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组织培养技术应用广泛,但是组织培养成本高,特别是小牛血清,即价格昂贵又日趋短缺。培养原代兔婴肾细胞通常用含10%小牛血清的199培养液,培养2天左右换液,4～7天长成单层细胞。我们在实践中观察到细胞贴壁和生长速度似乎与培养液中血清含量关系密切,特进行本实验,探索节省血清的培养方法。按常规取出兔婴双侧肾脏,将肾皮质组织放入10 ml小瓶中,剪成约1mm~3大小的碎块,加入0.25%胰酶-0.02%EDTA液,于37℃消化20～30分钟。然后倾去消化液,用Hank′s液洗一次,加2～3 m1培养液,以毛细吸管将碎块吹打成乳糜状,再加培养液至10ml,静置数分钟,待未吹散的组织块沉降后,收集上部的细胞悬液。按此方法重复2次后,弃去沉降物,将几次收集的细胞悬液混合均匀,计数。培养采用含15%小牛血清的199培养液,其中含100u/ml青霉素和10ug/m1的链霉素。用该生长液把混合均匀的细胞悬液稀释
Tissue culture techniques are widely used, but the cost of tissue culture is high, especially for calf serum, which is expensive and increasingly scarce. Primary rabbit kidney cell culture is usually cultured with 199 medium containing 10% fetal bovine serum, cultured for about 2 days to change fluid, 4 to 7 days into monolayers. In practice, we observed that cell attachment and growth rate appeared to be closely related to the serum content of the culture broth. This experiment was specially conducted to explore ways to save serum. Rabbits were removed according to conventional bilateral kidneys, renal cortical tissue into 10 ml vials, cut into pieces about 1mm ~ 3 size, adding 0.25% trypsin - 0.02% EDTA solution, digested at 37 ℃ for 20 to 30 minutes . Then digest the digestive juice, wash once with Hank’s liquid, add 2-3 ml of culture broth, pipette the pieces with a capillary pipette into a chylous, add the medium to 10 ml, let it stand for a few minutes until it is not blown After the tissue mass has settled, collect the upper cell suspension. Repeat this method 2 times, discard the sediment, the cell suspension collected several times mixed evenly, counting. The culture consisted of 199 medium containing 15% calf serum containing 100u / ml penicillin and 10ug / ml streptomycin. Dilute the mixed cell suspension with this growth solution