Figure 1

Pigmented REPS exhibit poor cell survival in response to cryopreservation. (a) Schematic of overall process to seed, cryopreserve, and thaw a mature monolayer of Retinal pigmented Epithelial cells adhered to a Parylene Scaffold (REPS). (b) Representative image of REPS exhibiting pigmentation at 28 days post-seeding (DPS) (Bright field; scale bar, 1 mm). (c–e) REPS were cryopreserved at 30 DPS. (c) Cryopreservation of pigmented REPS results in significantly reduced viability one day post-thaw (DPT) compared to the non-cryopreserved control. (d) Pigmented REPS exhibit significantly reduced metabolic activity 1 DPT compared to levels measured prior to cryopreservation. Relative Fluorescence Units (RFU). (e) Cryopreservation of pigmented REPS results in severe hyperpigmentation within the first 24 hours post-thaw (Bright field; scale bar, 100 µm). (f) Optimized parameters to cryopreserve REPS were identified by analysis of (i) post-seeding culture period, (ii) cryopreservation medium and freeze rate, and (iii) post-thaw rinse solution. Optimal parameters are identified in bold (see also Supplementary Figs. S1, S2). Error bars indicate standard deviation. ***P < 0.001, unpaired two-tailed t-test.