Dörschmann Philipp, von der Weppen Sina, Koyama Emi, Roider Johann, Klettner Alexa
Department of Ophthalmology, University Medical Center, Kiel University, Arnold-Heller-Str. 3, Haus B2, 24105 Kiel, Germany.
Cells. 2025 Jul 1;14(13):1007. doi: 10.3390/cells14131007.
Age-related macular degeneration (AMD) is the main cause of blindness in Western nations. AMD models addressing specific pathological pathways are desired. Through this study, a best-practice protocol for polarized porcine single-eye retinal pigment epithelium (RPE) preparation for AMD-relevant models of RPE barrier and polarity is established. Single-eye porcine primary RPE cells (from one eye for one well) were prepared in 12-well plates including Transwell inserts. Different coatings (laminin (Lam), Poly-ᴅ-Lysine (PDL), fibronectin (Fn) and collagens) and varying serum contents (1%, 5% and 10%) were investigated to determine optimal culture parameters for this model. Success rates of cultures, cell number (trypan-blue exclusion assay), morphology/morphometry (light and fluorescence microscopy), protein secretion/expression (ELISA, Western blot), gene expression (qPCR), transepithelial electric resistance (TEER) and polar location of bestrophin 1 (BEST1) by cryosectioning (IHC-Fr) were assessed. Cells seeded on Lam exhibited the highest level of epithelial cells and confluence properties. Fn resulted in the highest cell number growth. Lam and Fn exhibited the highest culture success rates. TEER values and vascular endothelial growth factor secretion were highest when Lam was used. For the first time, polar (Transwell) porcine single-eye RPE morphometry parameters were determined. RPE on Lam showed bigger cells with a higher variety of cell shapes. CIV displayed the lowest claudin 19 expression. The highest basolateral expression of BEST1 was achieved with Lam coating. The higher the serum, the better the cell number increase and confluence success. A reduction in serum on Lam showed positive results for RPE morphology, while morphometry remained stable. A five percent serum on Lam showed the highest culture success rate and best barrier properties. RPE65 expression was reduced by using 10% serum. Altogether, the most suitable coating of Transwell inserts was Lam, and a reduction in serum to 5% is recommended, as well as a cultivation time of 28 days. A protocol for the use of polar porcine single-eye cultures with validated parameters was established and is provided herein.
年龄相关性黄斑变性（AMD）是西方国家失明的主要原因。人们期望有针对特定病理途径的AMD模型。通过本研究，建立了一种用于制备与AMD相关的视网膜色素上皮（RPE）屏障和极性模型的偏振猪单眼视网膜色素上皮制备的最佳实践方案。在包括Transwell小室的12孔板中制备单眼猪原代RPE细胞（每孔来自一只眼睛）。研究了不同的包被（层粘连蛋白（Lam）、聚-D-赖氨酸（PDL）、纤连蛋白（Fn）和胶原蛋白）和不同的血清含量（1%、5%和10%），以确定该模型的最佳培养参数。评估培养成功率、细胞数量（台盼蓝排斥试验）、形态/形态测量（光学和荧光显微镜）、蛋白质分泌/表达（ELISA、蛋白质印迹法）、基因表达（qPCR）、跨上皮电阻（TEER）以及通过冷冻切片（免疫组化-Fr）检测的最佳rophin 1（BEST1）的极性定位。接种在Lam上的细胞表现出最高水平的上皮细胞和汇合特性。Fn导致最高的细胞数量增长。Lam和Fn表现出最高的培养成功率。使用Lam时，TEER值和血管内皮生长因子分泌最高。首次确定了极性（Transwell）猪单眼RPE形态测量参数。Lam上的RPE显示细胞更大，细胞形状种类更多样。IV型胶原蛋白显示claudin 19表达最低。使用Lam包被时，BEST1的基底外侧表达最高。血清越高，细胞数量增加和汇合成功率越好。Lam上血清减少对RPE形态显示出积极结果，而形态测量保持稳定。Lam上5%的血清显示出最高的培养成功率和最佳的屏障特性。使用10%血清会降低RPE65表达。总之，Transwell小室最合适的包被是Lam，建议将血清减少到5%，以及培养时间为28天。本文建立并提供了一种使用具有验证参数的极性猪单眼培养物的方案。
