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Y. Pan, D. O. Zamora, B. Appukuttan, A. V. Moses, J. T. Rosenbaum, S. R. Planck, J. R. Smith; Immortalization of Human Ocular Endothelial Cells for Study of Ocular Vascular Diseases. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2940.
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© ARVO (1962-2015); The Authors (2016-present)
Vascular endothelial cells (ECs) are critical participants in the development of blinding diseases, including diabetic retinopathy, age-related macular degeneration, uveitis and neovascular glaucoma. Previously we have reported methods to isolate ECs from human retina, choroid and iris. Primary cultures yield a limited quantity of ECs that are insufficient for some studies of disease mechanisms. We investigated the possibility of obtaining large numbers of ocular EC, without compromising the cell phenotype, by immortalizing the cells using a murine retroviral construct encoding the E6/E7 oncogenes of human papilloma virus 16 and a gene conferring G418 antibiotic resistance.
Ocular ECs were isolated separately from retina, choroid and/or iris of both eyes of human cadavers, by digestion with ≤ 3 mg/mL type II collagenase and ≤ 0.3 mg/mL Dispase, followed by separation using anti-human CD31 antibody-coated magnetic beads (Dynal-Invitrogen). Cells were cultured under standard conditions in MCDB-131 medium with 2-10% FBS and growth factors (Clonetics, EGM-2 SingleQuot) and re-purified with magnetic beads as necessary. Actively proliferating ECs were exposed for 24 hours to the murine amphotropic recombinant retrovirus, LXSN, harvested from PA317 packing cells, with 5 µg/mL polybrene in some cases, and subsequently cultured in the presence of G418 for 10 days. Selected transduced EC isolates were immunostained for CD31 and von Willebrand factor (VWF), and tested for formation of capillary-like tubules. PCR for E6/E7 was performed on DNA extracted from ECs.
After successful isolation, a total of 27 different human ocular EC isolates (9 retinal; 9 choroidal; 9 iridal) were subjected to the immortalization protocol. Of these isolates, we judged that 9 were successfully immortalized on the basis of growth to ≥ 7 passages in culture with ≥ 1:3 split. Multiple magnetic bead separations were needed to maintain 99% purity of the ECs. Immortalized ECs had cobblestone morphology, and growth was contact inhibited. Retention of EC phenotype was confirmed for selected isolates by expression of CD31 and/or VWF (n=8) and formation of capillary tubes on extracellular matrix (n=4). PCR verified the integration of E6/E7 into the EC genome.
Human ocular ECs may be expanded by a straightforward immortalization protocol, without loss of phenotype, although the success rate is less than 50%. We anticipate that application of this procedure will provide substantial numbers of ECs for use in studies of the pathogenesis of multiple ocular diseases.
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