title: Ex vivo gene electrotransfer to the endothelium of organ cultured human corneas.
abstract: OBJECTIVE
  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.
authors.affiliation: Laboratory Biology, Engineering and Imaging of Corneal Grafts, JE2521, IFR143, Faculty of Medicine, University Hospital of Saint-Etienne, Saint-Etienne, France.
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: He
authors.forename: Aurélien
authors.initials: A
authors.lastname: Pipparelli
authors.forename: Chloé
authors.initials: C
authors.lastname: Manissolle
authors.forename: Sophie
authors.initials: S
authors.lastname: Acquart
authors.forename: Olivier
authors.initials: O
authors.lastname: Garraud
authors.forename: Philippe
authors.initials: P
authors.lastname: Gain
authors.forename: Gilles
authors.initials: G
authors.lastname: Thuret
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D016923:Cell Death; D003316:Corneal Diseases / Q000628:therapy; D016039:Corneal Transplantation; D004566:Electrodes; D018274:Electroporation / Q000295:instrumentation / Q000379:methods*; D004728:Endothelium, Corneal / Q000166:cytology / Q000378:metabolism*; D005260:Female; D018014:Gene Transfer Techniques / Q000009:adverse effects / Q000295:instrumentation*; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D008297:Male; D008875:Middle Aged; D009924:Organ Culture Techniques; D015203:Reproducibility of Results; D019108:Tight Junctions / Q000648:ultrastructure; D013997:Time Factors; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase