Studies On The In Vitro Culture And Culture Condition Of Bull In Blue Fibroblasts

The present experiment aims to establish Bull in Bine skin cell line for to clone bull and othe biological research. The culture methods and characteristics of Bull in Blue skin fibroblast cell were also studied. The results as follows:1. Primary culture of Bull in Blue skin cell could be derived by outgrowth of migrating cells from fragment of tissue. Fibroblast and epithelial cells could be pured according to their different sensibility to trypsin. Complex cultures treated by 0.25% trypsin+ 0.02% EDTA for 5 min at 37 , the dispersed cells were mainly fibrollasts. The pured Fibroblast cell line could be obtained by 2-3passages.2. Bull in Blue Skin tissue digested by 0.25% trypin for 2h at 37癈 could provided a high yield of viable proliferating fibroblast for primary culture.3. Using RPMI1640 containing 10% NCS, 100Iu/ml Penicillin and 100Iu/ml Streptomycin as basic media, 5 g/ml FGF and 2 g/ml insulin were added and selected to culture Bull in Blue fibroblasts. The resule suggested that RPMI1640 supplemented with 2 g/ml Insulin had the better protective effect to the growth of fibroblasts. However, the fibroblasts growth in medium with and without FGF had no significant difference.4. After used medium in eluding 0.5% NCS and without NCS direct starvation, the numbers of fibroblast first in creased slightly, then gradually decreased. During the serum gradient serum stouration, after bing changed medium including4%, 2% and 1% NCS, the nembers of fibroblasts still rose a little; after being changed medium including 0.5% NCS, The numbers gradually declined.5. The fibroblasts of Bull in Blue were frozen in RPMI1640 with serum and antibiotics by adding 10% ethylene, 10% DMSO and 10% glycerol respeltively. The attachment rates of thawed cells after 36h were 70.4%, 80.85%, 86.53% individually. Thetreatment with 10% DMSO had the best effect, which can meet the needs of fisroblast passage and log-term preservation.6. Bull in Blue fibroblasts of passage 5 were cryo-preserved by method of embryofreezing with temperature decreasing rate of 0.3 , 0.6 0.9 and 1.2 per minute. The attacnment rates thawed was 76.8%~88.6%.The result suggests that the proper equilibrium duration is 2 hours.7. The commore method of embryo freeing is fit to fibroblarts.8. Bull in Blue thawed fibroblasts could be cryo-presened repeatedly. The attached ratesof the second thawed cells after 36h were 87.3%.