{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T02:24:36Z","timestamp":1760149476731,"version":"build-2065373602"},"reference-count":31,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2023,8,17]],"date-time":"2023-08-17T00:00:00Z","timestamp":1692230400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/ijms241612889","type":"journal-article","created":{"date-parts":[[2023,8,17]],"date-time":"2023-08-17T10:15:48Z","timestamp":1692267348000},"page":"12889","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Comprehensive Analysis of Photoreceptor Outer Segments: Flow Cytometry Characterization and Stress-Driven Impact on Retinal Pigment Epithelium Phagocytosis"],"prefix":"10.3390","volume":"24","author":[{"given":"Haoqian","family":"Liang","sequence":"first","affiliation":[{"name":"Cell Therapy Engineering and Development, Genentech, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qiang","family":"Wu","sequence":"additional","affiliation":[{"name":"Cell Therapy Engineering and Development, Genentech, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xinzheng Victor","family":"Guo","sequence":"additional","affiliation":[{"name":"Cell Therapy Engineering and Development, Genentech, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Linda","family":"Chan","sequence":"additional","affiliation":[{"name":"Cell Therapy Engineering and Development, Genentech, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-7808-5848","authenticated-orcid":false,"given":"Tin","family":"Mao","sequence":"additional","affiliation":[{"name":"Cell Therapy Engineering and Development, Genentech, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Cinzia","family":"Stella","sequence":"additional","affiliation":[{"name":"Protein Analytical Chemistry, Genentech, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6007-1618","authenticated-orcid":false,"given":"Axel","family":"Guilbaud","sequence":"additional","affiliation":[{"name":"Protein Analytical Chemistry, Genentech, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4020-2278","authenticated-orcid":false,"given":"Julien","family":"Camperi","sequence":"additional","affiliation":[{"name":"Cell Therapy Engineering and Development, Genentech, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,8,17]]},"reference":[{"key":"ref_1","first-page":"e29583","article-title":"Age-Related Macular Degeneration: Epidemiology, Pathophysiology, Diagnosis, and Treatment. Cureus","volume":"14","author":"Vyawahare","year":"2022","journal-title":"Cureus."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"652","DOI":"10.1111\/ceo.13949","article-title":"Neovascular age-related macular degeneration: A review of findings from the real-world Fight Retinal Blindness! registry","volume":"49","author":"Nguyen","year":"2021","journal-title":"Clin. Exp. Ophthalmol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1043","DOI":"10.1111\/ceo.13834","article-title":"Retinal pigment epithelium and age-related macular degeneration: A review of major disease mechanisms","volume":"48","author":"Somasundaran","year":"2020","journal-title":"Clin. Exp. Ophthalmol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"727870","DOI":"10.3389\/fphar.2021.727870","article-title":"Functions and Diseases of the Retinal Pigment Epithelium","volume":"12","author":"Yang","year":"2021","journal-title":"Front. Pharmacol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1186\/s12967-018-1434-6","article-title":"phagocytic function declines in age-related macular degeneration and is rescued by human umbilical tissue derived cells","volume":"16","author":"Inana","year":"2018","journal-title":"J. Transl. Med."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"13158","DOI":"10.1073\/pnas.0705963104","article-title":"Photoreceptor outer segment phagocytosis attenuates oxidative stress-induced apoptosis with concomitant neuroprotectin D1 synthesis","volume":"104","author":"Mukherjee","year":"2007","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"604205","DOI":"10.3389\/fimmu.2020.604205","article-title":"Phagocytosis by the Retinal Pigment Epithelium: Recognition, Resolution, Recycling","volume":"11","author":"Kwon","year":"2020","journal-title":"Front. Immunol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1111\/dgd.12704","article-title":"Stem-cell-based therapies for retinal degenerative diseases: Current challenges in the establishment of new treatment strategies","volume":"63","author":"Uyama","year":"2021","journal-title":"Dev. Growth Differ."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Maeda, T., Sugita, S., Kurimoto, Y., and Takahashi, M. (2021). Trends of Stem Cell Therapies in Age-Related Macular Degeneration. J. Clin. Med., 10.","DOI":"10.3390\/jcm10081785"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"396","DOI":"10.1016\/j.stem.2009.07.002","article-title":"Directed Differentiation of Human Embryonic Stem Cells into Functional Retinal Pigment Epithelium Cells","volume":"5","author":"Idelson","year":"2009","journal-title":"Cell Stem Cell"},{"key":"ref_11","first-page":"134","article-title":"Retinal pigment epithelium culture; a potential source of retinal stem cells","volume":"4","author":"Akrami","year":"2009","journal-title":"J. Ophthalmic Vis. Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1615\/CritRevBiomedEng.v37.i4-5.30","article-title":"Retinal Pigment Epithelium Differentiation of Stem Cells: Current Status and Challenges","volume":"37","author":"Uygun","year":"2009","journal-title":"Crit. Rev. Biomed. Eng."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Rastoin, O., Pag\u00e8s, G., and Dufies, M. (2020). Experimental Models in Neovascular Age Related Macular Degeneration. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21134627"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1186\/s13287-022-02894-0","article-title":"Human-induced pluripotent stem cells-derived retinal pigmented epithelium, a new horizon for cells-based therapies for age-related macular degeneration","volume":"13","author":"Dehghan","year":"2022","journal-title":"Stem Cell Res. Ther."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Duarri, A., Rodr\u00edguez-Bocanegra, E., Mart\u00ednez-Navarrete, G., Biarn\u00e9s, M., Garc\u00eda, M., Ferraro, L.L., Kuebler, B., Aran, B., Izquierdo, E., and Aguilera-Xiol, E. (2021). Transplantation of Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium in a Swine Model of Geographic Atrophy. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms221910497"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"551","DOI":"10.1007\/s12017-014-8308-8","article-title":"In-Depth Characterisation of Retinal Pigment Epithelium (RPE) Cells Derived from Human Induced Pluripotent Stem Cells (hiPSC)","volume":"16","author":"Brandl","year":"2014","journal-title":"NeuroMolecular Med."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"825","DOI":"10.1002\/stem.635","article-title":"Human Induced Pluripotent Stem-Derived Retinal Pigment Epithelium (RPE) Cells Exhibit Ion Transport, Membrane Potential, Polarized Vascular Endothelial Growth Factor Secretion, and Gene Expression Pattern Similar to Native RPE","volume":"29","author":"Kokkinaki","year":"2011","journal-title":"Stem Cells"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"4229","DOI":"10.1093\/hmg\/ddq341","article-title":"Molecular signature of primary retinal pigment epithelium and stem-cell-derived RPE cells","volume":"19","author":"Liao","year":"2010","journal-title":"Hum. Mol. Genet."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1016\/j.exer.2014.01.010","article-title":"Understanding photoreceptor outer segment phagocytosis: Use and utility of RPE cells in culture","volume":"126","author":"Mazzoni","year":"2014","journal-title":"Exp. Eye Res."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Voisin, A., Gaillard, A., Balbous, A., and Leveziel, N. (2022). Proteins Associated with Phagocytosis Alteration in Retinal Pigment Epithelial Cells Derived from Age-Related Macular Degeneration Patients. Antioxidants, 11.","DOI":"10.3390\/antiox11040713"},{"key":"ref_21","first-page":"245","article-title":"Analysis of Photoreceptor Rod Outer Segment Phagocytosis by RPE Cells In Situ","volume":"Volume 935","author":"Langmann","year":"2012","journal-title":"Weber BHF"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1083\/jcb.7.3.493","article-title":"Electron Microscopy of Retinal Photoreceptors","volume":"7","author":"Lasansky","year":"1960","journal-title":"J. Biophys. Biochem. Cytol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1016\/j.exer.2010.10.008","article-title":"Assessment of bovine rod outer segment disk membrane heterogeneity utilizing flow cytometry","volume":"92","author":"Corley","year":"2011","journal-title":"Exp. Eye Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"5.1.1","DOI":"10.1002\/cpim.40","article-title":"Flow Cytometry: An Overview","volume":"120","author":"McKinnon","year":"2018","journal-title":"Curr. Protoc. Immunol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1128\/CDLI.8.2.245-250.2001","article-title":"Comparison of a Classical Phagocytosis Assay and a Flow Cytometry Assay for Assessment of the Phagocytic Capacity of Sera from Adults Vaccinated with a Pneumococcal Conjugate Vaccine","volume":"8","author":"Jansen","year":"2001","journal-title":"Clin. Diagn. Lab. Immunol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1016\/j.preteyeres.2010.11.001","article-title":"The Cone-specific visual cycle","volume":"30","author":"Wang","year":"2011","journal-title":"Prog. Retin. Eye Res."},{"key":"ref_27","first-page":"293","article-title":"Cell-Specific Markers for the Identification of Retinal Cells and Subcellular Organelles by Immunofluorescence Microscopy","volume":"Volume 1834","author":"Langmann","year":"2019","journal-title":"Weber BHF"},{"key":"ref_28","first-page":"e52100","article-title":"Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells","volume":"94","author":"Parinot","year":"2014","journal-title":"J. Vis. Exp."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2654","DOI":"10.1021\/pr7006939","article-title":"Proteomic Analysis of the Retinal Rod Outer Segment Disks","volume":"7","author":"Panfoli","year":"2008","journal-title":"J. Proteome Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"4222","DOI":"10.1074\/jbc.M509769200","article-title":"Light-induced Oxidation of Photoreceptor Outer Segment Phospholipids Generates Ligands for CD36-mediated Phagocytosis by Retinal Pigment Epithelium","volume":"281","author":"Sun","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_31","unstructured":"Papermaster, D.S. (1982). Methods in Enzymology, Elsevier. Available online: https:\/\/linkinghub.elsevier.com\/retrieve\/pii\/S0076687982810100."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/16\/12889\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:35:45Z","timestamp":1760128545000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/16\/12889"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,8,17]]},"references-count":31,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2023,8]]}},"alternative-id":["ijms241612889"],"URL":"https:\/\/doi.org\/10.3390\/ijms241612889","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2023,8,17]]}}}