AIMS: To describe an innovative device that allows gene electrotransfer to human corneal endothelial cells (EC) during storage in organ culture. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. Copyright 2009 S. Karger AG, Basel.
AIMS: To describe an innovative device that allows gene electrotransfer to human corneal endothelial cells (EC) during storage in organ culture. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. Copyright 2009 S. Karger AG, Basel.
Authors: Mary Ann Stepp; James D Zieske; Vickery Trinkaus-Randall; Briana M Kyne; Sonali Pal-Ghosh; Gauri Tadvalkar; Ahdeah Pajoohesh-Ganji Journal: Exp Eye Res Date: 2014-03-04 Impact factor: 3.467