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A Simple Alkaline Method for Decellularizing Human Amniotic Membrane for Cell Culture
Human amniotic membrane is a standard substratum used to culture limbal epithelial stem cells for transplantation to patients with limbal stem cell deficiency. Various methods were developed to decellularize amniotic membrane, because denuded membrane is poorly immunogenic and better supports repopu...
Autores principales: | , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3827346/ https://www.ncbi.nlm.nih.gov/pubmed/24236148 http://dx.doi.org/10.1371/journal.pone.0079632 |
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author | Saghizadeh, Mehrnoosh Winkler, Michael A. Kramerov, Andrei A. Hemmati, David M. Ghiam, Chantelle A. Dimitrijevich, Slobodan D. Sareen, Dhruv Ornelas, Loren Ghiasi, Homayon Brunken, William J. Maguen, Ezra Rabinowitz, Yaron S. Svendsen, Clive N. Jirsova, Katerina Ljubimov, Alexander V. |
author_facet | Saghizadeh, Mehrnoosh Winkler, Michael A. Kramerov, Andrei A. Hemmati, David M. Ghiam, Chantelle A. Dimitrijevich, Slobodan D. Sareen, Dhruv Ornelas, Loren Ghiasi, Homayon Brunken, William J. Maguen, Ezra Rabinowitz, Yaron S. Svendsen, Clive N. Jirsova, Katerina Ljubimov, Alexander V. |
author_sort | Saghizadeh, Mehrnoosh |
collection | PubMed |
description | Human amniotic membrane is a standard substratum used to culture limbal epithelial stem cells for transplantation to patients with limbal stem cell deficiency. Various methods were developed to decellularize amniotic membrane, because denuded membrane is poorly immunogenic and better supports repopulation by dissociated limbal epithelial cells. Amniotic membrane denuding usually involves treatment with EDTA and/or proteolytic enzymes; in many cases additional mechanical scraping is required. Although ensuring limbal cell proliferation, these methods are not standardized, require relatively long treatment times and can result in membrane damage. We propose to use 0.5 M NaOH to reliably remove amniotic cells from the membrane. This method was used before to lyse cells for DNA isolation and radioactivity counting. Gently rubbing a cotton swab soaked in NaOH over the epithelial side of amniotic membrane leads to nearly complete and easy removal of adherent cells in less than a minute. The denuded membrane is subsequently washed in a neutral buffer. Cell removal was more thorough and uniform than with EDTA, or EDTA plus mechanical scraping with an electric toothbrush, or n-heptanol plus EDTA treatment. NaOH-denuded amniotic membrane did not show any perforations compared with mechanical or thermolysin denuding, and showed excellent preservation of immunoreactivity for major basement membrane components including laminin α2, γ1-γ3 chains, α1/α2 and α6 type IV collagen chains, fibronectin, nidogen-2, and perlecan. Sodium hydroxide treatment was efficient with fresh or cryopreserved (10% dimethyl sulfoxide or 50% glycerol) amniotic membrane. The latter method is a common way of membrane storage for subsequent grafting in the European Union. NaOH-denuded amniotic membrane supported growth of human limbal epithelial cells, immortalized corneal epithelial cells, and induced pluripotent stem cells. This simple, fast and reliable method can be used to standardize decellularized amniotic membrane preparations for expansion of limbal stem cells in vitro before transplantation to patients. |
format | Online Article Text |
id | pubmed-3827346 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-38273462013-11-14 A Simple Alkaline Method for Decellularizing Human Amniotic Membrane for Cell Culture Saghizadeh, Mehrnoosh Winkler, Michael A. Kramerov, Andrei A. Hemmati, David M. Ghiam, Chantelle A. Dimitrijevich, Slobodan D. Sareen, Dhruv Ornelas, Loren Ghiasi, Homayon Brunken, William J. Maguen, Ezra Rabinowitz, Yaron S. Svendsen, Clive N. Jirsova, Katerina Ljubimov, Alexander V. PLoS One Research Article Human amniotic membrane is a standard substratum used to culture limbal epithelial stem cells for transplantation to patients with limbal stem cell deficiency. Various methods were developed to decellularize amniotic membrane, because denuded membrane is poorly immunogenic and better supports repopulation by dissociated limbal epithelial cells. Amniotic membrane denuding usually involves treatment with EDTA and/or proteolytic enzymes; in many cases additional mechanical scraping is required. Although ensuring limbal cell proliferation, these methods are not standardized, require relatively long treatment times and can result in membrane damage. We propose to use 0.5 M NaOH to reliably remove amniotic cells from the membrane. This method was used before to lyse cells for DNA isolation and radioactivity counting. Gently rubbing a cotton swab soaked in NaOH over the epithelial side of amniotic membrane leads to nearly complete and easy removal of adherent cells in less than a minute. The denuded membrane is subsequently washed in a neutral buffer. Cell removal was more thorough and uniform than with EDTA, or EDTA plus mechanical scraping with an electric toothbrush, or n-heptanol plus EDTA treatment. NaOH-denuded amniotic membrane did not show any perforations compared with mechanical or thermolysin denuding, and showed excellent preservation of immunoreactivity for major basement membrane components including laminin α2, γ1-γ3 chains, α1/α2 and α6 type IV collagen chains, fibronectin, nidogen-2, and perlecan. Sodium hydroxide treatment was efficient with fresh or cryopreserved (10% dimethyl sulfoxide or 50% glycerol) amniotic membrane. The latter method is a common way of membrane storage for subsequent grafting in the European Union. NaOH-denuded amniotic membrane supported growth of human limbal epithelial cells, immortalized corneal epithelial cells, and induced pluripotent stem cells. This simple, fast and reliable method can be used to standardize decellularized amniotic membrane preparations for expansion of limbal stem cells in vitro before transplantation to patients. Public Library of Science 2013-11-13 /pmc/articles/PMC3827346/ /pubmed/24236148 http://dx.doi.org/10.1371/journal.pone.0079632 Text en © 2013 Saghizadeh et al http://creativecommons.org/licenses/by/4.0/ 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 properly credited. |
spellingShingle | Research Article Saghizadeh, Mehrnoosh Winkler, Michael A. Kramerov, Andrei A. Hemmati, David M. Ghiam, Chantelle A. Dimitrijevich, Slobodan D. Sareen, Dhruv Ornelas, Loren Ghiasi, Homayon Brunken, William J. Maguen, Ezra Rabinowitz, Yaron S. Svendsen, Clive N. Jirsova, Katerina Ljubimov, Alexander V. A Simple Alkaline Method for Decellularizing Human Amniotic Membrane for Cell Culture |
title | A Simple Alkaline Method for Decellularizing Human Amniotic Membrane for Cell Culture |
title_full | A Simple Alkaline Method for Decellularizing Human Amniotic Membrane for Cell Culture |
title_fullStr | A Simple Alkaline Method for Decellularizing Human Amniotic Membrane for Cell Culture |
title_full_unstemmed | A Simple Alkaline Method for Decellularizing Human Amniotic Membrane for Cell Culture |
title_short | A Simple Alkaline Method for Decellularizing Human Amniotic Membrane for Cell Culture |
title_sort | simple alkaline method for decellularizing human amniotic membrane for cell culture |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3827346/ https://www.ncbi.nlm.nih.gov/pubmed/24236148 http://dx.doi.org/10.1371/journal.pone.0079632 |
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