RPCs were harvested from fresh retinal tissue of postnatal-day-one GFP transgenic C57BL/6 mice (a gift from Dr. Masaru Okabe, University of Osaka, Japan). The cells were cultured with proliferation medium containing advanced Dulbecco′s Modified Eagle Media: Nutrient Mixture F-12 (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) [44 (link)]. The culture medium was replaced every 2 days, and the cells were passaged at regular intervals of 3 or 4 days. For differentiation, the cells were cultured in differentiation medium containing 10% fetal bovine serum (FBS) (Invitrogen) without EGF. The cultures were then allowed to grow for seven days. The culture medium was renewed three times a week.
All animals were handled according to the ARVO animal usage standards and following approval by the animal care and use committee of the Schepens Eye Research Institute, where the original derivation of the cells was performed.
All animals were handled according to the ARVO animal usage standards and following approval by the animal care and use committee of the Schepens Eye Research Institute, where the original derivation of the cells was performed.
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