The critical shortage of donor organs creates an urgent need for novel treatments for patients with inborn errors of metabolism and end stage liver failure. Conventional cell therapy and tissue engineering approaches for 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. Using the NovoGen Bioprinter® Platform, we have designed an implantable 3D bioprinted human liver tissue containing primary hepatocytes, liver endothelial cells, hepatic stellate cells, and HUVECs. Here we show data supporting functionality and engraftment of our implantable human liver patch in small animal disease models for up to 90 days.
Modeling NAFLD and TGF beta-induced fibrosis in ExVive™ human liver tissue
Nonalcoholic fatty liver disease (NAFLD) is the most common liver disorder with an estimated prevalence of over 25% worldwide and is projected to become the leading indication for liver transplant by 2025. Despite decades of research focused on NAFLD, an effective treatment has yet to be approved. This is due in part to the reliance on cell culture and animal models that present challenges in translation due to limited functional longevity and species differences, respectively. ExVive™ Human Liver Tissue, a clinically-translatable in vitro model, is ideal for studying the effects of drugs on liver disease progression, regression, and the mechanisms involved. Here we present results showing a nutrient overload induction of liver disease and TGFβ-induced fibrosis in ExVive™ Human Liver Tissue. A variety of disease-relevant phenotypes including steatosis, inflammation, and fibrosis can be demonstrated in the model.
Monocrotaline toxicity in ExVive 3D bioprinted human liver tissue
Monocrotaline (MCT), a pyrrolizidine alkaloid causes liver injury in animals similar to that of hepatic venoocclusive disorder in humans. MCT induced liver injury occurs through a complex set of cellular insults involving multiple cell types which can ultimately lead to fibrotic changes. In the study, we evaluated the effects of MCT in 3D-bioprinted human liver tissue comprising of primary hepatocytes, hepatic stellate cells, and endothelial cells (ExVive™ Human Liver Tissue). The bioprinted tissues were treated with MCT for fourteen days. MCT treatment led to time- and dose-dependent decreases in tissue health as measured by LDH leakage and albumin synthesis and by histopathologic changes in the tissues, as well as increases in the production of the pro-inflammatory cytokines IL-1β, IL-4, IL-8 and IL-10. Histologic assessment of formalin-fixed, paraffin-embedded tissue revealed signs of tissue damage, including dissociation of the network of hepatocytes and reduced cellularity within the tissues. Immunohistochemical analyses revealed a dose-dependent increase in CD31+ cells and a marked increase in the appearance of large, CD31+ bright cells that co-expressed smooth muscle actin (α-SMA), often forming clusters or complex multicellular structures. Changes in organization of CD31 expressing endothelial cells and appearance of α-SMA expressing cells are indicative of remodeling and initiation of fibrotic events. Observations which emerged from this study capture the spectrum of changes induced by MCT ranging from reduced hepatocellular function and vascular remodeling, which may involve endothelial cell migration, organization, proliferation, apoptosis, and endothelial-to-mesenchymal transformation to early fibrotic events.
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.
Modeling compound-induced fibrogenesis in vitro using 3D bioprinted human liver tissues
Compound-induced hepatotoxicity leading to fibrosis remains a challenge for human risk assessment. Latency to detection and lack of early biomarkers make it difficult to characterize the dynamic and complex intercellular interactions that occur during progressive liver injury. Thus in vitro systems that recapitulate this cellular complexity in a three dimensional context offer advantages over simple monoculture systems with respect to predictive modeling. Through the incorporation of key architectural features and primary cell types, 3D bioprinted liver can be maintained in culture for greater than four weeks, making it an ideal platform for studying phenotypes that arise during recurring exposure. Here, we discuss the utility of NovoView™ Human Liver Tissue for modeling chronic compound exposure using fibrosis as a case study and provide a comprehensive approach to examine key initiating events and progression of tissue injury.
Use of bioprinted 3D human tissues for the assessment of drug toxicity
Human tissue biology is strongly influenced by the unique interplay and extensive cross talk that exists between different resident cell populations. These cell types are most often spatially arranged in a specific architecture which defines their biological function and mechanistic response to drug treatment over time. Three-dimensional bioprinted tissues that model this cellular complexity and form offer major advantages over conventional in vitro systems with respect to predictive modeling. Our tissues incorporate key architectural features and primary cell types and can be maintained in culture on a timescale of several days to weeks. We can apply our technology to design various tissues, including liver, kidney, skin, and oncology models. Here, we present specific case studies using ExVive™ Human Liver Tissue to assess drug toxicity via various modalities including acute to chronic dosing, metabolite-driven toxicity, and fibrosis induced liver injury.
