人视网膜色素上皮细胞原代培养冻存和复苏方法改进

An improved primary culture and freezing and recovery method of human retina pigment epithelium

目的优化建立一种简单有效的人视网膜色素上皮细胞(RPE)的原代培养、冻存和复苏的方法,对细胞进行鉴定,为研究RPE的功能和治疗有关眼病提供细胞来源。方法采用0.25%胰酶+0.02%乙二胺四乙酸(EDTA)二次消化分离细胞并进行传代,以0.125%胰酶进行传代,用免疫组织化学检测进行角蛋白与S100表达鉴定RPE。利用-80℃冰箱对细胞冻存,复苏后观察细胞活性。结果 RPE体外生长旺盛、1 h贴壁。初期为圆型,胞壁内充满黑色色素,传代后细胞呈圆型、不规则形,细胞透亮。免疫组织化学角蛋白与S100均呈强阳性,证实培养的是RPE细胞,所获细胞纯度100%。冻存细胞复苏后生长旺盛,细胞形态与复苏前相比无区别。结论二次胰蛋白酶消化分离细胞数量多,能成功建立RPE的体外培养模式,用-80℃冻存RPE的方法简便,成本低,冻存复苏后细胞存活率及细胞形态不受影响,是目前较理想的RPE细胞保存方法。

Objective To establish the method of a simple, effective and optimized primary culture, freezing and recovery to human retina pigment epithelium （RPE） was established. By using this method, RPE was identified. This study yielded a large amount of RPE to investigate RPE function and treat related with ocular diseases. Methods RPE was isolated by twice digestions of 0.25% pancreatin and 0.02% EDTA, respectively. Following digestion, the obtained ceils were passaged by 0.125% pancreatin. Finally, RPE was identified by detecting the expressions of keratin and Sl00 using immunohistoc-hemical method. The harvested RPE was freezed in -80 ℃ freezer. The cellular activity was assessed following recovery.Results RPE showed vigorous growth when cultured in vitro, and cell attachment appeared 1 h later. During early culture stage, the cells were round and the cellular walls were filled with black pigment. After passage, the ceils became transparent with round or irregular shapes. The strong positive expressions of keratin and SIO0 protein were detected. Together with these findings, the results verified that the harvested cells were RPE with a purity of 100%. Even though RPE were pre-freezed, these cells still grew vigorously following recovery. No significant difference was noted regarding cellular morphology before and after recovery. Conclusion After twice digestions of trypsin, a large amount of RPE can be isolated, ensuring the successful establishment of RPE culture in vitro. The freezing method of RPE in -80 ℃ freezer is a simple and low-cost option. Moreover, there is no significant difference in cellular morphology before and after recovery. Therefore, it is a preferred storage way for RPE.