A New Bioprinted Dual-Layered Corneal Structure Using Collagen-Based Bioinks

Abstract

Bioengineered corneal constructs are a promising solution to the global shortage of donor tissue. However, most current models lack anatomical curvature and appropriate extracellular matrix (ECM) composition of the native cornea, limiting their relevance for studying graft integration and stromal–endothelial interactions. In this study, we developed a bioprinted, dual-layer corneal model comprising corneal stromal cells laden in type I collagen (Col-I) and a monolayer of corneal endothelial cells supported by collagen type IV (Col-IV). The construct was printed onto a curved support to replicate the posterior curvature of the native cornea. The use of ECM-specific, human-derived collagen bioinks supported high cell viability (>90%) and the formation of a continuous endothelial layer. Histological and immunofluorescence analyses confirmed distinct layering and appropriate cellular morphology and phenotypic marker expression for both corneal stromal and endothelial cells. The construct retained its curvature, transparency, and interfacial integrity over 3 weeks in culture and demonstrated adherence when positioned over an ex vivo corneal tissue. This anatomically curved, multilayered in vitro model offers a physiologically relevant platform for exploring stromal–endothelial architecture and cell interaction in corneal tissue engineering applications.

Graphical Abstract

Impact Statement

This study presents a dual-layer bioprinted corneal stromal–endothelial model fabricated using Col-I and Col-IV bioinks to replicate native ECM composition and curvature. The construct maintains optical clarity, cell viability, and correct stromal–endothelial layout, demonstrating the feasibility of printing anatomically relevant, multilayered corneal structures. By integrating two tissue-specific bioinks and cell types within a curved architecture, this work provides a more anatomically relevant corneal construct than existing flat or single-layer models. The CSE model provides a new in vitro system for studying stromal–endothelial interactions, graft design and advancing the development of complex bioengineered corneal tissues.