The hRPC line used in this study (GS089) was isolated from the retinae of a single human fetus at 16 weeks of gestation in compliance with the Declaration of Helsinki. Material was obtained from Advanced Bioscience Resources (Alameda, CA), under the quality and safety guidelines for Food and Drug Administration (FDA) Good Tissue Practice conditions. The isolation and culture of hRPC has been described in detail previously.
12,19,22 In brief, the neural retina was dissociated with collagenase I and expanded at 37°C under low oxygen conditions (3% O
2, 5% CO
2, 100% humidity) on human fibronectin-coated flasks in Ultraculture medium (Lonza, Walkersville, MD) supplemented with 20 ng/mL human epidermal growth factor (EGF; Peprotech, Rocky Hill, NJ), 10 ng/mL human basic fibroblast growth factor (bFGF; Peprotech), and 2 mM L-glutamine (Invitrogen, Carlsbad, CA). Cells were passaged at 80% confluence and reseeded at a density of 2 × 10
4 cells/cm
2.
The hRPC batches used for transplantation into rats were produced at ReNeuron's Guildford, UK laboratories from a P8 GMP working cell bank, which was thawed and cultured in human fibronectin-coated T500 flasks at 3% O2 as described above. After 72 hours in culture, hRPC were harvested with TrypZean (Sigma-Aldrich Corp., St. Louis, MO), followed by defined trypsin inhibitor (Gibco/Thermo Fisher Scientific, Waltham, MA) and Benzonase (Merck & Co., Rockville, MD). The P9 GS089 hRPC then were formulated at 1 × 105 cells/μL in vehicle (Ca2+, Mg2+-free HBSS containing 0.5 mM N-acetyl-L-Cysteine [HBSS-NAC]) on the morning of each transplantation day and shipped to UCL-Institute of Ophthalmology (UCL-IOO). This stock solution was either used undiluted for the 2 × 105 (200 K) dosage group (2 μL per rat), or diluted appropriately with vehicle to generate cell suspensions for the 1 × 105 (100 K), 5 × 104 (50 K), and 1 × 104 (10 K) dosage groups. On each surgery day, the cells were counted and assessed for viability upon completion of transplants. This was done using a hemocytometer, where cell viability was determined by calculating the percentage of living cells that exclude trypan blue. Using this method, cell viability was consistently >70% upon completion of transplants.