High-throughput 3D bioprinting of corneal stromal equivalents.

In this study we designed high-throughput 3D bioprinting of corneal equivalents which may address the need for in vitro models. In our digital 3D cornea model, average dimensions of adult cornea were converted to 3D shapes, then to G-code files which were printed by BIOX printer (CELLINK). To maintain the curvature of cornea, a support scaffold was designed using stereolithographic printer. The support scaffold could facilitate the printing of 6-12 corneas at a time thus enabling high-throughput printing. Human corneal keratocytes (HCKs) were incorporated in the optimized bio-ink, and cell-laden corneal stromal equivalents were printed. Printed structures were cross-linked by calcium chloride 100 mM, washed with Hanks' Balanced Salt Solution and incubated at 37°C in fibroblast media. Printed corneas were analyzed for live dead assay, Alamar assay, and expression of fibronectin and actin green markers. Printed corneas were able to maintain their structure, integrity, and clarity. Live dead assay and Alamar assay demonstrated that HCKs maintained high viability (>95%) for 2 weeks. HCKs in the printed corneas showed expression for fibronectin and actin green. In conclusion, high-throughput fabrication of 3D printed corneal stromal equivalents using a combination of stereolithography printing, extrusion based printing, and micro-transfer molding techniques was achieved.