| Embryonic stem (ES) cell lines are routinely derived from in vivo produced blastocysts.There has been considerable efforts to establish ES cell lines from other animals,so far,have been derived from mice and rat.Here,we examined effects of different conditions on the establishment of ES cells from rabbits,rats and bovine.Also to establish a suspension culture system of mouse embryonic stem cells (mES). Long-term maintenance of ES cells in their pluripotent undifferentiated state are provided by theirs system.We isolated the inner cell mass(ICM) from blastocysts of rabbit, rat and bovine manually,then cultured them by their conditions.ES colonies were passaged manually too.As a result, the rabbit ESCs (rES) can be passaged 4 times, rat ESCs (RES) were 10 times, bovine ESCs (bES) were 7 times. Meanwhile, the rES, RES and bES accord with morphological criteria of ES. RES and bES express alpaline phosphatase. ESCs of the murine cell line R1 were grown in feeder-free tissue culture flasks in mES medium A, which consisted of 15% FBS + 1000IU mLIF + 0.2mg/ml dispase + 84% DMEM/F12 + 1% MEM NAA + 2 mmol/L L-Glutamine+ 0.1 m mol/Lβ-mercaptoethanol. The mESCs were stained alkaline phosphatase and passaged more than 20 times. Teratomas were outgrowth triumphantly by hypodermic mESCs.We demonstrate that: in the system consisted of mouse embryonic endoderm cells inactivated as feeder, 78% D-MEM/F-12+20% Knockout SR+2mM/L-Glutamine+1% MEM NAA+0.1 mM/Lβ-mercaptoethanol+8 ng/ml hrbFGF+2% CEE as medium, the rabbit inner cell masses could grew into outgrowth with embryonic stem cell's typical morphology.RESCs were grown on a mitomycin-treated feeder layer which was 3T3 cell line. The RES cell medium was composed of: 86% DMEM + 10% SR + 3% CEE + 1% MEM NAA + 2 mmol/L L-Glutamine+ 0.1 mmol/Lβ-mercaptoethanol . In this condition, RES could maintain undifferentiated.When feeder was mitomycin-treated old feeder layer,bES could maintain undifferentiated. In the system of medium A, R1 could grow quickly.
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