Customized strategies for high-yield purification of retinal pigment epithelial cells differentiated from different stem cell sources
Abstract 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 m...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2022-09-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-19777-2 |
| _version_ | 1818021574349946880 |
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| author | Kakkad Regha Mayuri Bhargava Abdurrahmaan Al-Mubaarak Chou Chai Bhav Harshad Parikh Zengping Liu Claudine See Wei Wong Walter Hunziker Kah Leong Lim Xinyi Su |
| author_facet | Kakkad Regha Mayuri Bhargava Abdurrahmaan Al-Mubaarak Chou Chai Bhav Harshad Parikh Zengping Liu Claudine See Wei Wong Walter Hunziker Kah Leong Lim Xinyi Su |
| author_sort | Kakkad Regha |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-04-14T08:19:58Z |
| format | Article |
| id | doaj.art-3bd7339aa071480992c9732da3df4a0f |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-14T08:19:58Z |
| publishDate | 2022-09-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-3bd7339aa071480992c9732da3df4a0f2022-12-22T02:04:14ZengNature PortfolioScientific Reports2045-23222022-09-0112111110.1038/s41598-022-19777-2Customized strategies for high-yield purification of retinal pigment epithelial cells differentiated from different stem cell sourcesKakkad Regha0Mayuri Bhargava1Abdurrahmaan Al-Mubaarak2Chou Chai3Bhav Harshad Parikh4Zengping Liu5Claudine See Wei Wong6Walter Hunziker7Kah Leong Lim8Xinyi Su9Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR)Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR)Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR)Lee Kong Chian School of Medicine, Nanyang Technological University (NTU)Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR)Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR)Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR)Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR)Lee Kong Chian School of Medicine, Nanyang Technological University (NTU)Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR)Abstract 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.https://doi.org/10.1038/s41598-022-19777-2 |
| spellingShingle | Kakkad Regha Mayuri Bhargava Abdurrahmaan Al-Mubaarak Chou Chai Bhav Harshad Parikh Zengping Liu Claudine See Wei Wong Walter Hunziker Kah Leong Lim Xinyi Su Customized strategies for high-yield purification of retinal pigment epithelial cells differentiated from different stem cell sources Scientific Reports |
| title | Customized strategies for high-yield purification of retinal pigment epithelial cells differentiated from different stem cell sources |
| title_full | Customized strategies for high-yield purification of retinal pigment epithelial cells differentiated from different stem cell sources |
| title_fullStr | Customized strategies for high-yield purification of retinal pigment epithelial cells differentiated from different stem cell sources |
| title_full_unstemmed | Customized strategies for high-yield purification of retinal pigment epithelial cells differentiated from different stem cell sources |
| title_short | Customized strategies for high-yield purification of retinal pigment epithelial cells differentiated from different stem cell sources |
| title_sort | customized strategies for high yield purification of retinal pigment epithelial cells differentiated from different stem cell sources |
| url | https://doi.org/10.1038/s41598-022-19777-2 |
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