改良揭膜消化法分离培养恒河猴角膜内皮细胞

Modified “peel-and-digest”procedure for isolation and culture corneal endothelial cells in Rhesus monkeys

目的分析改良揭膜消化法分离培养恒河猴角膜内皮细胞的可行性。方法揭取恒河猴角膜后弹力层和内皮层,反复剪碎,用1 mg·m L^(-1)胶原酶A消化15 min,离心后重悬接种。待原代细胞长满80%~90%时,以40×106个·L^(-1)的密度传代。观察记录原代及传代细胞生长情况、传代时间,进行细胞鉴定,并与组织贴壁法相比较。结果改良揭膜消化法获取的原代细胞生长较慢,21 d融合成单层铺路石样结构,无基质细胞污染;传代细胞生长较快,5~6 d可传一代;神经元特异烯醇化酶表达阳性。组织贴壁法分离组织24h后组织片体积变小,透亮度明显下降,疑似溶解,高倍镜下观察显示,后弹力层纤维排列紊乱,细胞观察不清;继续培养至第5天,细胞完全溶解,组织片碎裂,轮廓不清,未见角膜内皮细胞爬出。结论改良揭膜消化法能简易高效地获得大量恒河猴角膜内皮细胞。

Objective To analyze the feasibility of a modified＂peel-and-digest＂procedure for the isolation and culture of corneal endothelial cells from Rhesus monkey corneas. Methods Descemet’s membranes were stripped from Rhesus monkey corneas,and then sheared and digested for 15 minutes with 1 mg · m L-1collagenase A. After centrifuge,the precipitation was performed to resuspend and culture. When cells reached 80%- 90% confluence,corneal endothelial cells were plated at a density of 40 × 106L-1. The grow th characteristics of primary and passaged corneal endothelial cells were investigated,and the time for spreading over dish was recorded. The cultured cells were identified. This modified method was compared with tissue-piece inoculation for the isolation of corneal endothelial cells. Results The primary corneal endothelial cells harvested from this modified ＂peel-and-digest＂procedure grew slowly,which could form an intact monolayer and consisted of paving-stone shape without stromal cells after 21 days Subcultured corneal endothelial cells proliferated fast,which could be passaged in 5- 6 days averagely. NSE was expressed in them. The corneal endothelial cells harvested from tissue-piece inoculation became small and dissolved,the fiber in the posterior elastic layer arranged disorder,and the cells could not be observed clearly. And the cells were all dissolved after culture for 5 days,the tissue slice was fractured,no corneal endothelial cells grew out. Conclusion The modified ＂peel-and-digest＂method can harvest many available corneal endothelial cells simply and efficiently.