Abstract
Purpose :
The porcine retina represents an optimal model system for inherited retinal dystrophies due to the anatomical similarities to the human retina, including a cone enriched visual streak. As large animal models require significant infrastructural and financial resources, the organotypic retina culture might pose a good model system to study gene therapeutic approaches prior to in vivo application. However, to this date it appeared to be difficult to reproduce good quality explants that can be kept in culture long enough to enable viral vector mediated genome editing experimentation. Our protocol allows to keep explants in culture for up to 20 days with good morphological preservation.
Methods :
Two to four retinal explants per eye were obtained from the visual streak and transferred onto a polycarbonate membrane insert with the photoreceptors facing down. They were cultured for up to 28 days with Neurobasal-A medium containing 100 or 450 mg/dl glucose at 37°C and 5% CO2. We supplemented the medium with combinations of fetal calf serum, B-27 with or without Insulin and N-2. Explants were analyzed using confocal laser scanning microscopy after immunofluorescent (IF) labeling with antibodies against glial fibrillary acidic protein, glutamine synthetase, protein kinase C alpha, rhodopsin, and short-wave-sensitive opsin 1, all counterstained with 4',6-Diamidino-2-phenylindol (DAPI).
Results :
We were able to keep explants in culture up to 20 days with only little degradation as seen after IF at different harvesting time points. Best results were attained using Neurobasal-A medium containing 100 mg/dl glucose, 2x B-27 containing Insulin, 1x N-2, 1x L-Glutamine and 1x antibiotic-antimycotic, and a medium change every 48 hours. Eyes treated with heat for decontamination purposes by the butcher showed significantly less good preservation compared to those obtained without heat treatment. Keeping the eyes in ice cold medium until right before preparation and minimizing transport time gave the best results.
Conclusions :
Using a standardized protocol, porcine retinal explants represent an easy to handle intermediate model between in vitro and in vivo experimentation. Using pig eyes from a local butcher renders this model system easily reproducible and contributes to the implementation of the 3R principle by Russell and Burch. This model system is currently tested with standard gene therapy vectors for efficient gene transfer.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.