Dörschmann Philipp, Prinz Marie, Schmitkall Greta, Roider Johann, Klettner Alexa
Department of Ophthalmology, University Medical Center, Kiel University, Arnold-Heller-Str. 3, Haus B2, 24105 Kiel, Germany.
Int J Mol Sci. 2025 Aug 29;26(17):8434. doi: 10.3390/ijms26178434.
The retinal pigment epithelium (RPE) is strongly involved in the pathogenesis of several retinal diseases, such as age-related macular degeneration (AMD). RPE models addressing specific pathological pathways are of high importance for understanding cellular pathomechanisms and pre-clinical screening of potential new therapeutics. The goal of this study is to establish standard operation protocols for single-eye porcine RPE preparation for AMD-relevant models of oxidative stress (RPE-Ox) and inflammation (RPE-Inf). Porcine primary RPE were prepared from one eye and seeded into one well of 12-well plates or, for polar differentiation, in transwell inserts. Different coatings (Poly-ᴅ-Lysine and laminin) and serum content of media (10%, 5%, and 1%) were tested to determine optimal culture parameters. For RPE-Ox, cells were treated with NaIO, CoCl, or erastin; cell viability (thiazolyl blue tetrazolium bromide, MTT), and gene expression (RT-qPCR) were determined. For RPE-Inf, cells were treated with lipopolysaccharide (LPS), polyinosinic/polycytidylic acid (Poly I:C), or tumor necrosis factor alpha (TNF-α); cell viability (MTT), cytokine secretion (ELISA), and gene expression (RT-qPCR) were determined. For transwell plates in RPE-Inf, cell viability (MTT), polar cytokine secretion (ELISA), gene expression (RT-qPCR), and transepithelial electrical resistance (TEER) for barrier assessment were conducted. For RPE-Ox, effective LD could be achieved by using 24 h stimulation with 25 µm erastin, seven days after preparation in 5% serum cultures, without coating. For gene expression assessment, the use of Poly-ᴅ-Lysine is recommended. For RPE-Inf, three days of LPS stimulation (1 µg/mL) showed effective cytokine activation with 5% serum on uncoated 12-well plates. Transwell plates are not recommended for cytokine secretion assessment. It can be used for cell barrier assays in which LPS also showed effective cell barrier decrease and gene expression assays. Two specific best practice protocols for the use of porcine single-eye cultures in AMD research concerning oxidative stress and inflammation with optimized parameters were established and are provided.
视网膜色素上皮（RPE）在多种视网膜疾病的发病机制中起着重要作用，如年龄相关性黄斑变性（AMD）。针对特定病理途径的RPE模型对于理解细胞病理机制和潜在新疗法的临床前筛选至关重要。本研究的目的是为用于氧化应激（RPE-Ox）和炎症（RPE-Inf）相关AMD模型的单眼猪RPE制备建立标准操作方案。从一只眼睛制备猪原代RPE，并接种到12孔板的一个孔中，或者为了进行极性分化，接种到Transwell小室中。测试了不同的包被（聚-D-赖氨酸和层粘连蛋白）和培养基的血清含量（10%、5%和1%），以确定最佳培养参数。对于RPE-Ox，用碘酸钠、氯化钴或埃拉斯汀处理细胞；测定细胞活力（噻唑蓝四唑溴盐，MTT）和基因表达（RT-qPCR）。对于RPE-Inf，用脂多糖（LPS）、聚肌苷酸/聚胞苷酸（Poly I:C）或肿瘤坏死因子α（TNF-α）处理细胞；测定细胞活力（MTT）、细胞因子分泌（ELISA）和基因表达（RT-qPCR）。对于RPE-Inf中的Transwell板，进行细胞活力（MTT）、极性细胞因子分泌（ELISA）、基因表达（RT-qPCR）和用于屏障评估的跨上皮电阻（TEER）测定。对于RPE-Ox，在无包被的5%血清培养物中制备七天后，用25μm埃拉斯汀刺激24小时可实现有效的半数致死剂量（LD）。对于基因表达评估，建议使用聚-D-赖氨酸。对于RPE-Inf，在未包被的12孔板上用1μg/mL LPS刺激三天显示在5%血清中可有效激活细胞因子。不建议使用Transwell板进行细胞因子分泌评估。它可用于细胞屏障测定，其中LPS也显示可有效降低细胞屏障和进行基因表达测定。建立并提供了两个关于在AMD研究中使用猪单眼培养物进行氧化应激和炎症研究的特定最佳实践方案，其参数经过优化。
