Ex vivo Gene Electrotransfer to the Endothelium of Organ Cultured Human Corneas

Abstract

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 (β-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 β-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 β-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.

Wounding the cornea to learn how it heals

“…Building on the solid background of research done using organ cultures, an in vitro organ culture system was recently described to study corneal scarring using donor human corneas (Janin-Manificat et al, 2012). Much attention has been given to optimizing conditions to allow using primate and human corneas in organ culture models (He et al, 2010; Janin-Manificat et al, 2012; Saghizadeh et al, 2010). The cornea derives its nutrients primarily from the aqueous humour that contains low levels of serum-derived proteins and growth factors.…”

“…The cornea derives its nutrients primarily from the aqueous humour that contains low levels of serum-derived proteins and growth factors. The media used for organ culture studies include those that have defined components and are serum–free, or have reduced serum (Stepp et al, 1993; Zheng et al, 2013; He et al, 2010; Pipparelli et al, 2013; Saghizadeh et al, 2010; Saghizadeh et al, 2011) although some groups use 8% FCS in their media (Kryczka et al, 2012). Most protocols maintain corneas at an air-liquid interface which is achieved by maintaining the height of the media in the dish at the limbal border and gently rocking it so that media intermittently moistens the surface of the cornea.…”

“…Growth factors, function blocking antibodies, and various activators and inhibitors can be added to organ cultures to assess their impact on cell migration, proliferation, and/or scarring. Transfection studies, using human corneas, have been performed in organ culture for times up to several weeks (He et al, 2010; Jessup et al, 2005; Saghizadeh et al, 2010; Saghizadeh et al, 2011). …”

Delivery of macromolecules into the endothelium of whole ex vivo human cornea by femtosecond laser-activated carbon nanoparticles

“…Alternative methods consist of applying physical stimuli to generate nanometric holes in the cell membrane, in order to deliver molecules directly into the cytoplasm, bypassing biological pathways. Electric pulses and ultrasound have already been tested on corneal cells, but showed a low delivery rate and/or low reproducibility 13 14. Another physical stimulus is laser irradiation, which perforates cells by focusing a laser beam on their surface.…”

Pore size assessment during corneal endothelial cells permeabilization by femtosecond laser activated carbon nanoparticles

“…Finally, physical methods consist to generate nanoscale holes in cell membranes by physical stimuli in order to deliver genes and drugs directly into cytoplasm, we called this phenomena cell permeabilization. Membrane pores can be generated efficiently by several types of physical stimuli: electric pulses [9], ultrasounds [10], laser irradiation.…”