Fig. 1

Sustained subconfluent culture results in the loss of capacity to differentiate. Fetal RPE cells were plated at 4,000 cells/cm² and passaged at subconfluence every 3–5 days. At each passage a portion of the cells were plated at 80,000 cells/cm² and maintained without further passaging. a Whole culture photographs of 64-day serially passaged RPE cells from three different donors (A, B, and C) plated on microporous inserts at 80,000 cells/cm². b Phase contrast micrographs of 3-day low density cultures of P0 (a) and P5 (c) cells plated at low density reveal only minor differences in phenotype. At 32 days P0 cultures (b) plated at high density have a prototypical pigmented cobblestone RPE phenotype, while P5 cells (d) are not pigmented and have a disorganized morphology. c Passage 0 cells were plated at 4,000 and 80,000 cells/cm² on tissue culture plastic and maintained without further passage. Phase contrast (Ph) and brightfield (Bf) micrographs of the cultures as function of time show that high density plating is a requirement for normal RPE differentiation