Isolation and Culturing of Retinal Progenitor Cells

RPCs were isolated from fresh retinal tissue of postnatal-day-one GFP transgenic C57BL/6 mice (a gift from Dr. Masaru Okabe, University of Osaka, Japan)27 (link)28 (link). The cells were seeded into flasks at a density of 2 × 10⁵ cells/ml with proliferation medium containing advanced DMEM/F12 (Invitrogen, Carlsbad, CA, USA), 1% N2 neural supplement (Invitrogen), 2 mM L-glutamine (Invitrogen), 100 U/ml penicillin-streptomycin (Invitrogen) and 20 ng/ml epidermal growth factor (EGF, Invitrogen)29 (link)30 (link). Every two or three days, the proliferation medium was changed, and the cells were passaged at regular intervals of three or four days. For differentiation, the cells were seeded at a density of 1 × 10⁵ cells/ml with differentiation medium, to which 10% fetal bovine serum (FBS) (Invitrogen) was added while EGF was removed; the cultures were then allowed to grow for 7 days. The culture medium was renewed every two days. All of the animals were handled according to the ARVO animal usage standards and following the approval by the animal care and use committee of the Schepens Eye Research Institute, where the original derivation of the cells was performed.