Abstract: For improving the efficiency of isolation and culture of fibroblast cells from Bos mutus, effects on the isolation culture and subculture of Bos mutus somatic cells by organize preservation method, primary culture method, culture media type,and cryopreservation media formula were assessed. The results showed that PBS, D/F12 and TCM199 media adapted to preserve the skin organize of Bos mutus. D/F12 and TCM199 media could be used to the culture of Bos mutus organize pieces, and the survival rate of organize pieces reached (86.29±4.62)%. So organize pieces culture could be successfully used to primary culture of Bos mutus fibroblast cells. Better effect was achieved by the culture of Bos mutus fibroblast cells in D/F12 media, and population doubling time as well as plateau phase density of the cells were 38.47 h and 2.075×10⁶/mL, respectively. The percentage of normal diploid karyotype of F7 cells was 84.33% in the D/F12 culture media. Obvious proliferation effect on the cells was produced with the supplement of EGF and bFGF into culture media, and plateaued phase density of the cells obviously rised. Two cryopreservation media (D/F12 supplemented with fetal bovine serum and DMSO, fetal bovine serum supplemented with DMSO) could be used to cryopreservation of Bos mutus cells, and the survival rates of the cells were 87.33% and 89.00%, respectively.

Key words: Bos mutus; cell culture; fibroblast cell; cell growth factor