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Abstract #21750 Published in IGR 10-3
Development and characterization of an adult retinal explant organotypic tissue culture system as an in vitro intraocular stem cell transplantation model
Johnson TV; Martin KRInvestigative Ophthalmology and Visual Science 2008; 49: 3503-3512
PURPOSE: To develop and characterize a retinal explant culture system to facilitate investigation of novel methods of improving retinal stem cell therapy. METHODS: Retinas explanted from adult rats were cultured in serum-free medium (B27/N2) or medium containing normal horse serum (NHS). Tissue viability was assessed by gross morphology, propidium iodide (PI) uptake, cell survival quantification, activated caspase-3 expression, and immunohistochemistry. Müller progenitor cells (hMIO-M1), or mesenchymal stem cells (MSC) were placed on explants, to model intravitreal cell transplantation. Explants were compared with whole eyes, with or without experimental glaucoma and/or intravitreal cell transplantation. RESULTS: Explants cultured in B27/N2 medium were viable for at least 17 days, as assessed by the aforementioned parameters. NHS medium was associated with obvious tissue degradation, greater/more diffuse PI uptake, significant cell loss over time, and temporal increase in activated caspase-3(+) cells. Explants in B27/N2 medium strongly expressed β-III-tubulin, neurofilament, NeuN, Brn3a, Thy-1, GFAP, vimentin, nestin, and glutamine synthetase, whereas immunoreactivity was weak in NHS medium and decreased further with time. Seven and 14 days after coculture or transplantation, glial reactivity (GFAP/vimentin expression) was highly upregulated in explants and eyes, respectively. Some grafted cells migrated into the retina, but most remained outside the inner limiting membrane. CONCLUSIONS: Retinal explants prepared using the described techniques and cultured in B27/N2 medium are viable for at least 2 weeks and mimic in vivo glial reactivity to transplantation while allowing few grafted cells to integrate. This system may be a useful in-vitro model for investigating methods of enhancing retinal stem cell therapy.
Dr. T.V. Johnson, Cambridge Centre for Brain Repair, University of Cambridge, Cambridge, UK
Classification:
2.17 Stem cells (Part of: 2 Anatomical structures in glaucoma)