Abstract
Purpose :
Neovascularization is a key feature in sight-threatening retinal diseases, such as proliferative diabetic retinopathy and neovascular age-related macular degeneration. However, the lack of a clinically relevant tissue model that accurately replicates neovascularization specific to these pathologies hinders the development of therapeutics. Therefore, the aim of this project was to develop and characterize a clinically relevant 3D human organotypic choroidal sprout model.
Methods :
Culture conditions (explant sizes, locations of excision relative to the optic nerve head, shape of Matrigel) and donor characteristics (age and post-mortem time) were optimized by assessing the rate of sprouting and total growth area . Immunohistochemistry was used to identify choroidal sprout cell types using specific markers: nerve/glial-antigen 2 (NG2) for pericytes, cluster of differentiation 31 (CD31) and isolectinB4 for endothelial cells, ionized calcium-binding adaptor molecule 1 (Iba-1) for microglia, alpha-smooth muscle actin (α-SMA) for fibroblasts, and RPE65 for retinal pigment epithelium. Using the optimized culture conditions, the choroidal cultures were exposed to hypoxia and oxidative stress in medium containing low (50mM) and high (100mM) concentrations of Cobalt Chloride (CoCl2).
Results :
Up to eight explants were obtained from each donor choroid. The optimum culture conditions were found to be 2 mm explants embedded in dome-shaped Matrigel as these conditions resulted in greater chances of sprouting (p<0.05) and larger area of sprouting (p<0.05)(n=16). No statistically different growth rate and area were found between explants extracted from central compared to peripheral choroid (n=16), nor between explants from different donor age and post-mortem time (n=16). The new vessels were positive for endothelial cell markers and negative for pericytes, fibroblasts, microglia, and retinal pigment epithelium markers. CoCl2 resulted in decreased growth rate, leaving avascular tubules with elevated regions of cells clumping.
Conclusions :
A 3D clinically relevant human choroidal model of neovascularization was successfully developed and characterized. CoCl2 exposure resulted in measurable changes in cell expression and phenotype suggesting that the model could be utilized for the study of vascular changes in the choroid in response to different pathologies.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.