人RPE细胞培养、冻存、复苏及传代研究

STUDY ON CULTURE, REFRIGERATED STORAGE, REVIVIFICATION AND PROLIFERATION OF HUMAN RPE CELLS

目的：建立人视网膜色素上皮（retinalpigigmentepithelium,RPE）细胞体外培养模式，为研究RPE细胞及有关的眼底病防治提供细胞模型和细胞来源。方法：自眼球锯齿缘前2mm处环形剖开眼球，酶消化分离RPE细胞，20％DMEM培养液培养，细胞接近融合状态时进行传代，将处于对数生长期细胞以普通冰箱进行梯度降温后液氮保存，采用改良的速溶方法复苏，1％台盼蓝确定细胞活力，免疫组化染色鉴定细胞来源，作生长曲线观察细胞增殖。结果：原代细胞生长良好，免疫组化染色阳性率为100％，改进冻存复苏方法后，细胞存活率提高20％以上。结论：基本建立了人RPE细胞体外培养模式，改进传统的培养方法后，效果理想。

Objective: To establish model culturing of humanretinal pigment epithelium (RPE) cells in vitro, and tosupply cellmodel for the study of RPE cells and the prophylaxis and treatment of relevant oculur fundus diseases.Method: Eyes were dissected cireumferentially 2 mm anterior to the ora serrate; RPE cells were separated with enzyme andwere cultured in 20% DMEM medium. Cells were subcultured when they grew near confluence. Cells in logarithmicgrowth phase were placedin refrigerator for gradient cooling and then were preserved in liquid Nitrogen(-196℃).proved instant thaw method was used to revive the RPE cells, and the viability of cells was determined by trypan-bluedye exclusion. The origin of cells was identifiedby immunohistochemical staining for cytoleratin and growth curve wasused to evaluate the proliferation of cells. Results: The primary cells grew good and all cells were stained for cytoeratins.Tth rate of survival of cells increased over 20% after improved freeze and instant thaw methods. Conclusion:: HumanRPE cell culture model was basically established and mere ideal results were obtained by improvement onthe traditionalfrozen and thaw methods.