Biomanufacturing of Kidney Organoids, Perfusable Proximal Tubules, and Kidney Tissues

Abstract

Human kidneys are vital organs that filter blood, regulate electrolyte homeostasis, and produce urine. These complex processes are carried out by nephron subunits composed of glomeruli and tubular segments that are responsible for filtration and reabsorption, respectively. While considerable efforts have focused on the fabrication of in vitro renal models that recapitulate nephron structure and function for studying nephrotoxicity and renal development, progress remains limited. Kidney organoids derived from human induced pluripotent stem cells (hiPSCs) are three dimensional (3D), multicellular structures that contain many of the cell types and architectures present in human kidneys. There is a growing interest in using kidney organoids as a platform for improved drug screening and as organ building blocks (OBBs) for the biomanufacturing of functional kidney tissues for modeling tissue development, and ultimately, renal replacement. The overarching goal of this Ph.D. thesis is to generate kidney organoids and explore their use as building blocks for biofabricating perfusable proximal tubules, and bulk kidney tissues. Specifically, this research focuses on the scalable 3D differentiation of kidney organoids in stirred bioreactors (STRs), the development of organoid-derived perfusable proximal tubules-on-chip, and the biofabrication of 3D kidney tissues for in vitro and in vivo assessment. We first investigated the effect of hiPSC seeding density and stir rate on embryoid body (EB) formation and differentiation efficiency. Their initial differentiation efficiency was roughly 70%, which motivated the development of an optical-based screening method to rapidly predict differentiation success. Next, the concentration and timing of differentiation reagents along with improved media preparation methods were implemented to further enhance the differentiation efficiency of nephron-rich kidney organoids to roughly 95%. The STR-generated kidney organoids exhibited glomerular, proximal tubule, distal tubule, stromal and vascular cell types and architectures. Compared to kidney organoids differentiated in static conditions, STR-generated kidney organoids demonstrated increased expression of tubular, ciliary, and vascular cell types. Most importantly, kidney organoid differentiation in stirred bioreactors greatly increased the organoid volume produced in a given time period relative to 2D (static) differentiation methods. Next, we developed an organoid-derived proximal tubule epithelial cell (OPTEC)-on-chip model that exhibits improved drug uptake compared to those based on tert1-immortalized proximal tubule (PTEC-TERT) cells. First, lotus tetragonolobus lectin (LTL+) proximal tubule cells are isolated from mature kidney organoids, that were dissociated into individual cells, using magnetic activated cell sorting (MACS) and expanded in vitro. These OPTECs are then seeded into cylindrical channels embedded within an optimized extracellular matrix (ECM) composed of gelatin-fibrin, where they form a confluent monolayer. A second bare channel is introduced adjacent to this 3D tubule within reusable multiplexed chips to mimic basolateral drug uptake. Our 3D OPTEC-on-chip model exhibits significant upregulation and improved polarization of organic cation 2 (OCT2) and organic anion 1/3 (OAT1/3) transporters, which resulted in higher drug uptake compared to PTEC-TERT-on-chip controls. Consequently, OPTEC-on-chip models also exhibited a higher normalized lactate dehydrogenase (LDH) release compared to those controls when exposed to known nephrotoxins, cisplatin and aristolochic acid. Importantly, LDH release could be diminished by adding known OCT2 and OAT1/3 inhibitors. This integrated multifluidic OPTEC platform paves the way for personalized kidney-on-chip models for drug screening and disease modeling. Finally, we investigated the biofabrication of 3D kidney tissues from OBBs with the goal of modeling in vitro tissue development and assessing their host integration in vivo. Kidney organoids differentiated from hiPSCs in STRs were mixed in a fibrinogen solution and compacted to form a cellularly dense tissue matrix. Sacrificial writing into functional tissue (SWIFT) is then used to print sacrificial ink channels into the OBB-ECM matrix. The SWIFT kidney tissues are perfused in vitro for 10 days, during which their fusion and longitudinal maturation are assessed. SWIFT kidney tissues maintained proper expression of glomerular, proximal tubule, distal tubule, stromal, and vascular cell types and architectures. Additionally, a progressive increase in nephron gene expression is observed via Nanostring analysis, highlighting the ability of SWIFT kidney tissues to undergo further maturation in vitro under flow. To explore their host integration and immune response, kidney tissue discs are fabricated by depositing the same OBB-ECM solution used for SWIFT into cylindrical molds. The kidney discs are cultured in vitro for 7 days to promote fusion, then implanted into NSG mice reconstituted with human allogeneic immune cells. Allogeneic immune cells infiltrated the discs and attacked nephron cell types within the transplanted tissues. We find that in vivo immune response towards the transplanted tissue discs exhibit a gene signature akin to clinical acute cellular rejection. Collectively, this work provides a foundation for the biofabrication and development of kidney tissues constructed from OBBs, insight into the immunological challenge of implanting OBB-based tissues, and a platform for future immunosuppressant drug development. In summary, a scalable approach for creating kidney organoids, perfusable organoid-derived proximal tubules, and bulk kidney tissues derived from human induced pluripotent stem cells has been established. The utility of each of these moieties (organoids, tubules, and tissues) have been validated through a combination of in vitro and in vivo studies. This PhD research provides a foundation for generating patient-specific kidney tissues for drug testing and therapeutic applications.