角膜内皮体外模型玻璃化法保存研究

Cryopreservation of in vitro model of corneal endothelia by vitrification

以牛角膜内皮细胞在培养至融合时的单层细胞作为角膜内皮的体外模型,采用两种玻璃化溶液,考察玻璃化过程对细胞活性的影响.首先以光学显微镜和扫描电镜考察了体外模型的细胞形态和胞间连接情况,发现培养的角膜内皮细胞融合后无论是细胞形态和胞间连接都与天然的角膜内皮相近.然后用低温显微镜考察了两种玻璃化溶液在升降温过程的结冰情况,发现两玻璃化溶液在100℃/min的降温和升温速率下都能玻璃化且反玻璃化较弱.设计了一套保护剂的导入和洗脱方案,使得内皮细胞的体积变化维持在-50%～40%.最后以CCK-8试剂盒考察了玻璃化溶液的毒性和细胞经冷冻后的活性,发现VS^2的毒性比VS^1强;经VS^1和VS^2冷冻后的细胞活率分别为61.3%和51.7%,这说明VS^1的保存效果比VS^2好.

As an in vitro model of natural corneal endotheliums, confluent monolayers of bovine corneal endothelial cells （BCECs） in culture were used in two unique vitrification solutions （VSs） to examine effect of vitrification processes on viability of cells. Investigations by the confluent monolayer of BCECs in culture was close to endothelium in situ microsco both in py showed that cell shapes and cell junctions. Observations by cryomicroseopy indicated that both the two VSs can vitrify during cooling at 100 ℃/min and weak devitrification occurred during warming also at 100℃/min. An optimal adding and removing change of cells within - 50 protocol of the VSs was designed by calculations to maintain %-40%. The toxicity test results of VSs measured by cell c volume oun kit-8（CCK-8） show that toxic injury of VS2 is more severe than VS^1. After preservation ting by vitrification, cell viabilities are 61.3% and 51.65% respectively for VS^1 and VS^2. Thus, cryoprotective effect of VS^1 is better than that of VS^2.