Culture Methods Of Mouse And Porcine Fibroblasts And Cell Cycle Control

The purposes of this experiment were to establish cuture systems of fibroblasts and methods of cell cycle synchronization so as to provide better donor cells for somatic nuclear transfer.In this experiment, the method of trypsin digestion was improved, and that several kinds of mouse fibroblasts and porcine fibroblasts were successfully dissociated and cultured.During the culture of the porcine ear skin fibroblasts, the successful rate was increased by selecting culture methods, contrasting donor age and improving culture condition.The rate of living cells in the method of combining trypsin cold treatment with trypsin heat treatment was much higher than that in the method of trypsin heat treatment.The results showed that the method of combining trypsin cold treatment with trypsin heat treatment had less adverse effects on cells than the method of trypsin heat treatment. The harvest quantity of cell in the method of combining trypsin cold treatment with trypsin heat treatment was higher than that in the method of trypsin heat treatment. The culture of young individual's cells was easier than that of adult individual's cells. The culture results of the porcine ear skin fibroblasts was good by using DMEM/F12 containing 20% fetal bovine serum. The apoptosis in the passage cells was detected by using flow cytometry.It was fmded that the apoptosis of passage 12 cells was not obvious. Cells were subcultured to sixteenth passage.It proved that the subculturing of porcine ear skin fibroblasts was stable by using this kind of method combining trypsin cold treatment with trypsin heat treatment.Normal development of nuclear transfer embryos was thought to be dependent on transferral of nuclei in G0 or G1 phases of the cell cycle. Therefore,the cell cycle characteristics and the apoptosis of porcine ear skin fibroblasts cultured under a variety of cell cycle-arresting treatments were investigated.This was achieved by using flow cytometry to simultaneously measure cellular DNA and protein content, enabling the calculation of percentages of cells in G0, G1, S and G2+M phases of the cell cycle. Compared to values in cycling cells, higher percentages of cells in GO+G1 and GO alone were apparent when cells were serum starved or cultured to confluency or treated with aphidicolin.Separate assessment of small-, medium- and large-sized cells showed that as the cell size decreased from large to small, percentages of cycling cells, starved cells and confluent cells in G0+G1 and G0 alone increased remarkably, percentages of aphidicolin treatment cells in G0 increased obviously. Percentages of small cells after confluency treatment in G0 increasedremarkably and were higher than those of other treatments obviously. The effects of these treatments were reversible, and their removal led to a rapid progression in the cell cycle.It was finded by using flow cytometry that the apoptosis rate of porcine ear skin fibroblasts cultured under a variety of cell cycle-arresting treatments was quite low. The apoptosis rate of confluent cells was lowest among a variety of cell cycle-arresting treatments.These data indicated that porcine ear skin fibroblasts could be effectively synchronized at GO-H31 and GO alone without compromising their proliferation capacity.