Recent advances in the directed differentiation of human pluripotent stem cells to kidney organoids advances the prospect of drug screening, disease modelling, and even restoration of renal function using patient-derived stem cell lines. Here, we demonstrate the successful adaptation of our directed differentiation protocol to the NovoGen Bioprinter® MMX technology to achieve automated, rapid fabrication of self-organizing kidney organoids. Bioprinted organoids were found to be equivalent to those previously reported via manual generation at both the level of morphology and component cell types, as well as gene expression patterns and cell clusters revealed by single cell transcriptional profiling. Utilization of a bioprinter allows for the generation of large numbers of uniform and highly reproducible organoids in reduced time (approximately 20x faster) compared to manual processes. Treatment of bioprinted kidney organoids cultured in conventional 6-well format with doxorubicin exhibited concentration-dependent morphological changes consistent with cell injury and degeneration. Consistent with clinical observations, doxorubicin showed distinct glomerular toxicity with marked increases in cleaved caspase 3 mRNA and protein, accompanied by loss of podocyte-specific cell markers. Proof of concept high-throughput toxicity screening was achieved with bioprinted kidney organoids in 96-well Corning® Transwell® plates treated for 72 hours with a range of doxorubicin concentrations (0.2 to 25 µM). Analysis of 6-well and 96-well cell viability data suggested that organoids printed in both multi-well plate formats were similarly sensitive to doxorubicin. The doxorubicin IC50 for organoids bioprinted in 6-well plates was 3.9 ± 1.8 µM (value ± S.E.), while the calculated IC50 for organoids bioprinted in a 96-well plate was 3.1 ± 1.0 µM. Collectively, these results suggest that bioprinted kidney organoids are functionally equivalent to those prepared manually and thus are likely to be useful for toxicity screening, the development of iPSC-based approaches for the interrogation of complex disease phenotypes, and the scaling needed for clinical restoration of renal function with patient-derived iPSCs.
Utilization of the ExVive human liver tissue model to assess drug-induced liver injury across a diverse set of chemical classes
One of the key challenges in the drug development process continues to be the early identification of compounds with adverse and potentially dose-limiting liver toxicity. Traditionally hepatotoxicity prediction has relied on two-dimensional hepatocyte monolayers, sandwich culture assays and non-human animal models. These systems are limited in their ability to accurately reflect in vivo human biology and lack of the cellular complexity required to model tissue level outcomes of toxicity. In this study, drug-induced liver injury (DILI) was assessed in 3D-bioprinted human liver tissues comprised of primary hepatocytes, hepatic stellate cells, and endothelial cells (ExVive™ Human Liver Tissues) treated with known high and low DILI risk compounds. Tissue response to compounds was evaluated using a range of biochemical, cytokine secretion, gene expression and histologic analyses. The high DILI risk compounds tolcapone, benzbromarone, danazol and tamoxifen were evaluated using a 28 day dosing regimen and compared to safer compounds entacapone, phentolamine, betahistine, nifedipine and chloramphenicol. Tissues treated with the known toxicants exhibited evidence of toxicity in at least two assays. A comparison of the clinically related compounds tolcapone and entacapone at concentrations of 1x, 3x and 10x Cmax revealed clear differences in their impact on the bioprinted tissues; tolcapone resulted in a dose dependent decrease in tissue viability at 10x Cmax while entacapone resulted in no significant changes in viability. Significant reductions in albumin secretion were seen at 3x Cmax with tolcapone treatment, vs. 10x Cmax with entacapone. Treatment of the 3D liver tissues with 5x and 20x Cmax concentrations of benzbromarone for 28 days resulted in decreased tissue viability with the highest concentration along with time and dose dependent decreases in albumin production beginning at treatment day 7 with the 5x and 20x Cmax concentrations. Histologic assessment of these tissues revealed significant loss of tissue and disruption of cellular cohesion at the 20x Cmax dose. These results suggest 3D bioprinted liver tissues are well suited to differentiate high risk from low risk DILI compounds and utilize both biochemical and histologic endpoints to assess multiple mechanisms of DILI in vitro, providing a comprehensive means of examining tissue injury.
Utilization of the ExVive human kidney tissue model of the proximal tubule to assess nephrotoxicity across a diverse set of chemical classes
Due to its exposure to high concentrations of xenobiotics, the kidney proximal tubule (PT) is the primary site of nephrotoxicity which results in late-stage attrition during drug development. We have developed a 3D bioprinted, fully human in vitro model of the proximal tubulointerstitial interface to enable more accurate prediction of tissue level clinical outcomes. We challenged this model with a diverse set of known renal toxicants and assessed their impact using biochemical and histologic endpoints. Treatment of 3D PT tissues with the chemotherapeutic agent, Cisplatin, induced dose and time-dependent loss of tissue viability and epithelial function over the 14 day time-course evaluated. Temporal evaluation of the soluble biomarkers N-acetyl-β-D-glucosamindade (NAG) and lactate dehydrogenase (LDH) demonstrated maximum NAG release earlier than maximum LDH release. Histologic assessment of the tissues showed primarily epithelial cell-specific effects at the 2.5 µM and 5 µM doses, while additional toxicity in the interstitium was observed at the 10 uM dose. Co-treatment of the tissues with 5uM Cisplatin and the OCT2 inhibitor Cimetidine (1 mM) attenuated the Cisplatin-induced toxicity. To assess another class of nephrotoxicants, 3D PT tissues were treated with the aminoglycoside antibiotics, Amphotericin B and Gentamicin. Amphotericin B induced a dose-dependent decrease in epithelial function without a corresponding decrease in overall tissue metabolic activity, while Gentamicin treatment resulted in decreases in both epithelial function and tissue metabolic activity. Examination of the soluble biomarkers NAG and LDH showed a rapid response of the tissues to even low concentrations of Amphotericin B early in the treatment period. Consistent with the biochemical findings, histologic assessment of the Amphotericin B-treated tissues demonstrated a flattening of the epithelial cells with no observable impact on the other cells within the tissue. Collectively, these results suggest that bioprinted kidney tissues are well-suited to assess multiple mechanisms of nephrotoxicity including fibrosis in vitro, following biologically-relevant dosing regimens, and that biochemical, transcriptional and histologic endpoints provide a comprehensive means of examining the progression of tissue injury on a mechanistic basis.
3D proximal tubule tissues recapitulate key aspects of renal physiology to enable nephrotoxicity testing
Due to its exposure to high concentrations of xenobiotics, the kidney proximal tubule is a primary site of nephrotoxicity and resulting attrition in the drug development pipeline. Current preclinical methods using 2D cell cultures and animal models are unable to fully recapitulate clinical drug responses due to limited in vitro functional lifespan, or species-specific differences. Using Organovo’s proprietary 3D bioprinting platform, we have developed a fully cellular human in vitro model of the proximal tubule interstitial interface comprising renal fibroblasts, endothelial cells, and primary human renal proximal tubule epithelial cells to enable more accurate prediction of tissue-level clinical outcomes. Histological characterization demonstrated formation of extensive microvascular networks supported by endogenous extracellular matrix deposition. The epithelial cells of the 3D proximal tubule tissues demonstrated tight junction formation and expression of renal uptake and efflux transporters; the polarized localization and function of P-gp and SGLT2 were confirmed. Treatment of 3D proximal tubule tissues with the nephrotoxin cisplatin induced loss of tissue viability and epithelial cells in a dose-dependent fashion, and cimetidine rescued these effects, confirming the role of the OCT2 transporter in cisplatin-induced nephrotoxicity. The tissues also demonstrated a fibrotic response to TGFβ as assessed by an increase in gene expression associated with human fibrosis and histological verification of excess extracellular matrix deposition. Together, these results suggest that the bioprinted 3D proximal tubule model can serve as a test bed for the mechanistic assessment of human nephrotoxicity and the development of pathogenic states involving epithelial-interstitial interactions, making them an important adjunct to animal studies.
A 3D bioprinted model of the renal proximal tubule for evaluation of drug-induced nephrotoxicity
The kidney proximal tubule (PT) is a primary site of nephrotoxicity and resulting drug attrition in the development pipeline. Using Organovo’s proprietary bioprinting platform, we have developed a fully-human in vitro model of the PT to potentially enable more accurate prediction of tissue-level clinical outcomes. ExVive™ Human Kidney Tissue is created in a standard 24-well Transwell® plate by spatially-controlled deposition of cell aggregates in the absence of exogenous scaffold. Tissues are composed of an interstitial layer of renal fibroblasts and endothelial cells supporting a monolayer of human primary PT epithelial cells.
A 3D bioprinted model of the renal proximal tubulointerstitial interface for evaluation of drug-induced toxicity
Due to its exposure to high concentrations of xenobiotics, the kidney proximal tubule (PT) is a primary site of nephrotoxicity, a leading cause of attrition in the drug development pipeline. Current preclinical methods using 2D cell cultures and animal models are unable to fully recapitulate clinical drug responses due to limited in vitro functional lifespan, or species-specific differences. Our NovoGen Bioprinter® Technology offers an opportunity to build in vitro tissue models to enable more accurate prediction of clinical outcomes.
Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis
The human kidney contains up to 2 million epithelial nephrons responsible for blood filtration. Regenerating the kidney requires the induction of the more than 20 distinct cell types required for excretion and the regulation of pH, and electrolyte and fluid balance. We have previously described the simultaneous induction of progenitors for both collecting duct and nephrons via the directed differentiation of human pluripotent stem cells. Paradoxically, although both are of intermediate mesoderm in origin, collecting duct and nephrons have distinct temporospatial origins. Here we identify the developmental mechanism regulating the preferential induction of collecting duct versus kidney mesenchyme progenitors. Using this knowledge, we have generated kidney organoids that contain nephrons associated with a collecting duct network surrounded by renal interstitium and endothelial cells. Within these organoids, individual nephrons segment into distal and proximal tubules, early loops of Henle, and glomeruli containing podocytes elaborating foot processes and undergoing vascularization. When transcription profiles of kidney organoids were compared to human fetal tissues, they showed highest congruence with first trimester human kidney. Furthermore, the proximal tubules endocytose dextran and differentially apoptose in response to cisplatin, a nephrotoxicant. Such kidney organoids represent powerful models of the human organ for future applications, including nephrotoxicity screening, disease modelling and as a source of cells for therapy
