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A proper protocol for isolation of retinal pigment epithelium from rabbit eyes

BACKGROUND: Retinal pigment epithelium (RPE) is a hexagonal monolayer of pigmented cells located between the neural retina and the choroid with an essential role for visual function. So, isolation, propagation and maintenance of their functional integrity of RPE are crucial for research in vitro whi...

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Autores principales: Amirpour, Noushin, Karamali, Fereshteh, Razavi, Shahnaz, Esfandiari, Ebrahim, Nasr-Esfahani, Mohammad Hossein
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Medknow Publications & Media Pvt Ltd 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3928817/
https://www.ncbi.nlm.nih.gov/pubmed/24592359
http://dx.doi.org/10.4103/2277-9175.124630
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author Amirpour, Noushin
Karamali, Fereshteh
Razavi, Shahnaz
Esfandiari, Ebrahim
Nasr-Esfahani, Mohammad Hossein
author_facet Amirpour, Noushin
Karamali, Fereshteh
Razavi, Shahnaz
Esfandiari, Ebrahim
Nasr-Esfahani, Mohammad Hossein
author_sort Amirpour, Noushin
collection PubMed
description BACKGROUND: Retinal pigment epithelium (RPE) is a hexagonal monolayer of pigmented cells located between the neural retina and the choroid with an essential role for visual function. So, isolation, propagation and maintenance of their functional integrity of RPE are crucial for research in vitro which next used for cell transplantation. The evaluation of features of RPE cells as a sheet after 14 days has not been reported yet. This study aimed to examine and compare three protocols for RPE isolation from rabbit eyes and obtain a proper protocol, which illustrated isolated RPE cells as a sheet cause to preserve their characterize even after 2 weeks. MATERIALS AND METHODS: RPE cells were prepared from eyes of 24 rabbit eyes. After enucleating of eyes, anterior segment discarded and posterior segment cut to small pieces. Two of these procedures are based on the enzymatic digestion, but third protocol based on mechanical dissection. The culture cells harvested and morphological feature of cells assessed by phase-contrast microscope and then analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry. RESULTS: Evaluation of morphological feature showed that isolation of RPE cells as a sheet lead to preserve their hexagonal morphology. Immunocytochemistry and RT-PCR assessment demonstrated RPE cell cultured in sheet maintained their phenotypic feature, tight junction and the distribution of actin and cytokeratin filament. Comparison of three protocols showed that dissociation of RPE cells as a sheet was superior in the preserve of RPE characteristic. CONCLUSIONS: Isolation of RPE cells as a sheet maintains the integrity of these cells, this procedure promising a therapeutic approach, which is important for some retinal diseases.
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spelling pubmed-39288172014-03-03 A proper protocol for isolation of retinal pigment epithelium from rabbit eyes Amirpour, Noushin Karamali, Fereshteh Razavi, Shahnaz Esfandiari, Ebrahim Nasr-Esfahani, Mohammad Hossein Adv Biomed Res Original Article BACKGROUND: Retinal pigment epithelium (RPE) is a hexagonal monolayer of pigmented cells located between the neural retina and the choroid with an essential role for visual function. So, isolation, propagation and maintenance of their functional integrity of RPE are crucial for research in vitro which next used for cell transplantation. The evaluation of features of RPE cells as a sheet after 14 days has not been reported yet. This study aimed to examine and compare three protocols for RPE isolation from rabbit eyes and obtain a proper protocol, which illustrated isolated RPE cells as a sheet cause to preserve their characterize even after 2 weeks. MATERIALS AND METHODS: RPE cells were prepared from eyes of 24 rabbit eyes. After enucleating of eyes, anterior segment discarded and posterior segment cut to small pieces. Two of these procedures are based on the enzymatic digestion, but third protocol based on mechanical dissection. The culture cells harvested and morphological feature of cells assessed by phase-contrast microscope and then analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry. RESULTS: Evaluation of morphological feature showed that isolation of RPE cells as a sheet lead to preserve their hexagonal morphology. Immunocytochemistry and RT-PCR assessment demonstrated RPE cell cultured in sheet maintained their phenotypic feature, tight junction and the distribution of actin and cytokeratin filament. Comparison of three protocols showed that dissociation of RPE cells as a sheet was superior in the preserve of RPE characteristic. CONCLUSIONS: Isolation of RPE cells as a sheet maintains the integrity of these cells, this procedure promising a therapeutic approach, which is important for some retinal diseases. Medknow Publications & Media Pvt Ltd 2014-01-09 /pmc/articles/PMC3928817/ /pubmed/24592359 http://dx.doi.org/10.4103/2277-9175.124630 Text en Copyright: © 2014 Amirpour. http://creativecommons.org/licenses/by-nc-sa/3.0 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Original Article
Amirpour, Noushin
Karamali, Fereshteh
Razavi, Shahnaz
Esfandiari, Ebrahim
Nasr-Esfahani, Mohammad Hossein
A proper protocol for isolation of retinal pigment epithelium from rabbit eyes
title A proper protocol for isolation of retinal pigment epithelium from rabbit eyes
title_full A proper protocol for isolation of retinal pigment epithelium from rabbit eyes
title_fullStr A proper protocol for isolation of retinal pigment epithelium from rabbit eyes
title_full_unstemmed A proper protocol for isolation of retinal pigment epithelium from rabbit eyes
title_short A proper protocol for isolation of retinal pigment epithelium from rabbit eyes
title_sort proper protocol for isolation of retinal pigment epithelium from rabbit eyes
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3928817/
https://www.ncbi.nlm.nih.gov/pubmed/24592359
http://dx.doi.org/10.4103/2277-9175.124630
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