Bioengineering Human Trabecular Meshwork for Glaucoma Therapeutic Screening

Abstract

 

Purpose:

 

To develop an "artificial" trabecular meshwork (TM) that can be used to study the physiology of the human TM.

 

Methods:

 

To bioengineer 3D "artificial" TM, primary HTM cells were cultured on porous biocompatible scaffolds that simulate TM microenvironment structurally and chemically. The scaffold composition and geometry, as well as seeding density were optimized to yield confluent, condensed TM cell meshwork to mimic the physiological function of those found in vivo. Cell morphology was assessed using scanning electron microscopy (SEM) and F-actin phalloidin staining. HTM-specific gene expression was confirmed by immunocytochemistry visualized using fluorescent microscopy and confocal laser scanning microscopy. A stand-alone perfusion chamber with pressure sensing system was constructed for investigating the outflow facility of the 3D "artificial" TM treated with anti-glaucoma agents. Sheets of confluent HTM cells grown on the optimal substrate were subjected to through flow and the resistance to flow was recorded.

 

Results:

 

Of tested substrates, SU8 appeared to be the most biocompatible as HTM cells attached to SU8 support beams and spread across it. Optimum cell seeding density was found to be 4x10⁴ cell/cm². Compared to grid spacing of 7 and 15 µm, 11 µm grid spacing provided the best substrate for HTM cell growth and 3-D sheet-like formation (figure below). These cells exhibited spindle-shape appearance characteristic of HTM cells and expressed α-SMA, myocilin and α-β crystalline suggesting that they maintain characteristic HTM cell phenotype. Confocal imaging (by z-stacking) showed HTM cells grown on the substrate overlap and form a 3-D meshwork along the thickness of the substrate. Resistance to through flow of artificial TM (40 mm² membrane area) was 7+0.69 mmHg at a flow rate of 2 ul/min, which is comparable to the in vivo conditions.

 

Conclusions:

 

This work confirms the feasibility to bioengineer an in-vitro HTM which can have future applications in TM physiology studies and as high-throughput screening of IOP lowering agents.  

 

Keywords: trabecular meshwork • outflow: trabecular meshwork • intraocular pressure