3D Natural Biopolymer Scaffold for In Vitro Modeling of the Trabecular Meshwork

Abstract

 

Purpose

 

To develop a 3D scaffold using natural biopolymers that mimics the trabecular meshwork (TM) structure to help improve our understanding of TM cell biology and the intraocular pressure system and aid in the screening and development of pharmacological and biological agents targeting the trabecular outflow facility.

 

Methods

 

Using a unidirectional freezing and lyophilization process, we engineered anisotropic porous scaffolds of two polymers naturally present in the TM extracellular matrix. This technique allowed us to develop collagen-glycosaminoglycan (GAG) scaffolds with uniaxially aligned pores. The scaffolds were characterized with scanning electron microscopy (SEM) and dynamic mechanical analysis. We assessed the ability of our scaffolds to support primary porcine TM (pTM) cells’ viability, proliferation, and migration using fluorescent imaging, a cell proliferation assay, and histology.

 

Results

 

Fabricated scaffolds displayed anisotropic, unidirectional pore alignment when imaged with SEM (Fig.1). The average pore diameter was 13.69±5.2 µm, and the average pore density was 2028 pores/mm². The storage modulus of hydrated scaffolds was 38.23±6.1 kPa. Total GAG content of scaffolds was measured with a quantification assay showing that 66% of the GAG was retained after fabrication and sterilization. pTM cells seeded on the surface of scaffolds and cultured for 2 weeks showed a 60% increase in metabolic activity over the time period. Fluorescent viability stains were used to confirm the presence of live cells in the scaffolds up to two weeks after initial seeding (Fig.2A). Histological sections stained with H&E revealed cell attachment and migration into the interior of the scaffolds (Fig.2B).

 

Conclusions

 

Our results confirm that pTM cells grown on collagen-GAG scaffolds demonstrate high viability and are capable of migrating into the scaffold structure. Further development will allow this cell culture system to be used to study the physiology and pathology of glaucoma and to aid in the screening and development of new therapeutic agents.  

 

Fig. 1 SEM images of a collagen-GAG scaffold A. Top surface B. Cross section (top of the scaffold at the right of the image)

 

 

Fig. 2 Images of pTM cells on collagen-GAG scaffolds at 14 days A. Confocal microscopy of pTM cells labeled with a live/dead fluorescent dye B. Light microscopy image of an H&E histological section