Kiel University, University Medical Center, Department of Ophthalmology, Arnold-Heller-Str. 3, Haus B2, 24105, Kiel, Germany.
Kiel University, University Medical Center, Department of Ophthalmology, Arnold-Heller-Str. 3, Haus B2, 24105, Kiel, Germany.
Exp Eye Res. 2022 Jun;219:109063. doi: 10.1016/j.exer.2022.109063. Epub 2022 Apr 3.
Properties of retinal pigment epithelium (RPE) are relevant for the development of cell culture models concerning an exact reproduction of the ocular cell biology. Here, we want to investigate how different carrier materials and coatings influence proliferation, differentiation and functions of RPE in regard to development of a three-dimensional cell culture model based on primary porcine RPE. Human RPE cell line ARPE-19 and primary porcine RPE were used. Cells were cultivated on plates which were coated with collagen I, collagen IV, laminin or fibronectin, respectively, and cell numbers were assessed after different time periods via trypan blue staining. Also, the ARPE-19 were cultivated on polydimethylsiloxane (PDMS), alginate, gelatin methacrylate (GelMA), poly-N-isopropylacrylamide (PNIPAM) and cells number were assessed. Primary RPE were cultured on PDMS material. Supernatants were collected and analyzed via ELISA for their vascular endothelial growth factor (VEGF) and transforming growth factor β (TGF-β) content. After day 14 cells were lysed and retinal pigment epithelium-specific 65 kDa protein (RPE65) and bestrophin-1 (BEST1) expression was investigated via Western blot. Cellular functions were tested on collagen I, collagen IV, laminin and fibronectin with and without PDMS. Scratch assay was performed to detect wound healing 24 and 48 h after scratch application. Immunolabeling was used to highlight tight junctions in concert with Hoechst staining and phalloidin to label cell nuclei and actin filaments, respectively. Phagocytosis of fluorescently labeled latex beads opsonized with photoreceptor outer segments (POS) was assessed via fluorescence microscopy. Transepithelial electrical resistance was measured for detection of cellular barrier. Gene expression of RDH11 (retinol dehydrogenase 11), BEST1 (bestrophin 1) and TGFB1 (transforming growth factor beta 1) was investigated via real-time PCR. Only PDMS carrier material was appropriate for primary RPE and ARPE-19 cell cultivation. Coating of PDMS with laminin led to increased proliferation. In primary RPE, VEGF secretion was increased if PDMS was coated with laminin or fibronectin compared to uncoated PDMS. No significant changes in phagocytic ability and generation of tight junctions were detected between different coatings, but RPE65 expression was reduced on fibronectin coated PDMS. Laminin coating decreased TGF-β and increased BEST1 protein expression. Also, RPE on collagen IV showed highest TEER on transwell plates. The genes RDH11 and TGFB1 were decreased when coated with collagen IV without PDMS as well as coated PDMS. Laminin and collagen IV coating led to an increased wound healing. Cultivation of RPE and ARPE-1 on PDMS is a possible alternative for cell culture models whereas alginate, GelMA and PNIPAM were not suitable. Coating with laminin increased the proliferation, wound healing and VEGF secretion of the cells. The results suggest that laminin coated PDMS as carrier material is suitable for the development of 3D culture model systems.
视网膜色素上皮（RPE）的特性对于开发细胞培养模型至关重要，因为这些模型需要精确复制眼部细胞生物学。在这里，我们希望研究不同的载体材料和涂层如何影响 RPE 的增殖、分化和功能，以期开发基于原代猪 RPE 的三维细胞培养模型。我们使用了人 RPE 细胞系 ARPE-19 和原代猪 RPE。将细胞培养在分别涂有胶原蛋白 I、胶原蛋白 IV、层粘连蛋白或纤维连接蛋白的平板上，并在不同时间点通过台盼蓝染色评估细胞数量。此外，还将 ARPE-19 培养在聚二甲基硅氧烷（PDMS）、藻酸盐、甲基丙烯酰化明胶（GelMA）、聚 N-异丙基丙烯酰胺（PNIPAM）上，并评估细胞数量。将原代 RPE 培养在 PDMS 材料上。收集上清液并通过 ELISA 分析其血管内皮生长因子（VEGF）和转化生长因子 β（TGF-β）含量。第 14 天后，裂解细胞并通过 Western blot 检测视网膜色素上皮特异性 65 kDa 蛋白（RPE65）和最佳蛋白 1（BEST1）的表达。在有和没有 PDMS 的情况下，在胶原蛋白 I、胶原蛋白 IV、层粘连蛋白和纤维连接蛋白上测试细胞功能。划痕实验用于检测划痕应用后 24 和 48 小时的伤口愈合情况。免疫标记用于与 Hoechst 染色一起突出紧密连接，并分别用鬼笔环肽标记细胞核和肌动蛋白丝。通过荧光显微镜评估用光感受器外段（POS）包被的荧光标记乳胶珠的吞噬作用。通过测量跨上皮电阻来检测细胞屏障。通过实时 PCR 研究 RDH11（视黄醇脱氢酶 11）、BEST1（最佳蛋白 1）和 TGFB1（转化生长因子β 1）的基因表达。只有 PDMS 载体材料适合原代 RPE 和 ARPE-19 细胞培养。用层粘连蛋白涂覆 PDMS 可促进增殖。与未涂覆 PDMS 的情况相比，在用层粘连蛋白或纤维连接蛋白涂覆 PDMS 的情况下，原代 RPE 中 VEGF 的分泌增加。在不同涂层之间未检测到吞噬能力和紧密连接形成的显著变化，但在纤维连接蛋白涂覆的 PDMS 上 RPE65 的表达减少。层粘连蛋白涂层降低了 TGF-β 并增加了 BEST1 蛋白的表达。此外，在 Transwell 板上，胶原蛋白 IV 上的 RPE 显示出最高的 TEER。当没有 PDMS 或涂有 PDMS 的情况下用胶原蛋白 IV 涂覆时，基因 RDH11 和 TGFB1 减少。层粘连蛋白和胶原蛋白 IV 涂层可促进伤口愈合。RPE 和 ARPE-1 在 PDMS 上的培养是细胞培养模型的一种可行替代方法，而藻酸盐、GelMA 和 PNIPAM 则不适合。用层粘连蛋白涂覆可增加细胞的增殖、伤口愈合和 VEGF 分泌。结果表明，涂有层粘连蛋白的 PDMS 作为载体材料适合开发 3D 培养模型系统。
