New Tools for Hydrogel-Based 3D Bioprinting

Abstract

The in vitro recapitulation of tissue and organ function represents one of the main objectives of tissue engineering. Developments in this field have numerous applications, from alleviating the shortage of donor organs to providingmore representative platforms for drug testing. As such, there is an increasingly significant body of literature devoted to the development of better strategies to reconstruct living matter outside of the body. The three-dimensional (3D) architecture of a tissue, its histoarchitecture, is a key aspect to consider when engineering a tissue. Indeed, tissue function not only arises from individual elements that compose the tissue, its different cell types and their extracellular matrices (ECM), but also fromtheir precise spatial arrangement and interactions. Bioprinting is considered as one of the most promising techniques to recapitulate the complexity of in vivo histoarchitectures. Early bioprinting examples have shown successful application of inkjet printing to geometrically arrange cells and biomolecules in 2D and, to a much lesser extent, in 3D. However, no group has shown yet a technological platformable to print a living tissue construct whose make-up could even closelymimic the histoarchitecture of a native tissue. In this thesis the most significant challenges in using inkjet printers for tissue engineering were tackled. Perhaps the most critical bottleneck of state-of-the-art bioprinting is the lack of suitable bioinks; precursor liquids with distinct physico-chemical properties that very rapidly transforminto solid hydrogels and also possess the necessary bioactive characteristics to guide cell development into a functional tissue. To this end, a novel double network hydrogel systemwas conceived which combines ultra-rapid cross-linking of alginate, a biologically inert polysaccharide network, with the slower enzymatic crosslinking of a synthetic but highly bioactive poly(ethylene glycol) (PEG) gel. The latter could be tailored virtually on demand tomimic a desired ECM in a tissue. Based on this bioink concept it was demonstrated that complex multicomponent 3D shapes could be printed. Importantly, using primary human fibroblasts as model system, the bioinks showed outstanding characteristics as cellular microenvironments, facilitating rapid 3D cell migration and self-organization into multicellular networks. To mimic the spatial complexity of living tissues, it is crucial to master the deposition of multiple, cell-containing bioinks into user-defined 3D structures. Each bioink could thus mimic a specific cell/ECMcomponent present in a native tissue. To be able to reproduce an appropriate histoarchitecture, bioinks have to be deposited precisely enough to approximate their natural 3D spatial arrangement down to a cellular scale. Technically, this implies that the dispensing units must be extremely well aligned and synchronized to one another in order to pattern according to a specified design. In order to address these challenges, several novel bioprinting concepts were developed, which allowed the patterning of multiple bioinks containing living cells into 3D tissue-like geometries. In the last part of this thesis, an important first step towards the engineering of macroscopic tissue models was taken. Accordingly, a fundamental requirement for the viability and maturation of a printed tissue construct is the supply of nutrients and growth factors through a microfluidic network, similar to the vascularization in a native tissue. To this end, a novel approach for the printing of 3D constructs with an interconnected channel network enabling rapid nutrient transport was devised. This was achieved by exploiting the multicomponent printing developed earlier. A channel network inside the construct was patterned using a degradable hydrogel that could be selectively removed to leave behind the perfusable microfluidic system. Moreover, it was possible to combine this sacrificial layer approach with cell-containing bioinks to generate a cellularized perfusable tissue model. Encouraging preliminary long-termculture experiments revealed that dynamic cell culture conditions could be maintained, leading to viable cell growth over more than one week under perfusion conditions. Taken together, this thesis presents several important steps toward an inkjet-based platform for in vitro tissue engineering. It is expected that the strategies developed here will provide a solid basis for future advances in the emerging bioprinting field.