Nuffield Laboratory of Ophthalmology, Department of Clinical Neurosciences, John Radcliffe Hospital, Oxford, United Kingdom.
Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom.
J Ocul Pharmacol Ther. 2020 May;36(4):257-266. doi: 10.1089/jop.2019.0116. Epub 2020 Feb 6.
Age-related macular degeneration leads to retinal pigment epithelium (RPE) cell death and loss of central vision. studies have shown that the RPE layer has an innate, but limited, ability to repopulate atrophic areas. We aimed to establish a semiautomated, , wound healing assay workflow for targeted screening of compounds able to influence RPE wound healing. The ARPE-19 phenotype was evaluated using bright-field microscopy, immunocytochemistry, and quantitative real-time polymerase chain reaction. ARPE-19 monolayers were simultaneously scratched in a 96-well format and treated with Hoechst-33342 and an array of compounds. Initial wound dimensions and wound healing were subsequently evaluated using the EVOS FL Auto 2.0 imaging platform combined with automated image analyses. Long-term cultured ARPE-19 cells displayed a more RPE-like phenotype compared with recently seeded or short-term cultured cells. No statistical difference of initial scratch width was observed between short-term and long-term cultured cells, but more wells were excluded from analyses in total in the latter case due to scratch width, scratch smoothness, and imaging errors. Furthermore, the previous time spent in continuous culture had an effect on the observation of an altered wound healing response to different treatment conditions. We have established a semiautomated, 96-well format, wound healing assay with a reproducible workflow. This would enable screening of a significant number of compounds and greatly advances the potential of identifying novel therapeutics that may enhance the innate ability of RPE cells to repopulate atrophic areas.
年龄相关性黄斑变性导致视网膜色素上皮 (RPE) 细胞死亡和中心视力丧失。研究表明，RPE 层具有内在的，但有限的，能够在萎缩区域重新填充的能力。我们旨在建立一种半自动的、，伤口愈合测定工作流程，用于靶向筛选能够影响 RPE 伤口愈合的化合物。ARPE-19 表型通过明场显微镜、免疫细胞化学和定量实时聚合酶链反应进行评估。ARPE-19 单层在 96 孔格式中同时划伤，并使用 Hoechst-33342 和一系列化合物进行处理。随后使用 EVOS FL Auto 2.0 成像平台结合自动图像分析评估初始伤口尺寸和伤口愈合。与最近接种或短期培养的细胞相比，长期培养的 ARPE-19 细胞显示出更典型的 RPE 表型。短期和长期培养的细胞之间初始划痕宽度没有统计学差异，但在后者情况下，由于划痕宽度、划痕平滑度和成像误差，总共有更多的孔被排除在分析之外。此外，在连续培养中之前花费的时间会影响对不同处理条件下改变的伤口愈合反应的观察。我们已经建立了一种半自动的、96 孔格式的、伤口愈合测定工作流程，具有可重复的工作流程。这将能够筛选大量的化合物，并极大地推进识别可能增强 RPE 细胞内在填充萎缩区域能力的新型治疗方法的潜力。
