RESUMO
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.
