A rapid and simple strategy for corneal tissue-engineering : assembly of corneal layers with Bi-liquid/liquid-solid interfaces

Abstract

The cornea is the most anterior structure of the eye, protecting the inner structures while allowing light to pass through onto the retina. Corneal transplant has a wide range of indications, and often the only options for patients with severe corneal diseases such as corneal scarring and corneal endothelial dystrophy. The global shortage of corneal grafts urges the development of novel treatments, including cell-based therapy. The Aqueous Two- Phase System (ATPS) is a rapid, simple, and inexpensive strategy in biomedical engineering that could be used to construct cell sheets. However, the lack of mechanical strength of corneal cell sheets formed by ATPS limits its application, especially in treating corneal endothelial dystrophy. Therefore, this study modified the ATPS method by providing a solid scaffold to the cell sheets, forming the bi-liquid/ solid-liquid system (BLSLS). This research aims to: 1) Create corneal cell layers attached to a collagen-based scaffold within the BLSLS, while ensuring cell viability after the fabrication process; 2) Investigate the transplantation of corneal cells by BLSLS constructs, including cell proliferation and attachment, and the mechanical strength of the collagen-based scaffold; 3) Demonstrate the feasibility of constructing multilayered corneal-like structures with BLSLS and explore the maintenance and culturing of multilayered corneal-like structures constructed by the BLSLS. Primary human corneal epithelial cell line, primary porcine corneal endothelial cells, and primary human corneal fibroblasts were used for the in vitro experiments. Corneal epithelial and corneal endothelial tissue-like planar constructs were constructed using the bi-liquid/solid-liquid system which comprised of dextran (DEX) and collagen. Optimization of the materials and methodology were performed. Corneal cells remain mostly viable after the construct fabrication process. Cell proliferation beyond the constructs and cell attachment onto transplanted surfaces were observed. An in vitro study was performed to demonstrate the increase in re-epithelialization rate of wounded corneal cell layers after cell transplantation with BLSLS constructs, when compared with the control group (n = 3, p < 0.005). Moreover, the durability of collagen-based constructs formed by BLSLS was illustrated by the insertion of acellular collagen layers into the anterior chamber of fresh porcine eyes, mimicking the transplantation of corneal endothelial tissue-like planar constructs. In addition, the methodology of constructing multilayered corneal-like structures with BLSLS was optimized. Corneal epithelial cells were cultured on the collagen construct while corneal fibroblasts were embedded into the collagen layer. We proposed culturing the multilayered corneal-like structures in suspension by DEX. An in vitro chemical-induced hypoxia model was also performed on corneal endothelial cells showing that Rho kinase inhibitor (ROCKi) or nicotinamide (NIC) could increase corneal endothelial cell survival (p = 0.0104 and p = 0.0165). Our current results demonstrate the potential for BLSLS to assemble corneal tissue-like planar constructs with collagen scaffold, which could be used to aid the transplantation of corneal epithelial and endothelial cells. By stacking up the layers in BLSLS, multilayered corneal-like structures could also be assembled. In the future, in vivo models are required to demonstrate the efficacy of BLSLS-formed constructs for the transplantation of corneal epithelial and endothelial cells. The protocol for multilayered corneal-like structure construction with BLSLS could be further optimized.