Abstract
Purpose :
The choroid, a vascularized tissue situated between the retina and the sclera, plays a crucial role in maintaining ocular homeostasis. Despite its significance, research on choroidal abnormalities and the establishment of effective in vitro models have been limited. It is necessary to establish an in vitro tissue model for effective regenerative medicine and drug screening for choroidal abnormalities, such as choroidal thinning in myopic eyes and choroidal atrophy. This study aims to establish a multi-layered vascular structure resembling the choroid for vascular response screening.
Methods :
Choroidal fibroblasts were isolated from C57BL6J mouse eyes and co-cultured with iPSC-derived endothelial cells (ECs) on the transwell insert. Additionally, iPSC-derived retinal pigment epithelium (RPE) cells were cultured on the bottom of the culture plate. After 1 week of culture, the self-constructed tissue by the cells was immunostained for endothelial cell markers or stromal markers. To evaluate the vascular response by the vasodilator or vasoconstrictor, bunazosin or phenylephrine was treated to the constructed model and evaluated its vasculatures using AngioTool (National Cancer Institute) software.
Results :
The choroid in vitro model was generated with the combinations of three cell types (fibroblasts, ECs, and RPE cells). The morphology of the model showed multi-layers and was similar to that of the mouse choroid by observation of immunohistochemistry and H&E staining. The upper side of the constructed model showed a strong expression of endomucin, indicating choriocapillaris-like vasculature. 5 h after drug treatment, compared to the control group (non-treated), vessel area, the number of junctions, vessel length, or the mean of lacunarity changed after treatment with bunazosin or phenylephrine. The normalized total number of junctions and vessel length increased in the bunazosin-treated group (1.00±0.03 vs 1.18±0.05, p<0.001 and 1.00±0.01 vs 1.08±0.03, p < 0.01). In the phenylephrine-treated group, the vessel area decreased (1.00±0.02 vs 0.97±001, p<0.05), and the mean of lacunarity increased (1.00±0.05 vs 1.09±0.05, p<0.05) as the non-vessel area expanded due to its vasoconstriction effect.
Conclusions :
Our study demonstrates that co-culturing iPSC-derived ECs and RPE cells with mouse choroidal fibroblasts can successfully construct an in vitro choroid complex system for vascular response screening.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.