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A more recent version of this article appeared on August 1, 2008
 (Investigative Ophthalmology and Visual Science. )
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.07-1601
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Article

Development and characterization of an adult retinal explant organotypic tissue culture system as an in vitro intraocular stem cell transplantation model

Thomas Vincent Johnson 1* and Keith Martin 2

1 Centre for Brain Repair, Cambridge University, The ED Adrian Building, Cambridge, CB2 1TQ, United Kingdom
2 Centre for Brain Repair, Cambridge University, Cambridge, United Kingdom

* To whom correspondence should be addressed. E-mail: tvj20{at}cam.ac.uk.


  Abstract

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 media (B27/N2) or media 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. To model intravitreal cell transplantation, Muller progenitor cells (hMIO-M1) or mesenchymal stem cells (MSC) were placed on explants. Explants were compared to whole eyes with or without experimental glaucoma and/or intravitreal cell transplantation. Results: Explants cultured in B27/N2 media were viable for at least 17 days as assessed by the aforementioned parameters. NHS media 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 media strongly expressed {beta}-III-tubulin, neurofilament, NeuN, Brn3a, Thy-1, GFAP, vimentin, nestin, and glutamine synthetase, whereas immunoreactivity was weak in NHS media and decreased further with time. Seven and fourteen days after co-culture or transplantation, glial reactivity (GFAP/vimentin expression) was highly upregulated in explants and eyes, respectively. Some grafted cells migrated into the retina, but the majority remained outside the inner limiting membrane. Conclusions: Retinal explants prepared using our techniques and cultured in B27/N2 media 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 to enhance retinal stem cell therapy.

Key Words: retinal transplantation, transplantation, retinal cell culture




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T. V. Johnson, N. D. Bull, and K. R. Martin
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F. M. Nadal-Nicolas, M. Jimenez-Lopez, P. Sobrado-Calvo, L. Nieto-Lopez, I. Canovas-Martinez, M. Salinas-Navarro, M. Vidal-Sanz, and M. Agudo
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