原代人视网膜色素上皮细胞的冻存和复苏培养

Cryopreservation and thawing of primary human retinal pigment epithelial cell

目的 :观察对原代人视网膜色素上皮 (retinalpig mentepithelium ,RPE )细胞进行冻存和复苏培养的效果 .方法 :将 6只尸供眼 ,常规酶消化法分离、纯化的人原代RPE细胞 ,分为两组 ,实验组为即刻进行常规梯度降温液氮冷冻保存 ,1wk后行常规复苏培养 ;对照组为细胞直接进行后续实验 .台盼蓝拒染观察细胞存活率及生长状态 ;计算细胞贴壁率 ;初步计算克隆形成率 .结果 :RPE细胞存活率分别为试验组 (90± 7) % ,对照组 (87± 1 5 ) % ,两组相比无显著性差异(P >0 .0 5 ) ;两组接种细胞贴壁率从 2 4h开始分别为 (5 .0±3.1 ) %～ (6 1 .0± 7.3) %和 (1 5 .0± 6 .3) %～ (6 0 .0± 3.3) % ;培养 4 8h之前 ,两组之间存在统计学差异 (P <0 .0 5 ) ,4 8h之后组间无显著性差异 (P >0 .0 5 ) ;培养 5 ,7和 1 0d初步克隆形成率两组分别为 (1± 0 ) % ,(1± 1 ) % ,(3± 1 ) %和 (1±0 ) % ,(3± 1 ) % ,(2± 1 ) % ,组间无显著性差异 (P >0 .0 5 ,n=3) .冻存后复苏培养的RPE细胞生长状态良好 ,增生活跃 ,与对照组类似 .结论 :对原代人RPE细胞进行冻存 ,复苏培养的RPE细胞活性、存活能力、单细胞分裂增生能力不受影响 .

AIM: To investigate the effect of resuscitaion culture on cryopreserved primary human retinal pigment epithelial (RPE) cell thawed. METHODS: The purified primary RPE cells, isolated from 6 eyes with trypsin digestion, were divided into 2 groups. The experimental RPE cells were immediately cryopreserved, after 1 wk, thawed routinely and then used for the next observations; While the control RPE cells were directly used for the subsequent experiments. Trypan blue rejection stainings were performed to evaluate the RPE cells viability; The attached RPE cell numbers and the remarkable cell clone formation ratios were counted at 5 d, 7 d, 10 d respectively, whilst the RPE cell growth were observed and described. RESULTS: The experimental and the control RPE cells viability ratios were (90±7)% and (87±15)% respectively ( P >0.05); After 24 h culture, the cells in 2 groups showed the adherent ratios (5.0±3.1)%-(61.0±7.3)% and (15.0±6.3)%-(60.0±3.3)% respectively, whilst the data before 48 h displayed significant differences ( P <0.05); At 5, 7 and 10 d, the cell clone formation ratios were (1±0)%, (1±1)%, (3±1)% and (1±0)%, (3±1)%, (2±1)% respectively in the two groups, no statistical significance was observed. CONCLUSION: The thawed primary RPE cells showed potential viabilities as well as the freshly dissociated RPE cell.