Abstract
Purpose :
To establish an ex vivo glaucoma model comparing different cell types in an artificial conventional outflow system (ACOS) which can be utilized to explore outflow pathophysiology and novel therapies for glaucoma.
Methods :
Primary human trabecular meshwork cells (HTMC) and glaucomatous HTMC (GTMC) were cultured and verified by dexamethasone (Dex) responsiveness. HTMC were transfected with lentiviral vectors encoding myocilin Y437H or Q368X mutation to generate induced-GTMC (IGTMC). Expression of myocilin and extracellular matrix (ECM) components collagen IV and fibronectin was examined by staining and Western blotting (WB). HTMC, GTMC and IGTMC were cultured on microfabricated, porous SU-8 scaffolds for 14 days and examined by optical imaging, immunostaining, and WB. Further, these cell-scaffold constructs in ACOS were perfused at various rates with pressure continuously recorded. Simulated outflow facility (µL/min/mmHg/mm2) was calculated from the inverse of the slope of pressure vs flow graph. Statistical analysis was performed using GraphPad Prism. p<0.05 was considered statistically significant.
Results :
Both HTMC and GTMC were responsive to Dex with significantly increased myocilin expression. IGTMC carrying MYOC Y437H or Q368X mutation had significantly increased expression of myocilin and fibronectin. HTMC cultured in ACOS recapitulated HTMC marker expression of myocilin, collagen IV and fibronectin and exhibited simulated outflow facility at levels comparable to those in cadaveric human eyes. HTMC in ACOS were responsive to Dex, showing significantly reduced outflow facility. GTMC in ACOS had significantly lower outflow facility than HTMC. GTMC had disorganized F-actin and fibronectin deposition and myocilin expression different from HTMC in ACOS. IGTMC in ACOS exhibited significantly reduced outflow facility as compared to normal HTMC. IGTMC in ACOS significantly increased expression of myocilin, fibronectin and ER stress marker GRP78, a similar phenomenon to cultured cells without ACOS.
Conclusions :
HTMC, GTMC and IGTMC can be cultured in vitro and used in ACOS. The three cell types deposit differential ECM components and exhibit differential outflow facility with perfusion. Use of HTMC, GTMC and IGTMC in ACOS provides a new ex vivo model to study glaucoma-related outflow pathophysiology that can be used in drug discovery and in developing cell therapies for glaucoma treatment.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.