Development and characterization of an adult retinal explant organotypic tissue culture system as an in vitro intraocular stem cell transplantation model.
OBJECTIVE
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 beta-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.
Johnson
Thomas V
TV
Cambridge Centre for Brain Repair, University of Cambridge, Cambridge, United Kingdom.
Martin
Keith R
KR
D000818:Animals; D030801:Animals, Genetically Modified; D015415:Biomarkers / Q000378:metabolism; D001854:Bone Marrow Cells; D053148:Caspase 3 / Q000378:metabolism; D018929:Cell Culture Techniques; D002470:Cell Survival; D017690:Cell Transplantation; D018920:Coculture Techniques; D019084:Fluorescent Antibody Technique, Indirect; D008297:Male; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D018613:Microscopy, Confocal; D023421:Models, Animal; D009457:Neuroglia / Q000166:cytology*; D009798:Ocular Hypertension / Q000601:surgery; D009924:Organ Culture Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000166:cytology* / Q000201:enzymology
D015415:Biomarkers; D053148:Caspase 3