Developing Clinically Compatible Bioinks and Novel Multi-material 3D Bioprinting Strategies: Applications in Human Stem Cell Based Cornea

The scarcity of tissues and organs needed for transplantations is a major reason why innovative biofabrication technologies are needed. Three-dimensional (3D) bioprinting is an additive manufacturing technology with the potential to revolutionize regenerative medicine by offering precision, automation and scalability. However, reliance on animal-derived and non-biodegradable biomaterials and donor-dependent primary cells is a major bottleneck hindering its clinical translation, highlighting the need for clinically compatible raw materials and cells. Furthermore, multi-material 3D bioprinting advances the current state-of-the-art by incorporating various biomaterials and cell types into one structure, shifting the paradigm of bioprinting from simple structures toward complex, biomimetic tissues.

Human cornea is one of the tissues where donor tissue shortage is severe. Hence, bioprinting corneal substitutes offers a promising solution to the scarcity of corneal transplants used for treating corneal blindness, with the potential of improving the lives of countless patients. Multi-material bioprinting surpasses the traditional tissue engineering (TE) methods in mimicking the multi-layer nature of the cornea as well as its complex microstructure. Moreover, human adipose tissue-derived stem cells (hASCs) and human induced pluripotent stem cells (hiPSCs) have shown tremendous potential in corneal TE, providing a clinically relevant and scalable cell sources. Overall, producing fully biomimetic corneal equivalents requires differentiating functional cells, designing bioinks to fulfill the cell type-specific requirements, and creating a 3D environment to support tissue maturation and integration after transplantation.

This dissertation explored the development of clinically relevant bioinks and multi-material bioprinting strategies to create biomimetic corneal structures, as well as transplantation strategies for these bioprinted structures using a porcine cornea ex vivo model. First, a novel cornea-specific bioink was developed without relying on donor corneas, leveraging the extracellular matrix (ECM) produced during the differentiation of hASCs toward corneal stromal keratocytes (hASC-CSKs). Second, a multi-material bioprinting strategy was designed to mimic the microstructure of corneal stroma by using two different bioinks and hASCs. This approach enabled cellular growth in the softer bioink, while the acellular stiffer bioink provided mechanical strength and guidance for the cells. Third, this multi-material strategy was employed to integrate the stromal and epithelial layers by combining the previously developed stromal microstructure and hASC-CSKs with hiPSC-derived limbal epithelial cells (hiPSC-LSCs) for the epithelium.

To conclude, this dissertation presents innovative, streamlined, and most importantly, clinically applicable approaches to advance 3D bioprinting toward clinical use. The results from this thesis provide important insight into producing tissue substitutes with greater complexity and biological relevance by utilizing clinically compatible raw materials and cells. Furthermore, these results contribute to the scientific advancements essential for clinical translation of bioprinting, particularly when the availability of donor tissues is limited.