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Immunopanning purification and long-term culture of human retinal ganglion cells
PURPOSE: To establish a robust method to isolate primary retinal ganglion cells (RGCs) from human fetal retina for long-term culture while maintaining neuronal morphology and marker protein expression. METHODS: A total of six human retinas were obtained from aborted fetuses at 10 to 12 weeks of gest...
Autores principales: | , , , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
Molecular Vision
2010
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3012647/ https://www.ncbi.nlm.nih.gov/pubmed/21203402 |
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author | Zhang, Xin-Mei Li Liu, David Ta Chiang, Sylvia Wai-Yee Choy, Kwong-Wai Pang, Chi-Pui Lam, Dennis Shun-Chiu Yam, Gary Hin-Fai |
author_facet | Zhang, Xin-Mei Li Liu, David Ta Chiang, Sylvia Wai-Yee Choy, Kwong-Wai Pang, Chi-Pui Lam, Dennis Shun-Chiu Yam, Gary Hin-Fai |
author_sort | Zhang, Xin-Mei |
collection | PubMed |
description | PURPOSE: To establish a robust method to isolate primary retinal ganglion cells (RGCs) from human fetal retina for long-term culture while maintaining neuronal morphology and marker protein expression. METHODS: A total of six human retinas were obtained from aborted fetuses at 10 to 12 weeks of gestation with informed consent from mothers. RGCs were isolated and purified by a modified two-step immunopanning procedure. The cells were maintained in a serum-free defined medium supplemented with brain-derived neurotrophic factor, ciliary neutrophic factor, and forskolin. The viable RGCs and the extent of neurite outgrowth were examined by calcein-acetoxymethylester assay. Expression of RGC markers was studied by immunocytochemistry. RESULTS: Primary RGCs from human fetal retinas were isolated and maintained in vitro for one month with substantial neurite elongation. In cell culture, almost 70% of the isolated cells attached, spread, and displayed numerous dendrites. They were immunoreactive to RGC-specific markers (Thy-1, TUJ-1, and Brn3a) and negative for glial fibrillary acidic protein and amacrine cells marker HPC-1. CONCLUSIONS: Human RGCs were successfully isolated and maintained in long-term culture. This can serve as an ideal model for biologic, toxicological, and genomic assays of human RGCs in vitro. |
format | Text |
id | pubmed-3012647 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2010 |
publisher | Molecular Vision |
record_format | MEDLINE/PubMed |
spelling | pubmed-30126472011-01-03 Immunopanning purification and long-term culture of human retinal ganglion cells Zhang, Xin-Mei Li Liu, David Ta Chiang, Sylvia Wai-Yee Choy, Kwong-Wai Pang, Chi-Pui Lam, Dennis Shun-Chiu Yam, Gary Hin-Fai Mol Vis Research Article PURPOSE: To establish a robust method to isolate primary retinal ganglion cells (RGCs) from human fetal retina for long-term culture while maintaining neuronal morphology and marker protein expression. METHODS: A total of six human retinas were obtained from aborted fetuses at 10 to 12 weeks of gestation with informed consent from mothers. RGCs were isolated and purified by a modified two-step immunopanning procedure. The cells were maintained in a serum-free defined medium supplemented with brain-derived neurotrophic factor, ciliary neutrophic factor, and forskolin. The viable RGCs and the extent of neurite outgrowth were examined by calcein-acetoxymethylester assay. Expression of RGC markers was studied by immunocytochemistry. RESULTS: Primary RGCs from human fetal retinas were isolated and maintained in vitro for one month with substantial neurite elongation. In cell culture, almost 70% of the isolated cells attached, spread, and displayed numerous dendrites. They were immunoreactive to RGC-specific markers (Thy-1, TUJ-1, and Brn3a) and negative for glial fibrillary acidic protein and amacrine cells marker HPC-1. CONCLUSIONS: Human RGCs were successfully isolated and maintained in long-term culture. This can serve as an ideal model for biologic, toxicological, and genomic assays of human RGCs in vitro. Molecular Vision 2010-12-28 /pmc/articles/PMC3012647/ /pubmed/21203402 Text en Copyright © 2010 Molecular Vision. http://creativecommons.org/licenses/by/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 work is properly cited. |
spellingShingle | Research Article Zhang, Xin-Mei Li Liu, David Ta Chiang, Sylvia Wai-Yee Choy, Kwong-Wai Pang, Chi-Pui Lam, Dennis Shun-Chiu Yam, Gary Hin-Fai Immunopanning purification and long-term culture of human retinal ganglion cells |
title | Immunopanning purification and long-term culture of human retinal ganglion cells |
title_full | Immunopanning purification and long-term culture of human retinal ganglion cells |
title_fullStr | Immunopanning purification and long-term culture of human retinal ganglion cells |
title_full_unstemmed | Immunopanning purification and long-term culture of human retinal ganglion cells |
title_short | Immunopanning purification and long-term culture of human retinal ganglion cells |
title_sort | immunopanning purification and long-term culture of human retinal ganglion cells |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3012647/ https://www.ncbi.nlm.nih.gov/pubmed/21203402 |
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