Development of a murine ocular posterior segment explant culture for the study of intravitreous vector delivery
Related Experiment Video
Updated: Apr 17, 2026
Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium
Published on: August 7, 2015
Nora Denk1, Vikram Misra1, Lynne S Sandmeyer1
1Department of Small Animal Clinical Sciences (Denk, Sandmeyer, Bauer, Grahn); Veterinary Microbiology (Misra); and Veterinary Biomedical Sciences (Singh, Forsyth), Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan.
Abstract:
The objective of this study was to develop a murine retinal/choroidal/scleral explant culture system to facilitate the intravitreous delivery of vectors. Posterior segment explants from adult mice of 2 different age groups (4 wk and 15 wk) were cultured in serum-free medium for variable time periods. Tissue viability was assessed by gross morphology, cell survival quantification, activated caspase-3 expression, and immunohistochemistry. To model ocular gene therapy, explants were exposed to varying transducing units of a lentiviral vector expressing the gene for green fluorescent protein for 48 h. Explant retinal cells remained viable for approximately 1 wk, although the ganglion cell layer developed apoptosis between 4 and 7 d. Following vector infusion into the posterior segment cups, viral transduction was noted in multiple retinal layers in both age groups. An age of donor mouse influence was noted and older mice did not transduce as well as younger mice. This explant offers an easily managed posterior segment ocular culture with minimum disturbance of the tissue, and may be useful for investigating methods of enhancing retinal gene therapy under controlled conditions.
Insights
This study developed a mouse eye explant culture for testing gene therapy delivery. Younger mice showed better viral transduction, suggesting age impacts retinal gene therapy effectiveness.
Area of Science:
- Ophthalmology
- Molecular Biology
- Cell Biology
Background:
- Developing effective ocular gene therapy requires reliable models for vector delivery and transduction.
- Current models may not fully replicate the complex posterior segment environment or allow for controlled experimental manipulation.
Purpose of the Study:
- To establish a murine retinal/choroidal/scleral explant culture system for studying intravitreous vector delivery.
- To assess the viability and transduction efficiency of this explant model for ocular gene therapy applications.
Main Methods:
- Posterior eye segments from young (4 wk) and adult (15 wk) mice were cultured in serum-free medium.
- Tissue viability was evaluated using morphology, cell counts, and apoptosis markers (activated caspase-3).
- Explants were exposed to lentiviral vectors expressing green fluorescent protein to model gene therapy.
Main Results:
- Retinal cells remained viable for approximately one week, with ganglion cell apoptosis observed between 4-7 days.
- Viral transduction occurred in multiple retinal layers across both age groups.
- Younger donor mice exhibited superior viral transduction compared to older mice, indicating an age-dependent effect.
Conclusions:
- The developed explant system provides a manageable model for investigating posterior segment ocular gene therapy.
- This model facilitates controlled studies on vector delivery and transduction efficiency.
- Donor age significantly influences transduction outcomes, a critical factor for optimizing retinal gene therapy strategies.
