Amniotic membrane maintains the phenotype of rabbit retinal pigment epithelial cells in culture
The success of surgical removal of choroidal neovascularisation followed by transplantation of autologous retinal pigment epithelial cells (RPE) for age-related macular degeneration (ARMD) may be limited by damage in Bruch's membrane. We investigated whether amniotic membrane (AM) might be used...
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Veröffentlicht in: | Experimental eye research 2005, Vol.80 (1), p.103-112 |
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creator | Stanzel, Boris V. Espana, Edgar M. Grueterich, Martin Kawakita, Tetsuya Parel, Jean-Marie Tseng, Scheffer C.G. Binder, Susanne |
description | The success of surgical removal of choroidal neovascularisation followed by transplantation of autologous retinal pigment epithelial cells (RPE) for age-related macular degeneration (ARMD) may be limited by damage in Bruch's membrane. We investigated whether amniotic membrane (AM) might be used as an alternative basement membrane-containing matrix to support RPE growth and differentiation.
Primary RPE plastic cultures were established from freshly enucleated Dutch belted rabbit eyes in DMEM/F12 containing 0·1
m
m Ca
++ and 10% dialysed FBS. Upon subconfluence, cells were subcultured at 5000–9000 cells
cm
−2 in the above-mentioned culture medium on intact AM (iAM), epithelially denuded AM (dAM) or plastic. After confluence, the Ca
++ concentration in the medium was increased to 1·8
m
m for 4 weeks. Growth and morphology were monitored by phase contrast microscopy, and the phenotype by immunostaining with antibodies against cytokeratin 18, tight junction protein ZO-1, and RPE65 protein, and by transepithelial resistance (TER) measurement. Immunostaining to cytokeratin 18 confirmed the epithelial origin of isolated cells in both primary culture and subcultures. Compared to plastic cultures, RPE increased pigmentation within 24
hr after seeding on AM, with iAM being more pronounced than dAM. RPE adopted a hexagonal epithelial phenotype with more organised pigmentation, strong expression of ZO-1 and RPE65, and a significantly higher TER 4 weeks after Ca
++ switch on dAM. Our results indicate that AM may be used as a basement membrane-containing matrix to maintain RPE phenotype in vitro, and may facilitate subsequent transplantation to treat ARMD. |
doi_str_mv | 10.1016/j.exer.2004.06.032 |
format | Article |
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Primary RPE plastic cultures were established from freshly enucleated Dutch belted rabbit eyes in DMEM/F12 containing 0·1
m
m Ca
++ and 10% dialysed FBS. Upon subconfluence, cells were subcultured at 5000–9000 cells
cm
−2 in the above-mentioned culture medium on intact AM (iAM), epithelially denuded AM (dAM) or plastic. After confluence, the Ca
++ concentration in the medium was increased to 1·8
m
m for 4 weeks. Growth and morphology were monitored by phase contrast microscopy, and the phenotype by immunostaining with antibodies against cytokeratin 18, tight junction protein ZO-1, and RPE65 protein, and by transepithelial resistance (TER) measurement. Immunostaining to cytokeratin 18 confirmed the epithelial origin of isolated cells in both primary culture and subcultures. Compared to plastic cultures, RPE increased pigmentation within 24
hr after seeding on AM, with iAM being more pronounced than dAM. RPE adopted a hexagonal epithelial phenotype with more organised pigmentation, strong expression of ZO-1 and RPE65, and a significantly higher TER 4 weeks after Ca
++ switch on dAM. Our results indicate that AM may be used as a basement membrane-containing matrix to maintain RPE phenotype in vitro, and may facilitate subsequent transplantation to treat ARMD.</description><identifier>ISSN: 0014-4835</identifier><identifier>EISSN: 1096-0007</identifier><identifier>DOI: 10.1016/j.exer.2004.06.032</identifier><identifier>PMID: 15652531</identifier><language>eng</language><publisher>England: Elsevier Ltd</publisher><subject>age-related macular degeneration ; Amnion ; amniotic membrane ; Animals ; basement membrane ; Basement Membrane - cytology ; Basement Membrane - drug effects ; Calcium - pharmacology ; Cell Differentiation - drug effects ; Cells, Cultured ; Culture Media ; differentiation ; epithelial cells ; Epithelial Cells - cytology ; Epithelial Cells - drug effects ; eye ; Eye Proteins - analysis ; in vitro ; Keratins - analysis ; Membrane Proteins - analysis ; Microscopy, Phase-Contrast - methods ; Phenotype ; Phosphoproteins - analysis ; Pigment Epithelium of Eye - cytology ; Pigment Epithelium of Eye - drug effects ; pigmentation ; Proteins - analysis ; rabbit ; Rabbits ; retinal pigment epithelium ; RPE65 ; tight junctions ; transepithelial resistance ; Zonula Occludens-1 Protein</subject><ispartof>Experimental eye research, 2005, Vol.80 (1), p.103-112</ispartof><rights>2004 Elsevier Ltd</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c354t-528ab4187393bbef5fb864ef46f15f470b04d4d8eb6b006c36db1ac4848f9e643</citedby><cites>FETCH-LOGICAL-c354t-528ab4187393bbef5fb864ef46f15f470b04d4d8eb6b006c36db1ac4848f9e643</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.exer.2004.06.032$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,4024,27923,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15652531$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Stanzel, Boris V.</creatorcontrib><creatorcontrib>Espana, Edgar M.</creatorcontrib><creatorcontrib>Grueterich, Martin</creatorcontrib><creatorcontrib>Kawakita, Tetsuya</creatorcontrib><creatorcontrib>Parel, Jean-Marie</creatorcontrib><creatorcontrib>Tseng, Scheffer C.G.</creatorcontrib><creatorcontrib>Binder, Susanne</creatorcontrib><title>Amniotic membrane maintains the phenotype of rabbit retinal pigment epithelial cells in culture</title><title>Experimental eye research</title><addtitle>Exp Eye Res</addtitle><description>The success of surgical removal of choroidal neovascularisation followed by transplantation of autologous retinal pigment epithelial cells (RPE) for age-related macular degeneration (ARMD) may be limited by damage in Bruch's membrane. We investigated whether amniotic membrane (AM) might be used as an alternative basement membrane-containing matrix to support RPE growth and differentiation.
Primary RPE plastic cultures were established from freshly enucleated Dutch belted rabbit eyes in DMEM/F12 containing 0·1
m
m Ca
++ and 10% dialysed FBS. Upon subconfluence, cells were subcultured at 5000–9000 cells
cm
−2 in the above-mentioned culture medium on intact AM (iAM), epithelially denuded AM (dAM) or plastic. After confluence, the Ca
++ concentration in the medium was increased to 1·8
m
m for 4 weeks. Growth and morphology were monitored by phase contrast microscopy, and the phenotype by immunostaining with antibodies against cytokeratin 18, tight junction protein ZO-1, and RPE65 protein, and by transepithelial resistance (TER) measurement. Immunostaining to cytokeratin 18 confirmed the epithelial origin of isolated cells in both primary culture and subcultures. Compared to plastic cultures, RPE increased pigmentation within 24
hr after seeding on AM, with iAM being more pronounced than dAM. RPE adopted a hexagonal epithelial phenotype with more organised pigmentation, strong expression of ZO-1 and RPE65, and a significantly higher TER 4 weeks after Ca
++ switch on dAM. Our results indicate that AM may be used as a basement membrane-containing matrix to maintain RPE phenotype in vitro, and may facilitate subsequent transplantation to treat ARMD.</description><subject>age-related macular degeneration</subject><subject>Amnion</subject><subject>amniotic membrane</subject><subject>Animals</subject><subject>basement membrane</subject><subject>Basement Membrane - cytology</subject><subject>Basement Membrane - drug effects</subject><subject>Calcium - pharmacology</subject><subject>Cell Differentiation - drug effects</subject><subject>Cells, Cultured</subject><subject>Culture Media</subject><subject>differentiation</subject><subject>epithelial cells</subject><subject>Epithelial Cells - cytology</subject><subject>Epithelial Cells - drug effects</subject><subject>eye</subject><subject>Eye Proteins - analysis</subject><subject>in vitro</subject><subject>Keratins - analysis</subject><subject>Membrane Proteins - analysis</subject><subject>Microscopy, Phase-Contrast - methods</subject><subject>Phenotype</subject><subject>Phosphoproteins - analysis</subject><subject>Pigment Epithelium of Eye - cytology</subject><subject>Pigment Epithelium of Eye - drug effects</subject><subject>pigmentation</subject><subject>Proteins - analysis</subject><subject>rabbit</subject><subject>Rabbits</subject><subject>retinal pigment epithelium</subject><subject>RPE65</subject><subject>tight junctions</subject><subject>transepithelial resistance</subject><subject>Zonula Occludens-1 Protein</subject><issn>0014-4835</issn><issn>1096-0007</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kMtKxDAUhoMoOl5ewIVk5a71pEnTDrgR8QaCG12HJj1xMvRmkorz9maYAXcuwoHD9__kfIRcMsgZMHmzzvEHfV4AiBxkDrw4IAsGS5kBQHVIFgBMZKLm5Qk5DWGdtlxU4picsFKWRcnZgqi7fnBjdIb22GvfDEj7xg0xvUDjCum0wmGMmwnpaKlvtHaReoxuaDo6uc8eh0hxcgntXFoZ7LpA3UDN3MXZ4zk5sk0X8GI_z8jH48P7_XP2-vb0cn_3mhleipiVRd1oweqKL7nWaEuraynQCmlZaUUFGkQr2hq11ADScNlq1hhRi9ouUQp-Rq53vZMfv2YMUfUubD-TLhrnoGTFq0IuIYHFDjR-DMGjVZN3feM3ioHaalVrtdWqtloVSJW0ptDVvn3WPbZ_kb3HBNzuAEw3frsUD8bhYLB1Hk1U7ej-6_8FvKaKrA</recordid><startdate>2005</startdate><enddate>2005</enddate><creator>Stanzel, Boris V.</creator><creator>Espana, Edgar M.</creator><creator>Grueterich, Martin</creator><creator>Kawakita, Tetsuya</creator><creator>Parel, Jean-Marie</creator><creator>Tseng, Scheffer C.G.</creator><creator>Binder, Susanne</creator><general>Elsevier Ltd</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>2005</creationdate><title>Amniotic membrane maintains the phenotype of rabbit retinal pigment epithelial cells in culture</title><author>Stanzel, Boris V. ; Espana, Edgar M. ; Grueterich, Martin ; Kawakita, Tetsuya ; Parel, Jean-Marie ; Tseng, Scheffer C.G. ; Binder, Susanne</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c354t-528ab4187393bbef5fb864ef46f15f470b04d4d8eb6b006c36db1ac4848f9e643</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>age-related macular degeneration</topic><topic>Amnion</topic><topic>amniotic membrane</topic><topic>Animals</topic><topic>basement membrane</topic><topic>Basement Membrane - cytology</topic><topic>Basement Membrane - drug effects</topic><topic>Calcium - pharmacology</topic><topic>Cell Differentiation - drug effects</topic><topic>Cells, Cultured</topic><topic>Culture Media</topic><topic>differentiation</topic><topic>epithelial cells</topic><topic>Epithelial Cells - cytology</topic><topic>Epithelial Cells - drug effects</topic><topic>eye</topic><topic>Eye Proteins - analysis</topic><topic>in vitro</topic><topic>Keratins - analysis</topic><topic>Membrane Proteins - analysis</topic><topic>Microscopy, Phase-Contrast - methods</topic><topic>Phenotype</topic><topic>Phosphoproteins - analysis</topic><topic>Pigment Epithelium of Eye - cytology</topic><topic>Pigment Epithelium of Eye - drug effects</topic><topic>pigmentation</topic><topic>Proteins - analysis</topic><topic>rabbit</topic><topic>Rabbits</topic><topic>retinal pigment epithelium</topic><topic>RPE65</topic><topic>tight junctions</topic><topic>transepithelial resistance</topic><topic>Zonula Occludens-1 Protein</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Stanzel, Boris V.</creatorcontrib><creatorcontrib>Espana, Edgar M.</creatorcontrib><creatorcontrib>Grueterich, Martin</creatorcontrib><creatorcontrib>Kawakita, Tetsuya</creatorcontrib><creatorcontrib>Parel, Jean-Marie</creatorcontrib><creatorcontrib>Tseng, Scheffer C.G.</creatorcontrib><creatorcontrib>Binder, Susanne</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Experimental eye research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Stanzel, Boris V.</au><au>Espana, Edgar M.</au><au>Grueterich, Martin</au><au>Kawakita, Tetsuya</au><au>Parel, Jean-Marie</au><au>Tseng, Scheffer C.G.</au><au>Binder, Susanne</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Amniotic membrane maintains the phenotype of rabbit retinal pigment epithelial cells in culture</atitle><jtitle>Experimental eye research</jtitle><addtitle>Exp Eye Res</addtitle><date>2005</date><risdate>2005</risdate><volume>80</volume><issue>1</issue><spage>103</spage><epage>112</epage><pages>103-112</pages><issn>0014-4835</issn><eissn>1096-0007</eissn><abstract>The success of surgical removal of choroidal neovascularisation followed by transplantation of autologous retinal pigment epithelial cells (RPE) for age-related macular degeneration (ARMD) may be limited by damage in Bruch's membrane. We investigated whether amniotic membrane (AM) might be used as an alternative basement membrane-containing matrix to support RPE growth and differentiation.
Primary RPE plastic cultures were established from freshly enucleated Dutch belted rabbit eyes in DMEM/F12 containing 0·1
m
m Ca
++ and 10% dialysed FBS. Upon subconfluence, cells were subcultured at 5000–9000 cells
cm
−2 in the above-mentioned culture medium on intact AM (iAM), epithelially denuded AM (dAM) or plastic. After confluence, the Ca
++ concentration in the medium was increased to 1·8
m
m for 4 weeks. Growth and morphology were monitored by phase contrast microscopy, and the phenotype by immunostaining with antibodies against cytokeratin 18, tight junction protein ZO-1, and RPE65 protein, and by transepithelial resistance (TER) measurement. Immunostaining to cytokeratin 18 confirmed the epithelial origin of isolated cells in both primary culture and subcultures. Compared to plastic cultures, RPE increased pigmentation within 24
hr after seeding on AM, with iAM being more pronounced than dAM. RPE adopted a hexagonal epithelial phenotype with more organised pigmentation, strong expression of ZO-1 and RPE65, and a significantly higher TER 4 weeks after Ca
++ switch on dAM. Our results indicate that AM may be used as a basement membrane-containing matrix to maintain RPE phenotype in vitro, and may facilitate subsequent transplantation to treat ARMD.</abstract><cop>England</cop><pub>Elsevier Ltd</pub><pmid>15652531</pmid><doi>10.1016/j.exer.2004.06.032</doi><tpages>10</tpages></addata></record> |
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subjects | age-related macular degeneration Amnion amniotic membrane Animals basement membrane Basement Membrane - cytology Basement Membrane - drug effects Calcium - pharmacology Cell Differentiation - drug effects Cells, Cultured Culture Media differentiation epithelial cells Epithelial Cells - cytology Epithelial Cells - drug effects eye Eye Proteins - analysis in vitro Keratins - analysis Membrane Proteins - analysis Microscopy, Phase-Contrast - methods Phenotype Phosphoproteins - analysis Pigment Epithelium of Eye - cytology Pigment Epithelium of Eye - drug effects pigmentation Proteins - analysis rabbit Rabbits retinal pigment epithelium RPE65 tight junctions transepithelial resistance Zonula Occludens-1 Protein |
title | Amniotic membrane maintains the phenotype of rabbit retinal pigment epithelial cells in culture |
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