摘要
试验对荷斯坦奶牛耳组织成纤雏细胞的分离、体外培养及5种冷冻保存方法进行了研究。结果表明：用DMEM／F12＋20％胎牛血清＋100IU／mL双抗的完全培养液进行组织块培养，能获得良好的荷斯坦奶牛耳组织成纤维细胞原代培养物；成纤维细胞混合培养物经Trypsin-EDTA（0．25％Trypsin，1mmol／LEDTA．4Na）消化所收集到的细胞主要为成纤维细胞，经2—3代传代，可得到纯化的荷斯坦奶牛耳组织成纤维细胞。纯化培养的成纤维细胞在冷冻保护剂和血清成分相同的每件下，选用5种不同的冷冻保存方法进行冷冻保存，其中使用70％细胞悬液＋20％胎牛血清＋10％DMSO的冷冻保存悬液，先在4℃预冷平衡0.5h，接着在液氮罐口的气态氮中悬挂4h，然后沉入液氮的方法冻存的细胞，解冻后，经台盼蓝染色后进行细胞活力分析，活细胞率为86．68％；培养48h后细胞贴壁率为86．40％，明显高于其他几种方法。
Through isolation and pure culture of Holstein milk cow ear fibroblasts and analyses research on several cryoproservation methods in this studing, the results show the satisfactory primary culture of Holstein milk cow ear fibroblasts had been obtained by small tissue cubes culture with complete medium （DMEM/F12 ＋20% FCS ＋ 1001U/mL（P/S）. Most fibroblasts could be collected in the digestion of primary fibroblast .mass cultures by Trypsin- EDTA （0.25% Trypsin, l mM EDTA. 4Na）, through 2 to 3 subcultures the pure culture of Holstein milk cow ear fibroblasts could be implemented . On the conditions of the same cryoprotectant and FCS concentration Holstein milk cow ear fibroblasts were cryopreservated in five cryopresorvation methods, through the contrast and analyses of cell vigor after five months cryopreservation , method 3 （70% cell suspension ＋20% FBS ＋ 10% DMSO ; cryopreservation procedure： First fibroblasts were pre -cooled and balanced for half hour at 4℃, and second hanged in gaseous nitrogen in close to the mouth of nitrogen canister for 4 hours ,and finally sunk into liquid nitrogen, ） was the best cryopreservation method , it could maintain 84.8% of living rate after cells were revived and stained by Trypan Blue and 85.4% of adherence rate after cells were revived and cultured for 48 hours.
出处
《黑龙江畜牧兽医》
CAS
北大核心
2006年第5期9-12,共4页
Heilongjiang Animal Science And veterinary Medicine
基金
国家自然科学基金项目(30471242)
中科院"百人计划"2002年第1期
