Fig 2: Cryopreserved REPS retain high viability, cellular identity, and secretory function post-thaw. (a) Schematic of overall research design indicates the characterization assays conducted on the designated day post-seeding (DPS) or day post-thaw (DPT). Non-cryopreserved REPS were maintained in culture to provide an age-matched control for each day of sample collection. (b) Cryopreserved REPS exhibit 95 ± 2.3% viability as measured by propidium iodide exclusion at 1 DPT (n = 26); viability of non-cryopreserved control REPS (n = 7) averaged to 97 ± 0.89% (mean ± SD., *P < 0.05, unpaired two-tailed t-test). (c) REPS exhibit epithelial, cuboidal morphology prior to cryopreservation (Pre-Cryo), immediately post-thaw (PT), and 1 DPT. (Phase contrast, scale bar, 100 µm.) (d) Amount of secreted PEDF by non-cryopreserved control REPS (black triangles) and cryopreserved/thawed REPS (gray circles) as measured by ELISA. Thawed REPS exhibited a significant increase in secreted PEDF at 7 DPT compared to Pre-Cryopreservation, and also at 21 DPT compared to 7 DPT (***P < 0.001). A significant difference was detected between thawed REPS and age-matched non-cryopreserved controls at 7 DPT (**P < 0.01) and at 21 DPT (*P < 0.05). (Pre-Cryo, n = 93; Non-Cryo, n = 10; Cryo, n = 33. Each data point represents the mean of two technical replicates. Horizontal red bar indicates mean of the biological replicates. Independent samples Kruskal–Wallis test with pairwise comparisons.) (e) Non-cryopreserved control REPS (Non-Cryo) and cryopreserved/thawed REPS (Cryo) display uniform pigmentation and similar coverage of the parylene scaffold over the course of the analysis (Bright field; scale bar, 1 mm). (f) RT-qPCR analysis of RPE marker genes (TYRP1, RPE65) and an EMT marker (S100A4) for non-cryopreserved control REPS (black triangles) and cryopreserved/thawed REPS (gray circles). No significant differences were observed between cryopreserved REPS and age-matched non-cryopreserved controls for each target gene (P > 0.05). A significant increase in RPE65 was detected for both cryopreserved/thawed REPS and non-cryopreserved control REPS by 21 DPT or 28 DPS, respectively, compared to prior to cryopreservation and immediately post-thaw (**P < 0.01, independent samples Kruskal–Wallis test with pairwise comparisons). (g) En face (left, middle) and orthogonal (right) renderings of immunostained apical (ZO-1) and basolateral (BEST1) markers in cryopreserved REPS after 7–10 DPT. (Confocal Z-stacks, scale bar, 50 µm.) Error bars indicate standard deviation.