A convenient protocol for establishing a human cell culture model of the outer retina. [version 1; referees: 2 approved]
The retinal pigment epithelium (RPE) plays a key role in the pathogenesis of several blinding retinopathies. Alterations to RPE structure and function are reported in Age-related Macular Degeneration, Stargardt and Best disease as well as pattern dystrophies. However, the precise role of RPE cells i...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
F1000 Research Ltd
2018-07-01
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| Series: | F1000Research |
| Online Access: | https://f1000research.com/articles/7-1107/v1 |
| _version_ | 1818778512554721280 |
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| author | Savannah A. Lynn Eloise Keeling Jennifer M. Dewing David A. Johnston Anton Page Angela J. Cree David A. Tumbarello Tracey A. Newman Andrew J. Lotery J. Arjuna Ratnayaka |
| author_facet | Savannah A. Lynn Eloise Keeling Jennifer M. Dewing David A. Johnston Anton Page Angela J. Cree David A. Tumbarello Tracey A. Newman Andrew J. Lotery J. Arjuna Ratnayaka |
| author_sort | Savannah A. Lynn |
| collection | DOAJ |
| description | The retinal pigment epithelium (RPE) plays a key role in the pathogenesis of several blinding retinopathies. Alterations to RPE structure and function are reported in Age-related Macular Degeneration, Stargardt and Best disease as well as pattern dystrophies. However, the precise role of RPE cells in disease aetiology remains incompletely understood. Many studies into RPE pathobiology have utilised animal models, which only recapitulate limited disease features. Some studies are also difficult to carry out in animals as the ocular space remains largely inaccessible to powerful microscopes. In contrast, in-vitro models provide an attractive alternative to investigating pathogenic RPE changes associated with age and disease. In this article we describe the step-by-step approach required to establish an experimentally versatile in-vitro culture model of the outer retina incorporating the RPE monolayer and supportive Bruch’s membrane (BrM). We show that confluent monolayers of the spontaneously arisen human ARPE-19 cell-line cultured under optimal conditions reproduce key features of native RPE. These models can be used to study dynamic, intracellular and extracellular pathogenic changes using the latest developments in microscopy and imaging technology. We also discuss how RPE cells from human foetal and stem-cell derived sources can be incorporated alongside sophisticated BrM substitutes to replicate the aged/diseased outer retina in a dish. The work presented here will enable users to rapidly establish a realistic in-vitro model of the outer retina that is amenable to a high degree of experimental manipulation which will also serve as an attractive alternative to using animals. This in-vitro model therefore has the benefit of achieving the 3Rs objective of reducing and replacing the use of animals in research. As well as recapitulating salient structural and physiological features of native RPE, other advantages of this model include its simplicity, rapid set-up time and unlimited scope for detailed single-cell resolution and matrix studies. |
| first_indexed | 2024-12-18T11:45:54Z |
| format | Article |
| id | doaj.art-806a7f95156b4dba8deadf7db67b425d |
| institution | Directory Open Access Journal |
| issn | 2046-1402 |
| language | English |
| last_indexed | 2024-12-18T11:45:54Z |
| publishDate | 2018-07-01 |
| publisher | F1000 Research Ltd |
| record_format | Article |
| series | F1000Research |
| spelling | doaj.art-806a7f95156b4dba8deadf7db67b425d2022-12-21T21:09:17ZengF1000 Research LtdF1000Research2046-14022018-07-01710.12688/f1000research.15409.116791A convenient protocol for establishing a human cell culture model of the outer retina. [version 1; referees: 2 approved]Savannah A. Lynn0Eloise Keeling1Jennifer M. Dewing2David A. Johnston3Anton Page4Angela J. Cree5David A. Tumbarello6Tracey A. Newman7Andrew J. Lotery8J. Arjuna Ratnayaka9Clinical and Experimental Sciences, Faculty of Medicine, MP 806, Tremona Road, University of Southampton, Southampton, Hampshire, SO16 6YD, UKClinical and Experimental Sciences, Faculty of Medicine, MP 806, Tremona Road, University of Southampton, Southampton, Hampshire, SO16 6YD, UKClinical and Experimental Sciences, Faculty of Medicine, MP 806, Tremona Road, University of Southampton, Southampton, Hampshire, SO16 6YD, UKBiomedical Imaging Unit, MP 806, Tremona Road, University of Southampton, Southampton, Hampshire, SO16 6YD, UKBiomedical Imaging Unit, MP 806, Tremona Road, University of Southampton, Southampton, Hampshire, SO16 6YD, UKClinical and Experimental Sciences, Faculty of Medicine, MP 806, Tremona Road, University of Southampton, Southampton, Hampshire, SO16 6YD, UKBiological Sciences, Faculty of Natural & Environmental Sciences, Life Sciences Building 85, University of Southampton, Southampton, Hampshire, SO17 1BJ, UKClinical and Experimental Sciences, Faculty of Medicine, MP 806, Tremona Road, University of Southampton, Southampton, Hampshire, SO16 6YD, UKClinical and Experimental Sciences, Faculty of Medicine, MP 806, Tremona Road, University of Southampton, Southampton, Hampshire, SO16 6YD, UKClinical and Experimental Sciences, Faculty of Medicine, MP 806, Tremona Road, University of Southampton, Southampton, Hampshire, SO16 6YD, UKThe retinal pigment epithelium (RPE) plays a key role in the pathogenesis of several blinding retinopathies. Alterations to RPE structure and function are reported in Age-related Macular Degeneration, Stargardt and Best disease as well as pattern dystrophies. However, the precise role of RPE cells in disease aetiology remains incompletely understood. Many studies into RPE pathobiology have utilised animal models, which only recapitulate limited disease features. Some studies are also difficult to carry out in animals as the ocular space remains largely inaccessible to powerful microscopes. In contrast, in-vitro models provide an attractive alternative to investigating pathogenic RPE changes associated with age and disease. In this article we describe the step-by-step approach required to establish an experimentally versatile in-vitro culture model of the outer retina incorporating the RPE monolayer and supportive Bruch’s membrane (BrM). We show that confluent monolayers of the spontaneously arisen human ARPE-19 cell-line cultured under optimal conditions reproduce key features of native RPE. These models can be used to study dynamic, intracellular and extracellular pathogenic changes using the latest developments in microscopy and imaging technology. We also discuss how RPE cells from human foetal and stem-cell derived sources can be incorporated alongside sophisticated BrM substitutes to replicate the aged/diseased outer retina in a dish. The work presented here will enable users to rapidly establish a realistic in-vitro model of the outer retina that is amenable to a high degree of experimental manipulation which will also serve as an attractive alternative to using animals. This in-vitro model therefore has the benefit of achieving the 3Rs objective of reducing and replacing the use of animals in research. As well as recapitulating salient structural and physiological features of native RPE, other advantages of this model include its simplicity, rapid set-up time and unlimited scope for detailed single-cell resolution and matrix studies.https://f1000research.com/articles/7-1107/v1 |
| spellingShingle | Savannah A. Lynn Eloise Keeling Jennifer M. Dewing David A. Johnston Anton Page Angela J. Cree David A. Tumbarello Tracey A. Newman Andrew J. Lotery J. Arjuna Ratnayaka A convenient protocol for establishing a human cell culture model of the outer retina. [version 1; referees: 2 approved] F1000Research |
| title | A convenient protocol for establishing a human cell culture model of the outer retina. [version 1; referees: 2 approved] |
| title_full | A convenient protocol for establishing a human cell culture model of the outer retina. [version 1; referees: 2 approved] |
| title_fullStr | A convenient protocol for establishing a human cell culture model of the outer retina. [version 1; referees: 2 approved] |
| title_full_unstemmed | A convenient protocol for establishing a human cell culture model of the outer retina. [version 1; referees: 2 approved] |
| title_short | A convenient protocol for establishing a human cell culture model of the outer retina. [version 1; referees: 2 approved] |
| title_sort | convenient protocol for establishing a human cell culture model of the outer retina version 1 referees 2 approved |
| url | https://f1000research.com/articles/7-1107/v1 |
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