maintained an undifferentiated state for 5 passages.2 The method of Continuous digestive for ES cells is better than The single digestive method, it improve the rate of forming ESC clones. Digestion with the feeder layer has the lowest rate of ES clones, ES cell had maintained an undifferentiated state for only 1 passages, not suitable for ES cells separation and passage in the early time.3 PGCs colonies can be isolated from 14-18 days of gestation embryos of rabbit, but the PGCs colonies isolate from 15, 16 days Fetus are more than 14, 17 and 18 days'; and One EG cell line in this two days maintained undifferentiation for 9 passages, suitable for cloning rabbits EG-like cells. Rat fetus cardiomyocyte cells condition medium is as good as EG cell medium with LIF, both are better than the medium contained no cytokine, it showed that rat fetus cardiomyocyte cell conditioned medium is fit for the isolation and culture of rabbit PGCs.4 Let the PGCs culture on mouse embryonic fibroblasts(MEF), rabbit embryonic fibroblasts ( REF) and co-cultured with the homogenous embryonic fibroblast from genital ridges(HEFgr)or co-cultured with Rabbit Sertoli cells(RSCs). The Result is that the manner of co-cultured was better than cultured depended on mitotically inactive feed layer. PGCs co-cultured with RSCs gained the more EG than other ways. They remained undifferentiated state for the longest period and cultured for 8 passages. There was not significant difference in PGCs co-cultured with the HEFgr from genital ridges and co-cultured with RSCs, but PGCs were just cultured for 6 passages. PGCs culture on MEF can also remained undifferentiated and cultured for 4 passages. PGCs culture on REF gained fewer EGs, they remained undifferentiated state were shorter and just cultured for 3 passages. So the manner of co-cultured with RSCs and co-cultured with the HEFgr can be used to isolation and cultivation rabbit PGCs.5 Comparing the effect of different methods on the subculture of Rabbit EG like cells, the method of Passage by hand as well as the methods of digestion together with freeder layer can obtain the more F1 clones, they are no obvious difference. But the method of Passage by hand in EG clones can cultured for 9 passages, The methods of digestion together with freeder layer just support the EG like cell for 4 passages, so it is not a good passaging methods for rabbit EG like cell.6 When isolated rabbit ESC and EGC were examined by observing morphology, AKP stain and in vitro differentiation capacity, it have a series of characters of Embryonic Stem Cells.