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Chunhua Jiao, Shemin Zeng, Michael Alexander Schelling, Ryson Stuart, Robert F Mullins, Elliott H Sohn; Novel organotypic culture model of pig choroid-scleral explant as a therapeutic screening tool. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2143.
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To establish a pig organotypic choroid-scleral explant culture system for use as an ex-vivo choroid tubulogenesis screening tool to study novel potential pharmaceutical therapies.
2mm explants of choroid/sclera without the RPE were obtained from adult porcine eyes and cultured on three-dimensional collagen matrix. After 2 days of culture in medium with 10% fetal bovine serum and 1 day of serum starvation in 4% serum medium, the explants were further cultured in the presence or absence of varying concentrations of VEGF (5ng/ml, 10ng/ml, 50ng/ml), complement 5a (10ug/ml, 50ug/ml), or tamoxifen (10ug/ml, 20ug/ml) for an additional 48 and 72 hour period. Vascular sprouting from the choroid-scleral complex was visualized using B. simplicifolia isolectin B4 (BSI-B4) and anti-CD31 antibody using immunofluorescence and confocal microscopy. Morphology of sprouting was assessed on toluidine blue stained semithin sections using light microscopy. Density of vascular elements in each condition was quantified using ImageJ software. Comparisons of controls and each treatment group were performed by one-way ANOVA using SPSS. Results were expressed as mean +/- SEM. P<0.05 was considered statistically significant.
Choroidal endothelial cells began to proliferate and migrate out of the explant after 24 hours; tubulogenesis began at 48 hours, with a marked abundance of vascular tubes by 72 hours. Cells comprising the tubes were labeled with endothelial cell markers BSI-B4 and CD31 antibody. Explants treated with VEGF showed dose dependence and 50ng/ml of VEGF (n=5) had a statistically significant increase in vascular area compared to controls (n=9, p=0.038) after 48 hours of culture; a VEGF directed antibody inhibited this effect of VEGF. In addition, after 72 hours of incubation, 50ug/ml C5a induced tubulogenesis; tamoxifen (10ug/ml, n=4; 20ug/ml, n=2) on the other hand significantly reduced vessel formation compared to control (n=6, p=0.007) in porcine choroid-scleral explant.
This adult pig choroid-sclera explant culture model is a short-term organotypic culture system that offers an opportunity to evaluate pro- and anti-angiogenic effects of pharmacologic compounds on choroid biology.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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