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

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. E...

Full description

Saved in:
Bibliographic Details
Main Authors: Regha, Kakkad, Bhargava, Mayuri, Al-Mubaarak, Abdurrahmaan, Chai, Chou, Parikh, Bhav Harshad, Liu, Zengping, Wong, Claudine See Wei, Hunziker, Walter, Lim, Kah Leong, Su, Xinyi
Other Authors: Lee Kong Chian School of Medicine (LKCMedicine)
Format: Article
Language:English
Published: 2023
Subjects:
Online Access:https://hdl.handle.net/10356/171295
Tags: Add Tag
No Tags, Be the first to tag this record!
Institution: Nanyang Technological University
Language: English
id sg-ntu-dr.10356-171295
record_format dspace
spelling sg-ntu-dr.10356-1712952023-10-22T15:37:50Z Customized strategies for high-yield purification of retinal pigment epithelial cells differentiated from different stem cell sources Regha, Kakkad Bhargava, Mayuri Al-Mubaarak, Abdurrahmaan Chai, Chou Parikh, Bhav Harshad Liu, Zengping Wong, Claudine See Wei Hunziker, Walter Lim, Kah Leong Su, Xinyi Lee Kong Chian School of Medicine (LKCMedicine) Science::Biological sciences Cell Differentiation Retinal Pigment Epithelium 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. Ministry of Education (MOE) National Research Foundation (NRF) Published version This study was supported by National Research Foundation (NRF), Singapore, under its Competitive Research Program (CRP) [NRF-CRP21-2018-0008] and Ministry of Education (MOE) for its MOE Academic Research Fund Tier 3 (STEM) [MOET32020-0001]. 2023-10-20T04:34:07Z 2023-10-20T04:34:07Z 2022 Journal Article Regha, K., Bhargava, M., Al-Mubaarak, A., Chai, C., Parikh, B. H., Liu, Z., Wong, C. S. W., Hunziker, W., Lim, K. L. & Su, X. (2022). Customized strategies for high-yield purification of retinal pigment epithelial cells differentiated from different stem cell sources. Scientific Reports, 12(1), 15563-. https://dx.doi.org/10.1038/s41598-022-19777-2 2045-2322 https://hdl.handle.net/10356/171295 10.1038/s41598-022-19777-2 36114268 2-s2.0-85138183087 1 12 15563 en NRF-CRP21-2018-0008 MOET32020-0001 Scientific Reports © 2022 The Author(s). This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Science::Biological sciences
Cell Differentiation
Retinal Pigment Epithelium
spellingShingle Science::Biological sciences
Cell Differentiation
Retinal Pigment Epithelium
Regha, Kakkad
Bhargava, Mayuri
Al-Mubaarak, Abdurrahmaan
Chai, Chou
Parikh, Bhav Harshad
Liu, Zengping
Wong, Claudine See Wei
Hunziker, Walter
Lim, Kah Leong
Su, Xinyi
Customized strategies for high-yield purification of retinal pigment epithelial cells differentiated from different stem cell sources
description 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.
author2 Lee Kong Chian School of Medicine (LKCMedicine)
author_facet Lee Kong Chian School of Medicine (LKCMedicine)
Regha, Kakkad
Bhargava, Mayuri
Al-Mubaarak, Abdurrahmaan
Chai, Chou
Parikh, Bhav Harshad
Liu, Zengping
Wong, Claudine See Wei
Hunziker, Walter
Lim, Kah Leong
Su, Xinyi
format Article
author Regha, Kakkad
Bhargava, Mayuri
Al-Mubaarak, Abdurrahmaan
Chai, Chou
Parikh, Bhav Harshad
Liu, Zengping
Wong, Claudine See Wei
Hunziker, Walter
Lim, Kah Leong
Su, Xinyi
author_sort Regha, Kakkad
title 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_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_sort customized strategies for high-yield purification of retinal pigment epithelial cells differentiated from different stem cell sources
publishDate 2023
url https://hdl.handle.net/10356/171295
_version_ 1781793798370623488