摘要
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。
PURPOSE:To establish methods for cryopreservation of human retinal pigment epithelial cells(RPEs)and cell culture from thawing of frozen cells.METHODS:Primary cultured RPEs or its first or second passages,added with 10% dimethylsulfoxide,were kept in -20℃ for 1 to 2 hours,and then further froze to -40℃ over night before being placed in liquid nitrogen.The frozen cells were thawed in 60℃ within 2 minutes.Trypan blue staining and immunocytochemical staining with anti human keratin were performed for cell viability and differentiation.The growth curve was also determined by calculating the total number of cells/well/day.RESULTS:The viable rate from frozen RPEs was 90%.No differences were observed for growth activity between cultures from frozen cells and controls.The cells were positive with anti human keratin staining.The logarithmic growth phase was during 1 to 4 days and the doubling time was 1.55 days.CONCLUSION: Cryopreservation of RPEs in liquid nitrogen can maintain biological activities of cells with normal growth and features after thawing.This will provide cell lines for in vitro experiments and possibly for cell banks for RPE transplantation for some fundus diseases.
出处
《中华眼底病杂志》
CAS
CSCD
北大核心
1997年第3期157-159,共3页
Chinese Journal of Ocular Fundus Diseases
关键词
色素上皮
组织保存
低温保存
复苏培养
视网膜
Pigment epithelium of eye freezing Tissue preservation Cryopreservation
