Printing collagen IV membrane for bioengineering human corneal endothelium

Abstract

Purpose : Corneal endothelium transplantation accounts for 40% of corneal grafts. However, an acute global shortage of donor corneal tissue continues to prevent access to treatment. Bioengineering a corneal endothelium to replace donor cornea is an alternative to solve this global challenge. Collagen IV (Col-IV) is the main contributor to the matrix network of Descemet’s membrane. In this study, we have developed a printable Col-IV ink to bioengineer the endothelium.

Methods : A printable Col-IV ink was developed by neutralised 12mg/mL Col-IV solution with calcium and riboflavin. The ink was then crosslinked by either UV or blue light and examined by a rheometer. Various printing parameters were tested to print a 2 layered Col-IV lattice by Edu3D printer (TRICEP, UOW), and the pore size was examined under light microscope. Pore size=1 indicated the optimal printing condition. Transparency of Col-IV membrane was examined by colour Quest XE spectrometer. B4G12 cells were seeded and cultured on the printed membrane. Cell count and immunostaining were conducted to examine morphology, Ki67, ZO-1 and Na⁺K⁺ATPase. All tests were repeated at least twice.

Results : The rheology results showed that with UV activation, the activation time for the Col-IV ink to fully crosslink was 2.69min, whereas only 41.73 seconds were needed with blue light (400-500nm). However, the storage modulus for hydrogel formed by UV was 3 times higher than blue light. The printability test showed that Col-IV lattice can reach a pore size of 0.93 when printed at room temperature with a printing speed of 150mm/min, extrusion rate of 0.8mm/min and through a 25GA needle tip. The transparency of the Col-IV membrane was at 90.43%. Full confluence was achieved in all membranes. Corneal endothelial cells showed classic hexagonal shapes and strong expression of Ki67, ZO-1, and Na⁺K⁺ATPase.

Conclusions : We have successfully generated a Col-IV based corneal endothelium. The Col-IV ink can be readily printed into a membrane that enable corneal endothelial cell to proliferate and showed typical endothelial characteristics. As Col-IV is the main matrix protein in Descemet’s membrane, this new bioengineered corneal endothelium has high potential to replace donor endothelium. The printable Col-IV ink required short light activation which can also have a wide range of potential applications such as a carrier for primary endothelial cell culture and for cell printing.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.