Customized strategies for high-yield purification of retinal pigment epithelial cells differentiated from different stem cell sources.

Autor: Regha K; Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, 138673, Singapore.; Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore (NUS), Singapore, 117597, Singapore., Bhargava M; Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, 138673, Singapore.; Department of Ophthalmology, National University Hospital (NUH), Singapore, 119074, Singapore., Al-Mubaarak A; Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, 138673, Singapore.; Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore (NUS), Singapore, 117597, Singapore., Chai C; Lee Kong Chian School of Medicine, Nanyang Technological University (NTU), Singapore, 308232, Singapore., Parikh BH; Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, 138673, Singapore., Liu Z; Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, 138673, Singapore.; Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore (NUS), Singapore, 117597, Singapore.; Singapore Eye Research Institute (SERI), Singapore, 169856, Singapore., Wong CSW; Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, 138673, Singapore., Hunziker W; Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, 138673, Singapore.; Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore (NUS), Singapore, 117593, Singapore., Lim KL; Lee Kong Chian School of Medicine, Nanyang Technological University (NTU), Singapore, 308232, Singapore., Su X; Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, 138673, Singapore. xysu@imcb.a-star.edu.sg.; Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore (NUS), Singapore, 117597, Singapore. xysu@imcb.a-star.edu.sg.; Department of Ophthalmology, National University Hospital (NUH), Singapore, 119074, Singapore. xysu@imcb.a-star.edu.sg.; Singapore Eye Research Institute (SERI), Singapore, 169856, Singapore. xysu@imcb.a-star.edu.sg.
Jazyk: angličtina
Zdroj: Scientific reports [Sci Rep] 2022 Sep 16; Vol. 12 (1), pp. 15563. Date of Electronic Publication: 2022 Sep 16.
DOI: 10.1038/s41598-022-19777-2
Abstrakt: Retinal pigment epithelial (RPE) cell dysfunction and death are characteristics of age-related macular degeneration. A promising therapeutic option is RPE cell transplantation. Development of clinical grade stem-cell derived RPE requires efficient in vitro differentiation and purification methods. Enzymatic purification of RPE relies on the relative adherence of RPE and non-RPE cells to the culture plate. However, morphology and adherence of non-RPE cells differ for different stem cell sources. In cases whereby the non-RPE adhered as strongly as RPE cells to the culture plate, enzymatic method of purification is unsuitable. Thus, we hypothesized the need to customize purification strategies for RPE derived from different stem cell sources. We systematically compared five different RPE purification methods, including manual, enzymatic, flow cytometry-based sorting or combinations thereof for parameters including cell throughput, yield, purity and functionality. Flow cytometry-based approach was suitable for RPE isolation from heterogeneous cultures with highly adherent non-RPE cells, albeit with lower yield. Although all five purification methods generated pure and functional RPE, there were significant differences in yield and processing times. Based on the high purity of the resulting RPE and relatively short processing time, we conclude that a combination of enzymatic and manual purification is ideal for clinical applications.
(© 2022. The Author(s).)
Databáze: MEDLINE
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