Abstract
Purpose :
Significant challenges with current techniques to reproducibly grow RPE monolayers remain, and it is unclear which models are the most physiologically relevant. Here, we studied various conditions of long-term primary human RPE (hRPE) culture to optimize techniques for reproducible development of functional, polarized RPE monolayers.
Methods :
Early passage (p3-5) hRPE cells (Lonza) were seeded at densities of 0.5, 1, and 2 x 105 cells per cm2 (n=2-4). For media, hRPE were initially fed RPE cell Growth Medium Bullet Kit (RtEGM, Lonza) or RPE culture medium (RPECM, alpha-MEM plus 1% N1 Supplement, 1% Glutamine-Penicillin-Streptomycin, 1% non-essential amino acids, 250 mg/L taurine, 20 mg/L hydrocortisone, 0.013 mg/L triiodo-thyronine) with 1, 5, 10% FBS on transwell plates coated with human fibronectin (2 μg/cm2). RPE cultures with RtEGM media were either left with same media type or switched to RPECM (1, 5, 10% FBS) at 2d while cells with RPECM were kept in that media. TER, ZO-1 immunofluorescence, and pigmentation were evaluated at various time-points (14d/21d/28d). Statistical analyses were performed with ANOVA and Mann-Whitney U test.
Results :
Long-term hRPE cultures with high-density seeding of hRPE isolates (2 x 105) yielded the most tightly packed hexagonal arrays with the peak TER values (P<0.05) by 21d. Cultures seeded in RtEGM and then switched to 1%, 5%, and 10% FBS in RPECM demonstrated significantly increased TER measurement (> 400 Ω cm2, P<0.05) compared with RtEGM (250 – 350 Ω cm2) or RPECM (20–50 Ω cm2). Cultures seeded in RtEGM initially and then switched to RPECM at 2d reproducibly formed monolayers with increased pigmentation (3x) compared to cultures in RPE culture media alone.
Conclusions :
Establishing reproducible long-term primary hRPE cultures is critical to experimental designs that propose to study RPE biology. Our data provide an optimized technique for long-term culture of hRPE with high density seeding in growth factor enriched media (RtEGM) followed by feeding with RPECM at 2d. Tight junctions, TER and pigmentation are significantly increased, and monolayers are well-formed by 21d. The utilization of these methods will greatly assist others in the field that are performing long-term RPE culture to achieve reliable models of RPE cell biology and AMD-like RPE degeneration in vitro.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.