Development of 3D bioprinted corneal stroma equivalent using GMP grade decellularized human cornea extracellular matrix hydrogel for prevention of corneal opacity

Abstract

Purpose : Corneal opacification, one of the major causes of blindness and requires corneal transplantation for its management. The challenges with corneal transplantation have led to development of alternative approaches including biomimetic cornea. The study aims to optimize fabrication of corneal stromal construct using decellularized human cornea extracellular matrix (hDECM) hydrogel for prevention of corneal scar in animal model of corneal injury.

Methods : The critical process parameter for manufacturing of decellularized cornea matrix (DECM) hydrogel from non-transplant grade human cadaveric corneas as per GMP guideline was developed and evaluated for 3 consecutive batch (15 cornea/batch). Manufactured hydrogel was tested for transparency, gelation, protein content, residual DNA content, bioburden, endotoxin and safety. For bioprinting, hDECM hydrogel was dispensed through extruder nozzles in a circular pattern on a customized 3D scaffold printed using a CAD model of cornea. The 3D construct was transplanted in rabbit corneal wound (n=3) generated by anterior lamellar keratectomy (3mm diameter and 200 μm depth) and the cornea were clinically evaluated using slit lamp imaging, AS-OCT, keratography and histopathology for 12 weeks.

Results : The average yield of manufactured DECM powder was 7.48±0.19 % per gram of cornea. The protein and residual DNA content of reconstituted DECM (2%) was 5.36±0.05 and 10.32±0.40, respectively. The pH-adjusted DECM formed translucent hydrogel at 37°C and passed bacterial endotoxin (<0.125 EU/mL), sterility, bioburden (no CFU) and safety (> 70% cell viability) evaluation. The cornea was printed as a single layer with peripheral thickness (615±13.22 µm), central thickness (360±13.22 µm) and average diameter of 13.56±0.22 mm. The constructs showed mechanical stability, optimal curvature, supported cell viability and demonstrated implantation feasibility in an ex-vivo study. The corneal stromal construct showed tissue integration with the native cornea, restored corneal transparency, supported corneal epithelial growth and migration of stromal keratocytes at 12 weeks post-transplantation in rabbit corneal wound.

Conclusions : The study demonstrates the therapeutic feasibility of the developed 3D corneal stroma equivalent for the prevention and management of corneal opacity in patients with cornea blindness.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.