Cell Therapy Engineering and Development, Genentech, South San Francisco, CA 94080, USA.
Protein Analytical Chemistry, Genentech, South San Francisco, CA 94080, USA.
Int J Mol Sci. 2023 Aug 17;24(16):12889. doi: 10.3390/ijms241612889.
Phagocytosis is one of the key functions of retinal pigment epithelium (RPE) cells, which maintain photoreceptor health by removing photoreceptor outer segments (POSs) that are regularly shed. A deficiency in RPE function to phagocytose POSs may lead to vision loss in inherited retinal diseases and eventually to age-related macular degeneration (AMD) with geographic atrophy. Significant progress has been made in the field of cell replacement therapy for AMD using stem-cell-derived RPE. To test their function, RPE cells are incubated with purified bovine POSs for the demonstration of efficient binding, internalization, and digestion of POSs. Here, we present an image-based method to measure phagocytosis activity by using POSs labeled with a pH-sensitive fluorescent dye, which has low fluorescence at neutral pH outside of the cell and high fluorescence at low pH inside the phagosome. Further, we introduce a unique flow-cytometry-based method for the characterization of POSs by measuring specific markers for POSs such as rhodopsin and opsin. Using this method, we demonstrated a comparable quality of several bovine POS isolation batches and a reliable assessment of POS quality on RPE phagocytosis assay performance when subjected to different stress conditions. This work provides new tools to characterize POSs and insight into RPE phagocytosis assay development for the functional evaluation of RPE cells in the field of cell replacement therapy.
吞噬作用是视网膜色素上皮 (RPE) 细胞的关键功能之一，它通过清除定期脱落的光感受器外节 (POS) 来维持光感受器的健康。RPE 吞噬 POS 的功能缺陷可能导致遗传性视网膜疾病的视力丧失，并最终导致年龄相关性黄斑变性 (AMD) 伴地图状萎缩。使用干细胞衍生的 RPE 进行 AMD 的细胞替代治疗领域已经取得了重大进展。为了测试它们的功能，将 RPE 细胞与纯化的牛 POS 一起孵育，以证明 POS 的有效结合、内化和消化。在这里，我们提出了一种基于图像的方法，使用用 pH 敏感荧光染料标记的 POS 来测量吞噬作用活性，该染料在细胞外的中性 pH 下荧光较弱，在吞噬体内部的低 pH 下荧光较强。此外，我们还引入了一种独特的基于流式细胞术的方法来通过测量 POS 的特定标记物（如视蛋白和视黄醛）来表征 POS。使用这种方法，我们证明了几种牛 POS 分离批次的质量相当，并且当受到不同应激条件时，对 RPE 吞噬作用测定性能的 POS 质量进行可靠评估。这项工作为 POS 特性分析提供了新的工具，并深入了解了 RPE 吞噬作用测定在细胞替代治疗领域中用于 RPE 细胞功能评估的发展。
