Novel bioink design for 3D bioprinting of human pluripotent stem cell derived corneal epithelial cells

Abstract

Objectives: The correct function and structure of cornea is essential for vision. Cornea is maintained by limbal epithelial stem cells (LESCs), and the lack of functional LESCs in a disease called limbal stem cell deficiency (LSCD) can lead to blindness. The traditional treatment for corneal blindness is a corneal transplant. However, there is a severe shortage of cornea donors and the transplants lack functional LESCs. Thus, a corneal transplant cannot be used as a treatment for LSCD. The field of tissue engineering aims to restore, replace and regenerate damaged native tissues, such as develop artificial corneas. Yet, the conventional methods fail to mimic the native-like cellular variety and specific microstructure. Moreover, they lack the ability for precise positioning of cells and materials into a three-dimensional (3D) environment. 3D bioprinting offers a possibility to overcome these issues due to its better control, accuracy and customizability. The main challenge in 3D bioprinting is the lack of bioprintable, cell-laden bioinks with suitable properties to guide the desired cell behaviour. The aim in this thesis was to design and optimize a novel bioink and bioprinting conditions in order to 3D bioprint human pluripotent stem cell (hPSC) -derived corneal epithelium mimicking tissue.

Materials and methods: A novel bioink composition was done combining human and recombinant sourced extracellular matrix proteins. The native human cornea was used as a source of inspiration in the development. Moreover, two crosslinking strategies were combined. First, the printability of the bioink and the printing parameters for extrusion-based bioprinting were tested and determined. The hPSC-derived LESCs (hPSC-LESCs) were produced, and the bioprinting conditions, including the ultra violet (UV) light exposure and printing substrate, were optimized. The response to different bioprinting conditions and behaviour of the bioprinted hPSC-LESCs were analysed with phase contrast microscopy, proliferation and live/dead assays, and immunofluorescence analysis. Finally, the bioink was characterized by analysing its swelling behaviour, transparency and rheological properties.

Results and conclusions: Overall, the developed bioink was well extrudable and had good transparency. The bioink supported the proliferation and maturation of the hPSC-LESCs. UV exposure did not decrease the cell viability (> 88%), however, it was observed to affect the crosslinking density and the stiffness of the material considerably. The bioprinted hPSC-LESCs preferred softer, highly viscous material, and bioprinting without UV exposure resulted in the most stratified epithelium. From the printing substrates, MatrigelTM coating provided the best results in regards to the cell proliferation and adhesion. However, due to the softness of MatrigelTM, the bioprinted epithelium showed shrinkage leading to partially ruptured epithelium. Therefore, further optimization of the printing substrate is required. Furthermore, due to the low crosslinking degree of the bioink without UV, rheological measurements were challenging to perform, and require further optimization in the future. This was the first study in which the stratification of hPSC-LESCs was observed after extrusion-based 3D bioprinting. Thus, the novel bioink showed great potential for 3D bioprinting corneal epithelium mimicking structures and should be further studied in ocular surface reconstruction.