兔視網膜色素上皮細胞的培養與保存

目的探索一种有效的体外分离兔视网膜色素上皮(RPE)细胞的良好方法,并观察RPE细胞冻存复苏前后的形态学特点。方法用两步酶法分离兔RPE细胞,第一步酶(透明质酸酶 胰蛋白酶),用于松解视网膜神经上皮细胞与RPE细胞之间的连接;第二步(胰蛋白酶)用于离散RPE细胞,制成细胞悬液;然后用慢冻速融方法对细胞进行冷冻保存及复苏,最后用4种方法(RPE片层观察、H-E染色、免疫组化法及光、电镜观察法)观察RPE细胞在冻存前后的形态特点。结果 用此方法分离培养的细胞其结构完整,纯度高,收获量多,在体外生长旺盛,达到铺满汇合的时间缩短,同时维持体内细胞的许多生物学和形态学特点。细胞冻存复苏后的存活率高达80％左右,且与冻存前细胞形态相比未见明显差别。结论 此分离培养方法及冻存复苏法是高效可行的。

Isolation, culture and preservation of rabbit retinal pigment epithelial cells invitro

Objective To investigate an effective method of isolating the rabbit retinal pigment epithelial (RPE) cells invitro and to observe the morphologic characteristics of the RPE cells before or after freezing and recovery. Methods Two-step enzyme method was used to isolate the rabbit RPE cells. The first enzyme (hyaluronidase and trypsin) was used to loosen the connection between RPE and the neural retina. The second enzyme (0.25% trypsin) was used to dissociate RPE cells. Then the RPE cells were frozen, preserved and recovered through slow freezing and quick melting. At last, we studied the morphologic characteristics of the cells before and after freezing and recovery through RPE sheet, hematoxylin-eosin stains, immunohistochemical method, light and electron microscopy. Results This procedure gave RPE cells intact morphology, higher purity, high quantity, vigorous growth, short converging time and maintained a lot of biologic and morphologic characteristics invivo. The survival after cell recovery was about 80%. The morphology of cells was almost the same before or after freezing and recovery. Conclusion This methods is efficient and feasible.