Laboratory Biology, Engineering and Imaging of Corneal Grafts, JE2521, IFR143, Faculty of Medicine, University Hospital of Saint-Etienne, Saint-Etienne, France.
Ophthalmic Res. 2010;43(1):43-55. doi: 10.1159/000246577. Epub 2009 Oct 14.
To describe an innovative device that allows gene electrotransfer to human corneal endothelial cells (EC) during storage in organ culture.
Customized electrodes without endothelial contact were developed. Two plasmids containing the cytomegalovirus promoter and reporter genes [enhanced green fluorescent protein (eGFP) or beta-galactosidase (beta-gal)] were electroporated in 2 series of human corneas with eight 1-Hz 100-ms pulses of 125 mA square current. Controls were exposed to naked DNA without electric pulses. eGFP-transduced corneas were used to determine the transgene expression kinetics, whereas beta-gal measured transfection efficiency using image analysis tools. Overall, endothelial toxicity was determined by: (1) cytotoxicity tests using triple staining with Hoechst 33342, ethidium homodimer III, and calcein AM, 3 h and 3 and 14 days after electroporation on the series of 15 eGFP-transfected paired corneas; (2) anti-ZO-1 staining to assess tight junctions' integrity.
All electroporated corneas carried transfected ECs, whereas the controls carried none. eGFP expression was observed 3 h after electrotransfer, and was then present from days 1 to 28. Transfection efficiency determined on 63 corneas transfected with beta-gal ranged from 0.1 to 54% of the transfected ECs (mean +/- SD: 7 +/- 11%, median: 2.9%) with significant reproducibility for paired corneas from the same donor. Electroporation produced low early EC death. Anti ZO-1 staining revealed no dramatic change in EC mosaic continuity, neither 1 and 3 nor 28 days after electroporation.
Gene electrotransfer to the endothelium of organ-cultured human corneas with custom-designed electrodes allows rapid and easy EC transfection. However, further optimization is required to ensure reproducible results.
描述一种创新的设备，可在器官培养过程中对人眼角膜内皮细胞（EC）进行基因电转。
开发了无内皮接触的定制电极。用 125 mA 方波电流 8 个 1 Hz、100 ms 的脉冲对 2 组人眼角膜进行电穿孔，两组均含有巨细胞病毒启动子和报告基因[增强型绿色荧光蛋白（eGFP）或β-半乳糖苷酶（β-gal）]。对照组仅暴露于无电脉冲的裸 DNA。使用 eGFP 转导的角膜确定转基因表达动力学，而使用图像分析工具测量β-gal 的转染效率。总体而言，通过以下方法确定内皮毒性：（1）在 15 对 eGFP 转染的配对角膜系列中，电穿孔后 3 小时、3 天和 14 天，用 Hoechst 33342、ethidium homodimer III 和 calcein AM 三重染色进行细胞毒性试验；（2）用抗 ZO-1 染色评估紧密连接的完整性。
所有电穿孔的角膜都携带转染的 EC，而对照组则没有。电转后 3 小时观察到 eGFP 表达，随后在第 1 天至第 28 天存在。对 63 个用β-gal 转染的角膜进行的转染效率测定范围为转染 EC 的 0.1%至 54%（平均值±标准差：7±11%，中位数：2.9%），来自同一供体的配对角膜具有良好的重现性。电穿孔导致早期 EC 死亡较低。电穿孔后 1 天、3 天和 28 天，抗 ZO-1 染色显示 EC 镶嵌连续性无明显变化。
用定制电极对器官培养的人眼角膜内皮进行基因电转可实现快速、简便的 EC 转染。然而，需要进一步优化以确保可重复的结果。
