IOVS Arteriosclerosis, Thrombosis, and Vascular Biology
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Originally published In Press as doi:10.1167/iovs.07-1601 on April 11, 2008
 (Investigative Ophthalmology and Visual Science. 2008;49:3503-3512.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.07-1601
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Development and Characterization of an Adult Retinal Explant Organotypic Tissue Culture System as an In Vitro Intraocular Stem Cell Transplantation Model

Thomas V. Johnson and Keith R. Martin

From the Cambridge Centre for Brain Repair, University of Cambridge, Cambridge, United Kingdom.

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.






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Copyright © 2008 by the Association for Research in Vision and Ophthalmology