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Development and characterization of an in vitro system of the human retina using cultured cell lines

Rachel Churm Orcid Logo, Gareth Dunseath Orcid Logo, Sarah Prior Orcid Logo, Frankie Thomas, Sanjiv Banerjee, David Owens Orcid Logo

Clinical & Experimental Ophthalmology, Volume: 47, Issue: 8, Pages: 1055 - 1062

Swansea University Authors: Rachel Churm Orcid Logo, Gareth Dunseath Orcid Logo, Sarah Prior Orcid Logo, Frankie Thomas, David Owens Orcid Logo

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DOI (Published version): 10.1111/ceo.13578

Abstract

Background: Previously developed in vitro cultures of the human retina have been solo or dual cell cultures. We developed a triple-cell culture in vitro model utilizing a membrane system to produce a better representation of a functional and morphological human retina. Methods: Retinal microvascular...

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Published in: Clinical & Experimental Ophthalmology
ISSN: 1442-6404 1442-9071
Published: Wiley 2019
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URI: https://cronfa.swan.ac.uk/Record/cronfa50970
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We developed a triple-cell culture in vitro model utilizing a membrane system to produce a better representation of a functional and morphological human retina. Methods: Retinal microvascular endothelial cells (HRMVEC/ACBRI181, Cell systems), retinal pigment epithelium cells (RPE/ARPE-19, ATCC) and M&#xFC;ller glial cells (MIO-M1, UCL) were grown in a triple-culture. Our optimized triple-culture media contained a mix of specific endothelial medium and high glucose Dulbecco's Modified Eagle's medium (DMEM), where all three layers were viable for up to 5 days. Co-culture effect on morphological changes (cell staining) and gene expression of functional genes (pigment epithelial derived factor (PEDF) and vascular endothelial growth factor (VEGF)) were measured from RNA via real time PCR. Expression of tight junction protein 1 (TJP1) was measured in RNA isolated from ARPE-19s, to assess barrier stability. Results: The triple-culture promotes certain cell functionality through up-regulation of TJP1, increasing PEDF and decreasing VEGF expression highlighting its importance for the assessment of disease mechanisms distinct from a solo culture which would not allow the true effect of the native microenvironment to be elucidated. Conclusion: This model's novelty and reliability allows for the assessment of singular cellular function within the retinal microenvironment and overall assessment of retinal health, whilst eliminating the requirement of animal-based models.</abstract><type>Journal Article</type><journal>Clinical &amp; Experimental Ophthalmology</journal><volume>47</volume><journalNumber>8</journalNumber><paginationStart>1055</paginationStart><paginationEnd>1062</paginationEnd><publisher>Wiley</publisher><issnPrint>1442-6404</issnPrint><issnElectronic>1442-9071</issnElectronic><keywords>cell culture, human retina, in vitro model, triple culture</keywords><publishedDay>21</publishedDay><publishedMonth>11</publishedMonth><publishedYear>2019</publishedYear><publishedDate>2019-11-21</publishedDate><doi>10.1111/ceo.13578</doi><url/><notes/><college>COLLEGE NANME</college><department>Sport and Exercise Sciences</department><CollegeCode>COLLEGE CODE</CollegeCode><DepartmentCode>STSC</DepartmentCode><institution>Swansea University</institution><apcterm/><lastEdited>2020-08-27T14:05:49.6104088</lastEdited><Created>2019-07-01T08:18:05.6180713</Created><authors><author><firstname>Rachel</firstname><surname>Churm</surname><orcid>0000-0001-9855-6282</orcid><order>1</order></author><author><firstname>Gareth</firstname><surname>Dunseath</surname><orcid>0000-0001-6022-862X</orcid><order>2</order></author><author><firstname>Sarah</firstname><surname>Prior</surname><orcid>0000-0001-8703-8092</orcid><order>3</order></author><author><firstname>Frankie</firstname><surname>Thomas</surname><orcid/><order>4</order></author><author><firstname>Sanjiv</firstname><surname>Banerjee</surname><order>5</order></author><author><firstname>David</firstname><surname>Owens</surname><orcid>0000-0003-1002-1238</orcid><order>6</order></author></authors><documents><document><filename>0050970-17072019111927.pdf</filename><originalFilename>50970.pdf</originalFilename><uploaded>2019-07-17T11:19:27.4930000</uploaded><type>Output</type><contentLength>724829</contentLength><contentType>application/pdf</contentType><version>Accepted Manuscript</version><cronfaStatus>true</cronfaStatus><embargoDate>2020-06-29T00:00:00.0000000</embargoDate><copyrightCorrect>true</copyrightCorrect><language>eng</language></document></documents><OutputDurs/></rfc1807>
spelling 2020-08-27T14:05:49.6104088 v2 50970 2019-07-01 Development and characterization of an in vitro system of the human retina using cultured cell lines c6cd8267ff0b13f2ea333bbfefdae144 0000-0001-9855-6282 Rachel Churm Rachel Churm true false fccbba9edcaee08a839a3c5cff8cbe19 0000-0001-6022-862X Gareth Dunseath Gareth Dunseath true false cdda101035997acfaa6fdf17097f52b2 0000-0001-8703-8092 Sarah Prior Sarah Prior true false e95cfcc1431ad789b4af8b9a689117ab Frankie Thomas Frankie Thomas true false 2fd4b7c3f82c6d3bd546eff61ff944e9 0000-0003-1002-1238 David Owens David Owens true false 2019-07-01 STSC Background: Previously developed in vitro cultures of the human retina have been solo or dual cell cultures. We developed a triple-cell culture in vitro model utilizing a membrane system to produce a better representation of a functional and morphological human retina. Methods: Retinal microvascular endothelial cells (HRMVEC/ACBRI181, Cell systems), retinal pigment epithelium cells (RPE/ARPE-19, ATCC) and Müller glial cells (MIO-M1, UCL) were grown in a triple-culture. Our optimized triple-culture media contained a mix of specific endothelial medium and high glucose Dulbecco's Modified Eagle's medium (DMEM), where all three layers were viable for up to 5 days. Co-culture effect on morphological changes (cell staining) and gene expression of functional genes (pigment epithelial derived factor (PEDF) and vascular endothelial growth factor (VEGF)) were measured from RNA via real time PCR. Expression of tight junction protein 1 (TJP1) was measured in RNA isolated from ARPE-19s, to assess barrier stability. Results: The triple-culture promotes certain cell functionality through up-regulation of TJP1, increasing PEDF and decreasing VEGF expression highlighting its importance for the assessment of disease mechanisms distinct from a solo culture which would not allow the true effect of the native microenvironment to be elucidated. Conclusion: This model's novelty and reliability allows for the assessment of singular cellular function within the retinal microenvironment and overall assessment of retinal health, whilst eliminating the requirement of animal-based models. Journal Article Clinical & Experimental Ophthalmology 47 8 1055 1062 Wiley 1442-6404 1442-9071 cell culture, human retina, in vitro model, triple culture 21 11 2019 2019-11-21 10.1111/ceo.13578 COLLEGE NANME Sport and Exercise Sciences COLLEGE CODE STSC Swansea University 2020-08-27T14:05:49.6104088 2019-07-01T08:18:05.6180713 Rachel Churm 0000-0001-9855-6282 1 Gareth Dunseath 0000-0001-6022-862X 2 Sarah Prior 0000-0001-8703-8092 3 Frankie Thomas 4 Sanjiv Banerjee 5 David Owens 0000-0003-1002-1238 6 0050970-17072019111927.pdf 50970.pdf 2019-07-17T11:19:27.4930000 Output 724829 application/pdf Accepted Manuscript true 2020-06-29T00:00:00.0000000 true eng
title Development and characterization of an in vitro system of the human retina using cultured cell lines
spellingShingle Development and characterization of an in vitro system of the human retina using cultured cell lines
Rachel Churm
Gareth Dunseath
Sarah Prior
Frankie Thomas
David Owens
title_short Development and characterization of an in vitro system of the human retina using cultured cell lines
title_full Development and characterization of an in vitro system of the human retina using cultured cell lines
title_fullStr Development and characterization of an in vitro system of the human retina using cultured cell lines
title_full_unstemmed Development and characterization of an in vitro system of the human retina using cultured cell lines
title_sort Development and characterization of an in vitro system of the human retina using cultured cell lines
author_id_str_mv c6cd8267ff0b13f2ea333bbfefdae144
fccbba9edcaee08a839a3c5cff8cbe19
cdda101035997acfaa6fdf17097f52b2
e95cfcc1431ad789b4af8b9a689117ab
2fd4b7c3f82c6d3bd546eff61ff944e9
author_id_fullname_str_mv c6cd8267ff0b13f2ea333bbfefdae144_***_Rachel Churm
fccbba9edcaee08a839a3c5cff8cbe19_***_Gareth Dunseath
cdda101035997acfaa6fdf17097f52b2_***_Sarah Prior
e95cfcc1431ad789b4af8b9a689117ab_***_Frankie Thomas
2fd4b7c3f82c6d3bd546eff61ff944e9_***_David Owens
author Rachel Churm
Gareth Dunseath
Sarah Prior
Frankie Thomas
David Owens
author2 Rachel Churm
Gareth Dunseath
Sarah Prior
Frankie Thomas
Sanjiv Banerjee
David Owens
format Journal article
container_title Clinical & Experimental Ophthalmology
container_volume 47
container_issue 8
container_start_page 1055
publishDate 2019
institution Swansea University
issn 1442-6404
1442-9071
doi_str_mv 10.1111/ceo.13578
publisher Wiley
document_store_str 1
active_str 0
description Background: Previously developed in vitro cultures of the human retina have been solo or dual cell cultures. We developed a triple-cell culture in vitro model utilizing a membrane system to produce a better representation of a functional and morphological human retina. Methods: Retinal microvascular endothelial cells (HRMVEC/ACBRI181, Cell systems), retinal pigment epithelium cells (RPE/ARPE-19, ATCC) and Müller glial cells (MIO-M1, UCL) were grown in a triple-culture. Our optimized triple-culture media contained a mix of specific endothelial medium and high glucose Dulbecco's Modified Eagle's medium (DMEM), where all three layers were viable for up to 5 days. Co-culture effect on morphological changes (cell staining) and gene expression of functional genes (pigment epithelial derived factor (PEDF) and vascular endothelial growth factor (VEGF)) were measured from RNA via real time PCR. Expression of tight junction protein 1 (TJP1) was measured in RNA isolated from ARPE-19s, to assess barrier stability. Results: The triple-culture promotes certain cell functionality through up-regulation of TJP1, increasing PEDF and decreasing VEGF expression highlighting its importance for the assessment of disease mechanisms distinct from a solo culture which would not allow the true effect of the native microenvironment to be elucidated. Conclusion: This model's novelty and reliability allows for the assessment of singular cellular function within the retinal microenvironment and overall assessment of retinal health, whilst eliminating the requirement of animal-based models.
published_date 2019-11-21T04:02:41Z
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