Directed differentiation of human pluripotent stem cells to kidney organoids brings the prospect of drug screening, disease modelling and the generation of tissue for renal replacement. Currently, these applications are hampered by organoid variability, nephron immaturity, low throughput and limited scale. Here we apply extrusion-based 3D cellular bioprinting to deliver rapid and high throughput generation of kidney organoids with highly reproducible cell number and viability. We demonstrate that manual organoid generation can be replaced by 6- or 96-well organoid bioprinting and evaluate relative toxicity of aminoglycosides as a proof of concept for drug testing. In addition, 3D bioprinting enabled precise manipulation of biophysical properties including organoid size, cell number and conformation, with modification of organoid conformation substantially increasing nephron yield per starting cell number. This facilitated the manufacture of uniformly patterned kidney tissue sheets with functional proximal tubular segments. Hence, automated extrusion-based bioprinting for kidney organoid production deliver improvements in throughput, quality control, scale and structure, facilitating in vitro and in vivo applications of stem cell-derived human kidney tissue.

Recent advances in the directed differentiation of human pluripotent stem cells to kidney brings with it the prospect of drug screening and disease modelling using patient-derived stem cell lines. Development of such an approach for high content screening will require substantial quality control and improvements in throughput. Here we demonstrate the use of the NovoGen MMX 3D bioprinter for the generation of highly reproducible kidney organoids from as few as 4,000 cells. Histological and immunohistochemical analyses confirmed the presence of renal epithelium, glomeruli, stroma and endothelium, while single cell RNAseq revealed equivalence to the cell clusters present within previously described organoids. The process is highly reproducible, rapid and transferable between cell lines, including genetically engineered reporter lines. We also demonstrate the capacity to bioprint organoids in a 96-well format and screen for response to doxorubicin toxicity as a proof of concept for high content compound screening.

The vessel wall is an important barrier modulating drug delivery to the underlying diseased tissue. Oscillating microbubbles can be used to locally enhance vascular permeability and sonoporate cells. As the mechanism is not fully understood, our aim was to grow 3D human vessels-on-a-chip in the OrganoPlate® 40 and use this model to investigate the effect of αvβ3 -targeted microbubble and different ultrasound pressures (2 MHz, 100–850 kPa peak negative pressure) and cycle lengths (10×10 or 10×1000 cycles) on vascular permeability and sonoporation. The vascular permeability of 122 microvessels in 14 different conditions was quantified by microscopy imaging using the leakage pattern of a 150 kDa FITC-dextran dye. Furthermore, sonoporation was assessed using propidium iodide (PI). Upon microbubble and ultrasound treatment, an increase in vascular permeability was observed. Higher pressures and longer cycle length treatment showed a significantly higher vascular permeability and significant increase in PI uptake compared to all control groups (sham, ultrasound only, microbubble only), suggesting a simultaneous increase in vascular permeability and sonoporation correlating with higher pressure and longer cycle insonifications. In conclusion, the vessel-on-chip model is a suitable model to investigate how insonification with different ultrasound settings affects the microbubble-mediated vascular permeability increase and sonoporation.