Fig. 2.

Expansion and differentiation of polarized RPE cells on transwell membranes. a Dissociation of RPE cells obtained after the RPE induction step into single-cell suspension. (i) Samples were washed twice in DPBS and resuspended in Accutase, (ii) detached from the culture plate surface, and (iii) broken into small pieces using pipette tips. (iv) Small clusters of samples were resuspended thoroughly using a P1000 pipette, (v) transferred to a 15-ml Falcon tube, and incubated for 10 min at 37 °C. (vi) Samples were pipetted vigorously and incubated for other 10 min at 37 °C, resuspended several times, filtered through a 40-μm cell strainer, and (vii) centrifuged to recover the cellular pellet. b Representative images of RPE cells during expansion and differentiation on transwell membranes. The seeded cells adhere to the transwell membrane surface and start dividing (week 1). Cells expand until confluence is reached (week 2). RPE progenitors begin to assume the typical RPE morphology and pigmentation level (week 3). Mature RPE cells display highly pigmented cobblestone-like morphology (week 4). c Representative images of 6.5 mm diameter (upper panel) and 24 mm diameter (lower panel) transwell membranes after 4 weeks of culture. d Transmission electron microscopy images of highly pigmented samples highlighting cellular organization into a monolayer of tightly connected columnar-shaped cells with nuclei on the basal side and melanosomes on the apical side (left panel) and details of RPE cell ultrastructure. de = desmosome, mel = melanosome, mv = microvilli, tj = tight junction