Fig. 2

Frozen RPE at P2D5 showed highly expressed and properly localized RPE markers after thawing. A The bright-field images of thawed RPE cells frozen at indicated times at D14 (upper row) and D28 (lower row) post thaw, respectively. B RT-qPCR analysis of relative mRNA expression levels of RPE marker genes BEST1, MITF, RLBP1, and PMEL17 at D28 post thaw. GAPDH expression was used as internal control, and the values were normalized to the P2D5 group for each marker gene. n = 3 biological replicates. Data are represented as mean ± SD. Statistical differences are evaluated with two-way ANOVA with Bonferroni's post hoc test. *P < 0.05. C Immunostaining analysis for RLBP1 and ZO-1 expression in thawed RPE cells that were frozen at indicated time points at D28 post thaw, respectively. D Immunostaining analysis for BEST1 and F-actin (stained with Phalloidin) along the apical-to-basal axis of thawed RPE cells that were frozen at indicated time points at D28 post thaw, respectively. Nuclei were counter-stained with DAPI. Scale bars: 100 μm (A), 25 μm (C, D)