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.
Cells isolated from donors with nonalcoholic fatty liver disease exhibit disease phenotypes in 3D bioprinted human liver tissue
The identification of targets and biomarkers and development of therapeutics for nonalcoholic fatty liver disease (NAFLD) may be accelerated by the use of well-characterized primary cell and tissue reagents, as well as improved in vitro human cell-based disease models, including three-dimensional (3D) bioprinted liver tissue. The characteristics of donors from which the cells are isolated, and especially their stage on the NAFLD continuum, are likely to influence the resulting performance in two-dimensional (2D) and 3D models. Evaluation of individual cell type characteristics, and their performance when combined in a tissue coculture model, could enable development of in vitro models more representative of specific patient populations and disease phenotypes.
RNA sequencing (RNA-seq) was performed on (5) non-diseased and (5) NAFLD liver tissues with NAFLD Activity Score (NAS) of 3 or more, revealing clear separation of non-diseased vs. NAFLD tissues and differential expression of fibrosis related genes. Histological analyses performed on tissue microarrays revealed consistent altered distribution patterns of hepatic stellate cells (HSC), with differential activation of HSC. Hepatocytes and non-parenchymal cells (NPC) (HSC, endothelial cells, and Kupffer cells), were isolated from non-diseased donors and from donors with NAS of 3 or more. The isolated cells were characterized with respect to viability, growth kinetics, cytokine production, and phenotype. 3D bioprinted liver tissue was generated using either NPCs isolated from diseased donors combined with non-diseased hepatocytes, or hepatocytes isolated from diseased donors combined with non-diseased NPCs. 3D bioprinted liver tissue generated using NPCs from a diseased donor exhibited accelerated collagen deposition (by trichrome stain) and HSC activation (by α-SMA staining) in comparison to bioprinted liver tissue generated with non-diseased tissue donors. Tissue generated using hepatocytes from a diseased donor exhibited steatosis induction.
Characteristics of the tissue of origin for cells used for in vitro models, including disease status, influence the performance of the cells and the utility of the resulting model. Thus, characterization of cell donors could enable development of in vitro models more representative of specific patient populations and disease phenotypes.
Modeling NAFLD using 3D bioprinted human liver tissue
Nonalcoholic fatty liver disease (NAFLD) is a chronic condition that originates as lipid accumulation within hepatocytes (steatosis) and progresses into nonalcoholic steatohepatitis (NASH), characterized by lipid accumulation, inflammation, oxidative stress, and fibrosis. NAFLD is now recognized as the most common cause of chronic liver disease in the western world, with an estimated prevalence of 25% worldwide, and is projected to become the leading indication for liver transplant by 2025. Despite decades of research, the mechanisms of NAFLD progression, therapeutic approaches and non-invasive diagnostics are still resoundingly absent. The study of steatosis and NASH has traditionally utilized rodent models, which are time consuming to generate and do not fully recapitulate the complex phenotypes associated with the human disease. Furthermore, current 2D cell culture models lack relevant liver cell types, do not accurately display diseased phenotypes, and have limited utility due to rapid loss of cell viability and function. To date, there are no current models exploring the role of cell donor heterogeneity and its impact on disease phenotype and the progression of disease. Thus, there is a significant need for a more predictive human multicellular 3D in vitro model to study the progression of steatosis into NASH.
A human in vitro three-dimensional bioprinted liver tissue system can be used to model nutritional damage and protective effects of MSDC-0602K, a novel modulator of the mitochondrial pyruvate carrier (MPC)
The growing global incidence of NASH mirrors the availability of nutrients. Over-nutrition also results in insulin resistance and type-2 diabetes, which are often co-morbidities associated with NASH and are known to drive more adverse outcomes. MSDC-0602K, a modulator of the mitochondrial pyruvate carrier (MPC), is in clinical trials as a potential treatment for NASH. Preclinical studies have shown that the mitochondrial pyruvate carrier is increased in expression in animals fed a high fat diet. Moreover, selective knockouts of each of the mitochondrial proteins that make up the carrier have shown that the MPC is a key driver of both NASH pathology and pharmacology of MSDC-0602K. Diet-induced disease using a three-dimensional multicellular human tissue model provides the potential to reconstruct the effects of overnutrition in vitro and to potentially model the actions of an agent like MSDC-0602K.
Utilization of a 3D bioprinted liver tissue model to evaluate the antifibrotic effects of an ALK5 inhibitor in a TGFβ-induced model of hepatic fibrosis
Compound induced chronic liver injury can lead to initiation of profibrotic processes resulting in sustained production of growth factors and profibrotic cytokines where inflammation, tissue remodeling and repair pathways are activated simultaneously to counteract the injury. Evaluation of potential antifibrotic therapies are limited using conventional non-human animal models, due to their inability to accurately reflect complex in vivo human biology, while 2D models lack the multicellular complexity and life span required to study fibrosis progression and regression. Utilization of a human 3D-bioprinted liver tissue model (ExVive™ Human Liver Tissue) comprised of primary hepatocytes, hepatic stellate cells (HSCs), and endothelial cells, which can model TGFβ induced fibrosis [Norona, et al. (2016) Tox Sci. 154(2):354-367], enables a mechanistic interrogation with an anti-fibrotic compound. In this study, galunisertib, a small molecule ALK5 (TGFβR1 kinase) inhibitor, was used to evaluate pathway-specific blockage of TGFβ-induced fibrogenesis. The coadministration of galunisertib with TGFβ prevented the characteristic features of TGFβ-induced fibrosis, including upregulation of collagen deposition, phosphorylated SMAD2/3, and TIMP-1. Increased HSC activation was observed only in the TGFβ-induced fibrosis model, demonstrated by α-smooth muscle actin (αSMA) labeling and upregulation of ACTA2 transcript. Tissue and hepatocellular health remained stable following treatment with galunisertib, as shown by LDH release, viability, and albumin production which remained similar to vehicle levels, suggesting prevention of TGFβ induced tissue damage. These results demonstrate that a progressive in vitro model of liver fibrosis can be utilized to interrogate disease-associated pathways, and establish proof of concept for application of the model for preclinical evaluation of certain classes of antifibrotic drugs.
Modeling NAFLD using 3D bioprinted human liver tissue
Nonalcoholic fatty liver disease (NAFLD) is a chronic condition that originates as lipid accumulation within hepatocytes (steatosis) and progresses into nonalcoholic steatohepatitis (NASH), characterized by lipid accumulation, inflammation, oxidative stress, and fibrosis. NAFLD is now recognized as the most common cause of chronic liver disease, with a prevalence of 25% worldwide, and is projected to become the leading indication for liver transplant by 2025. Despite decades of research, the mechanisms of NAFLD progression, therapeutic approaches and non-invasive diagnostics are still resoundingly absent. The study of steatosis and NASH has traditionally utilized rodent models, which are time consuming to generate and do not fully recapitulate the complex phenotypes associated with the human disease. Furthermore, current 2D cell culture models lack relevant liver cell types and have limited utility due to rapid loss of cell viability and function. Thus, there is a significant need for a more predictive human multicellular 3D in vitro model to study the progression of steatosis into NASH.
Long-term performance of implanted bioprinted human liver tissue in a mouse model of human alpha-1 antitrypsin deficiency
Conventional cell therapy and tissue engineering approaches to treating liver diseases and injury are limited by low cell retention, poor engraftment, poor graft durability and complications including portal hypertension. Integration of next generation technologies such as 3D bioprinting is an essential step towards the clinical success of these promising approaches and has the potential for broad applicability ranging from treatment of inborn errors of metabolism to acute on chronic liver failure. Here, we report fabrication, implantation and engraftment of a human bioprinted therapeutic liver tissue (BTLT) containing human umbilical vein and liver endothelial cells, hepatic stellate cells (HSC) and hepatocytes (Heps) in a transgenic mouse model of alpha-1 antitrypsin deficiency (AATD). Following BTLT implantation on the surface of the liver in mice expressing the mutant human form of alpha-1 antitrypsin (PiZ mouse), human albumin, transferrin, alpha-1 antitrypsin (AAT) and fibrinogen were detected in the circulation as early as 7 days, with increasing levels of human albumin detected for at least 90 days post-implantation. Histopathologic evaluation of implanted BTLT and underlying host tissue revealed integration of the fabricated tissues with the underlying host liver, with the implanted graft having defined areas of parenchymal and non-parenchymal (NPC) zones. The non-parenchymal zones contained perfused human CD31-lined vasculature and desmin-positive HSC. Adjacent to the NPC-rich regions were areas of dense, polarized Heps, closely supported by cells phenotypically consistent with HSC. The human hepatocytes in the BTLT also stained positive for albumin, AAT, fibrinogen and ornithine transcarbamylase. When compared to sham-operated, age-matched control animals, BTLT implantation in the PiZ mice resulted in an improvement of the pathological features associated with accumulated, misfolded protein within the mouse hepatocytes. There was an observed reduction in the accumulation of PAS-stained globule-containing hepatocytes adjacent to the implanted tissue. The reduction in hepatocytes containing large ER-bound globules was also confirmed by a decrease in ATZ11-positive cells in the host tissue. The rapid vascularization, durable tissue engraftment, target cell retention, and improvement in tissue pathology evince a promising novel approach to treating AATD and other liver diseases.
Modeling NAFLD using 3D bioprinted human liver tissue
Nonalcoholic fatty liver disease (NAFLD) is a chronic condition that originates as lipid accumulation within hepatocytes (steatosis) and progresses into nonalcoholic steatohepatitis (NASH), characterized by lipid accumulation, inflammation, oxidative stress, and fibrosis. NAFLD is now recognized as the most common cause of chronic liver disease in the western world, with an estimated prevalence of 25% worldwide, and is projected to become the leading indication for liver transplant by 2025. Despite decades of research, the mechanisms of NAFLD progression, therapeutic approaches and non-invasive diagnostics are still resoundingly absent. The study of steatosis and NASH has traditionally utilized rodent models, which are time consuming to generate and do not fully recapitulate the complex phenotypes associated with the human disease. Furthermore, current 2D cell culture models lack relevant liver cell types, do not accurately display diseased phenotypes, and have limited utility due to rapid loss of cell viability and function. To date, there are no current models exploring the role of cell donor heterogeneity and its impact on disease phenotype and the progression of disease. Thus, there is a significant need for a more predictive human multicellular 3D in vitro model to study the progression of steatosis into NASH.
3D bioprinted human liver tissue for modeling progressive liver disease
Translation of preclinical data to clinical outcomes remains an ongoing challenge in drug development. 3D bioprinted tissues exhibiting physiological tissue-like responses can be created to bridge this gap, through spatially controlled, automated deposition of tissue-specific cell types. These multicellular, human in vitro tissue models enable improved cellular interactions and assessment of biological responses at the biochemical, genomic, and histological levels over extended time in culture. Organovo’s ExVive™ Human Liver can be used to model chronic liver disease relevant phenotypes including steatosis, inflammation, and fibrosis. Incorporation of Kupffer cells and stimulation with inflammatory signals induces inflammatory cytokine release. Chronic exposure to drugs known to induce steatosis such as valproic acid, or to nutrient overload (free fatty acids) induces formation of lipid droplets. Chronic exposure to chemical inducers of fibrosis or TGFβ stimulation leads to stellate cell activation and fibrosis. Finally, inflammatory inducers and nutrient overload together lead to steatosis and fibrosis. These results suggest that ExVive™ Human Liver Tissue holds promise for the study of complex, chronic conditions such as NASH, enabling the discovery of novel therapeutics, biomarkers and safety assessment of drugs in a disease-relevant background.
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.
