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Immunopanning purification and long-term culture of human retinal ganglion cells

Zhang, Xinmei and Li Liu, David Ta and Chiang, Sylvia Wai-Yee and Choy, Kwong-Wai and Pang, Chi-Pui and Lam, Dennis Shun-Chiu and Yam, Gary Hin-Fai (2010) Immunopanning purification and long-term culture of human retinal ganglion cells. Molecular vision, 16. pp. 2867-2872. ISSN 1090-0535

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Abstract

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

Item Type: Article
Uncontrolled Keywords: retinal ganglion cells, RGCs, cell viability, immunocytochemistry,
Subjects: FOR Classification > 0601 Biochemistry and Cell Biology
FOR Classification > 1101 Medical Biochemistry and Metabolomics
Faculty/School/Research Centre/Department > Institute of Sport, Exercise and Active Living (ISEAL)
Depositing User: VUIR
Date Deposited: 21 Mar 2013 02:59
Last Modified: 21 Mar 2013 02:59
URI: http://vuir.vu.edu.au/id/eprint/21485
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Citations in Scopus: 6 - View on Scopus

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