Yanbian Cattle Fibroblast Cell Culture, Cryopreservation And Nuclear Transfer Efficiency

We set up YanBian Cattle ear skin's fibroblast line, and went to freezing and preserving fibroblast by liquid nitrogen, get the best preserving system about YanBian Cattle ear skin's fibroblast, based on the tissue culture. Ovaries were obtained at YanBian slaughterhouse, and recovered the COC's, nuclear receptor were enucleated oocytes after maturation, nuclear donor were fibroblast, we investigated the biology characteristic of YanBian Cattle's fibroblast, the freezing effect and the application of cloning technique. The results were as follows:1, YanBian Cattle ear skin's fibroblast of subculture were derived by the lump of tissue, and developed by adherence with the wall of culture flask in vitro, the primary and earlier culture cells were the mixture of epithelium cells and fibroblast.2 The study demonstrated the repeated adherence method could pure fibroblast and epithelial cells line because the mixture of growing cells were treated in the medium with Trysin(0.25%) and EDTA(0.02%) for 2～3min at 37~C under atmosphere of 5% CO~2 in air with high humidity. A majority of floating cells were fibroblast.It showed that this method could obtain pure fibroblast through repeated adherence.3 The proliferation regulation of YanBian cattle ear skin's fibroblast was researched into drawing growth curve. It showed that the cells cultured by DMEM were in lag phase about one day, the cells came into logarithm growth about six days continuely. The cells took into abut inhibition and began death, and came into plateau phase about three days, cells fell off and died gradually, the amount was descend.4 The chromosome examination showed that YanBian Cattle's fibroblast could retain normal chromosome of double character at least fifth passage in feasible culture system.5 In freezing study of different concentration of glycerol and serum, the most appropriate concentration of glycrol was 10%, and serum was 20%, the thawing rate reached to 96%.6 In freezing study of different density and cryoprotectant, the most appropriate cell density was 2×10~6cells/mL, and the cryoprotectant was glycerol, the thawing rate could reach to 98%.7 In thawing fibroblast by 20~C and 37~C, the result showed that the thawing rate in 37~C was more than 20~C.8 This nuclear tranfere used cumulus cells and oviduct epithelium cells and skin's fibroblast, the results showed the development ratios have no differences, but the cleavage ratios have differences each other(P<0.05).9 Injected 5～7era and 11～13era fibroblast, the cleavage ratio by 5～7era was higher than by 11～13era(P<0.05), but the development ratio by 11～13era was higher than by 5～7era(P>0.05).10 Injected no-freezing and freezing fibroblast, the result showed that the cleavage ratio by lived cell was higher (P<0.05), but the development ratios were no differences (P>0.05).