Several approaches have been used to enrich, purify, and expand hPSC-RPE from the mixture for further applications. Manually picking out pigmented clusters and reattaching them to the adherent surface for expansion and passage in serum-free medium is commonly used when pigmented foci become abundant in culture in about 8 to 13 weeks after differentiation.
14,32,38,39 This procedure has been described in detail in a study reported by Vugler and colleagues,
39 indicating that it was feasible only when the sizes of the pigmented foci were over 1 mm in diameter with every effort made to dissect the surrounding and non-pigmented material for acquiring high purity of RPE cells under inverted microscope. Further, many research groups applied the same conditions—serum-free or low serum media, high plating density (approximately 1 × 10
5/cm
2), and 30-day passage (SH30)—for both expanding and maturing hiPSC-RPE cells, taking a couple of months to produce a large population of RPE cells. For instance, Singh and colleagues
32 picked and dissociated deeply pigmented hiPSC-RPE sheets in day 60 to day 90 after differentiation, concomitantly generated with NR on an optic vesicle induction platform. By passaging the pigmented mature hiPSC-RPE cells roughly every month, these cells were subcultured only 3 times, which took about 90 days after RPE selection, implying the timeframe of producing large population of hiPSC-RPE cells would be 150 to 180 days after differentiation, similar to the timeline reported by Leach et al.
38 Hence, this time-consuming and labor-intensive method requires skillful, experienced researchers. Slightly modified from the above approach, Julien et al.
9 enriched hPSC-RPE cells by direct passage from week 8 after differentiation. The culture conditions (serum-free medium, high plating density, and 3-week passage) were similar to the earlier-described studies. However, with this method, the RPE cells were passaged twice as reported, so it is unclear how far they could go. In our study, a novel procedure was developed to enrich and purify hiPSC-RPE from the mixture by formation and dissection of RPE spheroids, similar to isolating hRPE from human eyeballs (see “Methods” and “Results” sections).
23–25 Since the definite non-retinal tissues or cell masses could be routinely cleared from the mixture after detachment, our method readily enriched RPE spheroids and significantly reduced the medium consumption and cost. In addition, RPE spheroids could grow for a long time and achieve high degrees of maturation with proper pigmentation, specific markers expression, and cell polarity. RPE spheroids or aggregates were also observed in previous studies, which were often reattached to the adherent surface to allow RPE cells to migrate out and expand for 3 to 5 weeks.
23,37,40,41 However, in our study, RPE sheets were directly dissected from the spheroids and digested into single cells for quick expansion in our optimized culture conditions with serum medium, low seeding density (2.0 to 5.0 × 10
4/cm
2) and 7-day passage (SL7). Under these conditions, singularized RPE cells from RPE spheroids at varying timepoints after differentiation (day 53 to day 139) were all successfully expanded and routinely passaged at least 5 times. For BC1 and BC1-GFP hiPSC-RPE cells, 8 to 10 passages were achieved, yielding large quantities of RPE cells in a relatively short period. For example, one 6-well plate of hiPSCs could produce at least 3 × 10
11 hiPSC-RPE cells over 5 passages in about 1 month after cell dissociation, or in about 80 to 90 days after differentiation if day 53 RPE-spheroids were used in the dissociation. Therefore, our SL7 expansion method significantly reduced the time for cell preparation compared to those SH30, which might take about 140 to 150 days after differentiation to provide a similar output if RPE clusters were selected at the same timepoint.
32,38 In a subset of parallel experiments, we expanded RPE cells with the SH30. The cell number after 1-month culture was roughly equivalent to that in 1-week culture with our SL7 expansion method, which implies that the prolonged culture did not increase cell yield (data not shown). The cell contact inhibition and decreased proliferative activity in SFM might be related to the low yield of a 1-month culture. In summary, this optimized method for RPE enrichment and expansion is simple, practical and highly efficient, which could be applied in retinal organoid induction platform for producing RPE and retinal neural cells together, facilitating the basic and translational research of retinal diseases.