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目的：建立人视网膜色素上皮（ｒｅｔｉｎａｌｐｉｇｍｅｎｔｅｐｉｔｈｅｌｉｕｍ，ＲＰＥ）细胞的冻存和复苏方法。方法：根据慢冻速融的细胞冻存原则，将原代或传代１～２次培养的人ＲＰＥ细胞，加入１０％二甲基亚砜作为保护剂，液氮中冻存。复苏时将冻存细胞在６０℃、２分钟内融化，用台盼蓝染色测定细胞存活率，用抗人角蛋白抗体免疫细胞化学染色作细胞鉴定，每日计算细胞数以确定细胞生长曲线。结果：经冻存复苏后的ＲＰＥ细胞的存活率达９０％，生长状态与对照组无差异，抗人角蛋白染色阳性，细胞对数生长期在１～４天，群体细胞倍增期约为１．５５天。结论：人ＲＰＥ细胞液氮冻存可保持生物活性，复苏后生长良好，保持了细胞特征。这可提供细胞系，方便了体外实验，并可能作为ＲＰＥ细胞移植治疗一些眼底病的细胞库。
Objective: To establish a method of cryopreservation and resuscitation of human retinal pigment epithelium (RPE) cells. Methods: According to the principle of cryopreservation, the primary or passage cultured human RPE cells were added with 10% dimethylsulfoxide (DMSO) as protective agent, and stored in liquid nitrogen. During resuscitation, frozen cells were thawed at 60 ° C for 2 minutes. Cell viability was measured by trypan blue staining. Immunocytochemical staining with anti-human keratin antibody was used to identify the cells. The number of cells was calculated daily to determine the cell growth curve. Results: The survival rate of RPE cells after cryopreservation and resuscitation was 90%. There was no difference between the growth conditions and the control group, and the anti-human keratin staining was positive. The logarithmic growth phase was between 1 and 4 days and the population doubling period was about 1 .55 days. CONCLUSION: Human RPE cells remain viable in liquid nitrogen and grow well after resuscitation, maintaining cell characteristics. This provides cell lines, facilitates in vitro experiments, and may serve as RPE cell transplantation for the treatment of some fundus fundus cell bank.