Faculty of Medicine and Health Technology, BioMediTech, Tampere University, Tampere, Finland.
Institute for Multiphase Processes, Leibniz University of Hannover, Hannover, Germany.
Sci Rep. 2021 Jan 13;11(1):933. doi: 10.1038/s41598-020-79638-8.
Human pluripotent stem cell-derived retinal pigment epithelium (RPE) transplantation is currently under evaluation as treatment for macular degeneration. For therapeutic applications, cryostorage during cell production is typically needed with potential consequences to cell functionality. We have previously shown that the culture substrate affects human embryonic stem cell-derived RPE (hESC-RPE) properties in fresh cultures. Here, we aimed to further identify the role of RPE basement membrane proteins type IV collagen (Col-IV), laminin (LN), and nidogen-1 in the maturation and functionality of hESC-RPE after cryopreservation. In addition to cell attachment and morphology, transepithelial electrical resistance, expression of key RPE proteins, phagocytosis capacity and Ca signalling were analysed. After cryostorage, attachment of hESC-RPE on culture surfaces coated with Col-IV alone was poor. Combining Col-IV and LN with or without nidogen-1 significantly improved cell attachment and barrier properties of the epithelium. Furthermore, functional homogeneity of the hESC-RPE monolayer was enhanced in the presence of nidogen-1. Our results suggest that the choice of coating proteins for the cell culture may have implications to the functional properties of these cells after cryostorage cell banking.
人多能干细胞衍生的视网膜色素上皮（RPE）移植目前正在作为黄斑变性的治疗方法进行评估。对于治疗应用，细胞生产过程中的冷冻储存通常是必要的，这可能对细胞功能产生潜在影响。我们之前已经表明，培养底物会影响新鲜培养中的人胚胎干细胞衍生的 RPE（hESC-RPE）特性。在这里，我们旨在进一步确定 RPE 基底膜蛋白 IV 型胶原（Col-IV）、层粘连蛋白（LN）和巢蛋白-1 在 hESC-RPE 冷冻保存后的成熟和功能中的作用。除了细胞附着和形态外，还分析了跨上皮电阻、关键 RPE 蛋白的表达、吞噬能力和 Ca 信号。冷冻储存后，仅用 Col-IV 涂层的培养表面上 hESC-RPE 的附着不良。将 Col-IV 和 LN 与或不与巢蛋白-1 结合使用可显著改善上皮细胞的附着和屏障特性。此外，巢蛋白-1 的存在增强了 hESC-RPE 单层的功能均一性。我们的研究结果表明，细胞培养中涂层蛋白的选择可能会对冷冻储存细胞库中这些细胞的功能特性产生影响。
