Factors Influencing The Isolation And Culture Of Mouse Embryonic Stem Cells And Bovine Embryonic Germ Cells

The major object of this paper is to isolate and culture embryonic stem (ES) cells from BALB/c strain mouse using a method of immunosurgery and to isolate and culture bovine embryonic germ (EG) cells derived from primordial germ cells (PGCs) as well as to demonstrate pluripotent of mouse ES and bovine EG cells from various aspects. Some factors influencing the efficiency of the isolation and culture mouse ES cells and bovine EG cells have been discussed. Our work may be useful to further establish mouse ES cell and bovine EG cell lines. The results obtained were as follows:1. 20 pure ICMs were obtained from 45 embryos of BALB/c strain mouse using immunosurgical method. ICMs were plated on mitomicin-inactivated MEF feeders and cultured in a humidified environment of 5% CO2 in air, 37 癈. ES-like colonies were observed from 17 of 20 pure ICMs and one ES-like cell line had maintained undifferentiation for 10 passages. In addition, ES-like colonies were also obtained from 66 of 102 embryos of BALB/c strain mouse using a method of intact embryo culture. Both methods used mouse ES culture medium composed of DMEM (high glucose) containing 15% FBS, 0.1 mM p-mercaptoethanol, 0.01 mM non-essential amino acid, 10 ng/ml LIF, 100 lU/ml penicillin, and 100 IU/ml streptomycin.2. Primordial germ cells were isolated and cultured from gonads, genital ridges or urogenital ridges of 46 fetuses with the age of 29-100 days of gestation. Bovine EG cell culture medium contains DMEM (high glucose), 15% NBS (or FBS), 0.1 mM P-mercaptoethanol, 0.01 mM non-essential amino acid, 2 mM glutamine, 100 lU/ml penicillin, and 100 lU/ml streptomycin. One EG cell line had maintained an undifferentiated state for 6 passages. 29-45 days of gestation embryos are optimum for the isolation and culture of bovine EG cells. It is difficult to isolate and culture bovine EG cells if the age of fetuses is more than 70 days of gestation.3. Bovine fetal fibroblasts were isolated from a 45-day-old fetus, cultured in vitro, and passaged 28 passages. We find that a -MEM is a good cell culture medium for bovine fetal fibroblasts.4. MEF, STO and BEF feeders all can sustain mouse ES cells, there are no distinct difference in the rate of embryo attaching and ICM colonies formig (P>0.05). MEF, STO, BEF and HEF feeders all can promote growth of bovine EG cells in vitro, but STO is the best, HEF is the last and MEF, BEF is in the middle.5. DMEM medium is better than F12 medium in the isolation and culture of bovine PGCs and the formation rate of EG cells colonies. 15% NBS is better than 10% NBS in the rate of embryo attanching and ICM colonies forming in mouse (P<0.05). 15% FBS is more beneficial to isolate and culture mouse ES cells and bovine EG cells than 15% NBS (P<0.05).6. Supplement with LIF (10 ng/ml) is advantage to the isolation and culture of mouse ES cells (PO.05) and bovine EG cells. Moreover, the influence was distinct for bovine EG cells if LIF, SCF and bFGF (each of them is 10 ng/ml) are all in presence.7. BALB/c strain mouse ES cells is susceptive to trypsin. 0.05% trypsin-0.008% EDTA is a good dispersed liquid for mouse ES cells, which is relative gentle to ES cells and is better for forming new ES colonies (P<0.05).The mouse ES cells and bovine EG cells which are pluipotential cells have been indentified by colony morphology, AKP (or PAS) staining, in vitro differentiation and karyotyping.