| The major object of this paper is to establish feeder layer culture system of Kunming mouse embryonic fibroblast cells (MEF) for isolation and culture of embryonic stem cells (ES cells) in vitro, screening to be more suitable to culture system of Kunming mouse embryonic fibroblast cells for isolation and culture of embryonic stem cells in vitro.Established the foundation for further work on establishing of Kunming mouse embryonic stem cells cells lines.1. The fetus collected respectively from the female mouse, which had been pregnant for 11, 14.5 and 16 days, were used to prepare the fibroblast cells. Isolated MEF on different day ages were compared by proliferation and purification. Under difference of cell density, growth condition of MEF in vitro was observed. The result showed, the mouse foetus of 14.5 days was the best for culturing fibroblast cells. It can isolate a number of MEF, including a few parenchyma cells, and MEF proliferated rapidly. Therefore, vigorous growth and purification of the third passages of MEF suited to be a feeder layer of embryonic stem cells.2. By using intact embryo cultural method in the isolation and culture of mouse ES cells, mouse blastocysts and morulas were compared to the effects of the formation of ES Cell Colonies. Mouse blastocysts and morulas were cultured respectively on feeder layer. As far as the following indexes attaching ratio, ICM growth ratio and ES clone formation ratio are concerned, the choice of blastocysts is much better than morulas (P<0.01). Therefore, mouse blastocysts were more suitable than morulas to get ES cells. The rate of blastocysts collected at the 4th day was high.3. Three concentration of the digestion being used to digest ICM and the ES cells respectively was 0.25 % trypsin+0.04 % EDTA, 0.05 % trypsin+0.04 % EDTA and 0.02 % trypsin+0.01 % EDTA. The result showed, 0.02 % trypsin+0.01 % EDTA was used in the optical concentration for Kunming mouse ES cells, which is relative gentle to ES cells and is better for forming new ES colonies. Some of large cell aggregates can be separated by mechanical dissection.4. Using Generation 3 of MEF were selected.After treated with Mitomycin-C, living cell were inoculated at concentrations of 1x104 cells/mL, 1x106 cells /mL and 1x108 cells /mL after their number counted as the feeder layers of ES cells. The growth behavior of different density of MEF cells was observed. The blastulas of 4 days from Kunming mouse were collected, cultured on medium with different density of MEF. The growth of the blastulas, ICM and ES cells were observed. The result showed, the blastulas cultured on MEF with a density of 1×106 cells /mL showed that attachment rate and ICM growing rate were (97±3.606) % and (96.3±2.887) % respectively, which expressed more than those cultured on the other two kinds of feeders. The clone forming efficiency of ES cells on 1×106 cells /mL MEF were higher than those of on 1x104 cells /mL MEF (P﹤0.01) and on 1x108 cells /mL MEF (P﹤0.05). While the clone forming efficiency of ES cells on 1x104 cells /mL MEF and 1x108 cells /mL MEF were not significant (P﹥0.05). Therefore, as feeder celI of MEF with a density of 1×106 cells /mL is the best for isolation and culturing of embryonic stem cells in vitro, which can be beneficial for the development of blastulas, the proliferation of ICM, inducing the proliferation of ES cells, and inhibiting their diferentiation.5. The ES cells of Kunming mouse which were pluripotential cells have been identified by morphology of cells, colony form, alkaline phosphatase staining, karyotype analysis and the formation of embryoid body which are typical characteristics of ES cells. We found that these cells aggregated to form colonies with a clear borderline between colonies and feeder layer cells. Colonies were alkaline phosphatase-positive staining and had normal diploid karyotype. We also got embryoid bodies by non-adherent culture.
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