3D Bioprinting of Human Cornea Mimicking Structures with Stroma and Epithelium

Abstract

There is an immense need for corneal transplants to restore vision after injury or disease. Due to the shortage of donor corneas, three dimensional (3D) bioprinting has emerged as a possible solution to create cornea mimicking structures. The cornea consists of five layers, with the stroma and epithelium forming the two outermost cellular layers. To be able to 3D bioprint these layers, bioinks, and appropriate cell types are needed. For the epithelial layer, human pluripotent stem cell -derived corneal limbal epithelial stem cells (hPSC-LSCs) have great potential. For the stromal layer, human adipose stem cells -derived corneal stromal keratocytes (hASC-CSKs) have been explored together with a hyaluronic acid -based dopamine-containing (HA-DA) stromal bioink. To be able to create cornea mimicking structures with stroma and epithelium, the structures must be co-cultured after 3D bioprinting. However, the HA-DA stromal bioink is not stable in limbal epithelial stem cell (LSC) medium required by the hPSC-LSCs. To address this issue, this thesis aimed to enhance the stiffness and stability of the HA-DA stromal bioink in the LSC medium co-culture condition to enable the co-culture of 3D bioprinted cornea mimicking structures with stroma and epithelium.

To enhance the stiffness and stability of the HA-DA stromal bioink, the bioink composition was modified by incorporating a methacrylate hyaluronic acid (HAMA) component into the bioink to create a photocrosslinkable HA-DA HAMA stromal bioink. First, the composition of this HA-DA HAMA stromal bioink was optimized by evaluating the printability and shape fidelity of the bioink as well as handling of bioprinted structures after incubation in LSC medium. Then, the optimized HA-DA HAMA stromal bioink was compared to the original HA-DA stromal bioink by analyzing printability, shape fidelity, viscosity, swelling behavior, handling, mechanical properties, and transparency of the bioinks. After this, the cytocompatibility of the bioinks with hASC-CSKs was assessed. In the final part of this thesis, human cornea mimicking structures with stroma and epithelium were bioprinted, and two different co-culture conditions were tested.

HA-DA HAMA stromal bioink with good printability and shape fidelity was developed, and bioprinted stromal structures demonstrated good stability after incubation in LSC medium. Based on the comparison of the HA-DA and HA-DA HAMA stromal bioinks, the bioinks had similar printability, shear-thinning behavior, and transparency. Moreover, after incubation in LSC medium, the HA-DA HAMA stromal bioink had improved characteristics regarding handling, swelling, and shape fidelity. However, the HA-DA HAMA stromal bioink lacked the necessary cytocompatibility required for bioinks and demonstrated inferior cytocompatibility compared to the HA-DA stromal bioink. This underlined the importance of conducting cytocompatibility tests as part of bioink characterization. Furthermore, it was confirmed that the LSC medium co-culture condition significantly influenced the properties of the HA-DA stromal bioink. Therefore, bioink characterization should always consider the final application of the 3D bioprinted construct. Finally, cornea mimicking structures with stroma and epithelium were successfully bioprinted for the first time using extrusion-based bioprinting. A suitable co-culture condition was also found which combined the cell culture medium of both cell types used in the structure. This fulfilled better the needs of both cell types and prolonged the stability of the HA-DA stromal bioink during cell culture. In the future, further research is required to optimize cell density in the epithelial layer and analyze the effect of the co-culture medium on both cell types. Based on these findings, cornea mimicking structures with improved stability and cellular functionality can be 3D bioprinted in the future.