Engineering the (vascularized) Proximal Tubule, a Guide Towards Functionality

Abstract

This thesis focused on the fabrication of kidney proximal tubule models. A coaxial printing system allowed the robust and straightforward fabrication of coiled perfusable microfibers, replicating the kidney proximal convoluted tubules. The formed alginate-gelatin microfibers offer a complex tubular shape in which the cells exhibit mature markers, such as functional transporters and polarized monolayers. Moreover, this model has proven to support both healthy and cystinotic proximal tubule cell lines and allows for mechanistically studying tubulopathies. Though distinctive 3D-printing strategies offer opportunities to mimic the structure of the convoluted proximal tubule, printing resolution remains restricted for soft hydrogel materials. Therefore, a unique method was studied, whereby post-printing treatment induced resolution enhancement of methacrylated hyaluronic acid / gelatin methacryloyl (HAMA/GelMA) based hydrogels. This shrinking technique opens a wide application to create higher resolutions for 3D printing. However, we also found that the polycationic shrinking reagents used were relatively toxic for our cells of interest. Therefore, more research is needed to prevent the direct exposure of cells to the shrinking reagents. Using melt electrowriting, scaffolds were fabricated that are self-supportive, yet small-sized and highly porous. These scaffolds enable direct access to the basolateral and luminal cell sides to facilitate solute exchange with vasculature in immediate proximity, which is critical for functional proximal tubule constructs. The polymer applied, polycaprolactone, is used to prepare biomaterials that generally show good biocompatibility and is compatible with implantation in the future. Kidney proximal tubule cells and glomerular endothelial cells form polarized monolayers in our tubules, form their own extracellular matrix, and show functionality for important kidney transporters. In our preliminary results we showed initial work with induced pluripotent stem cell-derived kidney organoids, which is very promising for the development of implantable proximal tubule grafts. The aim was to develop vascularized kidney tubules for studying the clearance of protein bound uremic toxins, while gaining knowledge for the development of implantable kidney tubules. The developed models allow for studying transepithelial secretion of uremic toxins, an overarching application for these constructs. This will further advance our understanding of the kidney secretion processes and aid in studying interventions to main proximal tubule function in chronic kidney disease conditions in vitro. Furthermore, while fundamental by nature, our research deliverables enhance mechanistic insight in additive manufacturing and kidney development processes. Altogether, our findings will further advance the field of kidney engineering, while working towards kidney replacement therapies.