Bioengineering functional organoid models across scales

The last two decades have seen the development of organoid models for many different tissues and organs. Organoids are three-dimensional organ-mimetics derived from stem or progenitor cells comprising various specialized cell types, resembling the architecture of their native organ on a smaller scale, and recapitulating some of its functions. They completely changed the cell culture world, offering highly interesting insights into basic research and beginning to demonstrate their potential for clinical applications. Despite all this, organoid growth relies on poorly controllable stem cell self-organization, which limits their reproducibility and size. Moreover, organoids often mimic only one compartment (e.g. the epithelium) of their native counterparts, and for some organs organoid models do not yet exist. In this thesis, I introduce several bioengineering approaches to help overcome some of these limitations and obtain more robust and functional organoid models. Such engineering approaches were applied at different biological scales. In chapter I, we induced for the first time the formation pathophysiologically relevant tumors in vitro in a highly observable setting based on cell responsiveness to light. With an optogenetic system driving Cre recombinase expression, we controlled the induction of cancer driver mutations in space and time in Apcfl/fl;KrasLSL-G12D/+;Trp53fl/f (AKP) colon organoids. A colon organoid-on-a-chip platform was then used to obtain a defined topology compatible with long-term studies and imaging. These bioengineering approaches provided us with insights into tumor initiation and development processes that would not have been possible with conventional cancer models. In chapter II, I developed a new functional organoid model by applying knowledge gained from other organoids to the thymus, and, in particular, to thymic epithelial cells. The organoids were characterized in detail to show that the newly established culture conditions have the potential to preserve the functional ability of thymic epithelial cells to mediate thymopoiesis. I have demonstrated this by reaggregating thymic epithelial cells cultured as organoids with T cell progenitors and showing that T cell development in these reaggregates recapitulates to a large extent the process that occurs in the native thymus. In addition, when transplanted into mice, the reaggregates were capable of attracting new T cell progenitors, thus reproducing another important feature of the thymus in vivo. In general, this part aimed to bring the fields of organoid and immunology closer together. In chapter III, we used bioprinting as a bioengineering strategy to create larger organoid constructs with a shape and cell type composition resembling their native organ. Specifically, centimeter-scale tissue constructs were printed that assembled different parts of the gastrointestinal tract in one functional epithelial tube. This chapter illustrates the possibility of harnessing