Studies Related To Culture Of Buffalo Fetal Fibroblasts In Vitro

1. In vitro culture system of buffalo fetal fibroblasts (BFF) was investigated in this study. Line of buffalo fetal fibroblasts was established by explant culture technique and was subcultured to 32 passages with pre-cold 0.25% trypsin. Moreover, there was 70-80% of cells survived after frozen and thawed. Karyotype analysis indicated that 80-90% of cells had normal karyotype, which did not show significant difference among the 5th, 10th and 15th passages (p>0.05). NESTED-PCR analysis showed that the 9th, 18th, 28th passage cells had no mycoplsma contamination, with the exception of the 32th passage cells. In conclusion, this culture system was suitable and relatively safe to culture buffalo fetal fibroblasts, and keep relatively stable karyotype.2. The effects of different additives on the proliferation of low densities BFF (1, 2, 4, 6, 8 cells/50 L) were studied. The results showed that the average proliferation index of 2IU/mL insulin was significantly higher than that of conditioned medium and control (3.49 0.07 vs 1.47 0.29, 2.97 0.17; p<0.05), and was increased gradually with the increasing of cultured cells density. The average proliferation index of 8 cells/50ul was significantly higher than that of 1, 2 cells/50uL (3.11 ?.46 vs 2.09?.83, 2.48+0.61; p<0.05).This result shows that insulin at appropriate concentration can promote the proliferation of buffalo fetal fibroblasts, and single cell can be cultured in vitro, but the proliferation speed is lower than that of group culture.3. The cell cycle of BFF treated by different methods was examined. Treatment of BFF with serum direct starvation or gradient starvation resulted in a significant increase in percentage of cells arrested at G0/G1 stage in comparision with that of control group (p<0.05), but there was no significant difference between the serum direct starvation and gradient starvation groups. When BFF was treated with serum direct starvation for 3d, the percentage of cells in G0/G1 stage are significantly higher than that of 0d, 1d, 2d, 6d (p<0.05). These results indicate that BFF treated with serum direct starvation or gradient starvation can efficiently arrest them at Go/Gi stage, and the optimum duration of serum starvation is 3d.