Comparison of non-viral methods to genetically modify and enrich populations of primary human corneal endothelial cells.
OBJECTIVE
To compare different techniques of transfection of primary human corneal endothelial cells (HCECs) by non-viral methods and to enrich genetically modified cells to a highly pure population.


METHODS
HCECs were cultured following previously published methods. Dissection of the Descemet membrane (DM) was performed by tearing off strips from corneal buttons with forceps or by hydrodissection. Confirmation of HCECs identity was performed by reverse transcriptase polymerase chain reaction (RT-PCR) for alpha2 collagen VIII. For transfection, non-viral methods such as lipid-/liposome-mediated reagents and electroporation techniques were compared. Genetically modified cells were enriched by use of selection antibiotics and flow cytometry.


RESULTS
Viability of primary HCECs was lower in hydrodissected corneas. The rate of transfection varied from approximately 5%-30%. Highest rates of transfection were obtained with the Amaxa electroporation method. The next highest rate was yielded by the lipid-mediated reagent GenCarrier2, followed by electroporation with the BTX apparatus. Toxicity was moderate and manageable by adjusting the concentration of reagents, incubation times, and electrical parameters. Selection by flow cytometry was superior to antibiotic selection and produced nearly 100% genetically modified cells.


CONCLUSIONS
Electroporation of HCECs yields higher transfection efficiency than chemically mediated methods. It is possible to select genetically modified HCECs to high levels of homogeneity. Techniques to genetically modify and select HCECs as shown in this study could lead to improved success of future endothelial transplant procedures.
Wilmer Eye Institute, The Johns Hopkins Medical Institutions, Baltimore, MD 21287, USA.
Christoph
C
Engler
Clare
C
Kelliher
Karl J
KJ
Wahlin
Caroline L
CL
Speck
Albert S
AS
Jun
D000293:Adolescent; D000328:Adult; D000368:Aged; D002478:Cells, Cultured; D024102:Collagen Type VIII / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism*; D004728:Endothelium, Corneal / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D008875:Middle Aged; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014162:Transfection / Q000379:methods*; D014780:Viruses / Q000235:genetics*
C443712:COL8A2 protein, human; D024102:Collagen Type VIII; D018076:DNA, Complementary