Abstract
Purpose :
Abnormal lipid metabolism and increased transcytosis have shown to be an essential player in the pathology of Diabetic Retinopathy (DR). Mfsd2a contributes to the integrity of the blood-brain-barrier (BBB) and blood-retinal-barrier (BRB) through regulation of transcytosis and lipid metabolism. It has been demonstrated that co-culturing endothelial cells and pericytes together at a 1:1 ratio in vitro is more representative of the BRB. Historically, commercially available transwells have been used to study both of these cell types. However, transwells lack the necessary porosity to enable cross-talk between these cells. To overcome this, we engineered a 3D model of the human BRB on highly porous and uniform biocompatible scaffolds with enhanced expression of Mfsd2a, as well as junctional marker VE-cadherin and endothelial cell marker CD31, to demonstrate a tight microvascular barrier in our model.
Methods :
Scaffolds coated with hyaluronan-based hydrogel were seeded with primary human retinal microvascular endothelial cells (HRMVECs) and allowed to grow for 7 days. Scaffolds were inverted, primary human retinal pericytes (PCs) were added, and were grown for an additional 6 days until confluent after which lysates were harvested. These tissue constructs were compared side-by-side with cells cultured on commercially available transwells. Expression of MFSd2a, VE-cadherin and CD31 was evaluated via western blot. Knockdown of Mfsd2a was accomplished using siRNAs and the effects on transcytosis were observed using a clathrin- mediated transcytosis assay.
Results :
The bioengineered 3D model demonstrated higher levels of Mfsd2a (p<0.001, N=3), VE-cadherin (p<0.001, N=3) and CD31 (p<0.05, N=3) when compared to commercially available transwells. Furthermore, 3D cultures of HRMVECs alone showed lower expression of Mfsd2a when compared to its coculture counterpart (HRMVEC-PC) (2±0.6 fold increase, p>0.01, N=3). Mfsd2a knock down in our 3D model resulted in significantly higher transcytosis (p<0.01, N=3) when compared to vehicle samples.
Conclusions :
These results demonstrate the superiority of our 3D bioengineered BRB model compared to commercially available transwells. This model serves as a tool to study transcytosis at the BRB; opening the gate for the development of new therapies for DR and DME.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.