Disease Model, Downloadable Publications, Liver Tissue, Posters

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.

Disease Model, Downloadable Publications, Liver Tissue, Peer-Reviewed

Bioprinted liver provides early insight into the role of Kupffer cells in TGF-β1 and methotrexate-induced fibrogenesis

Hepatic fibrosis develops from a series of complex interactions among resident and recruited cells making it a challenge to replicate using standard in vitro approaches. While studies have demonstrated the importance of macrophages in fibrogenesis, the role of Kupffer cells (KCs) in modulating the initial response remains elusive. Previous work demonstrated utility of 3D bioprinted liver to recapitulate basic fibrogenic features following treatment with fibrosis-associated agents. In the present study, culture conditions were modified to recapitulate a gradual accumulation of collagen within the tissues over an extended exposure time frame. Under these conditions, KCs were added to the model to examine their impact on the injury/fibrogenic response following cytokine and drug stimuli. A 28-day exposure to 10 ng/mL TGF-β1 and 0.209 μM methotrexate (MTX) resulted in sustained LDH release which was attenuated when KCs were incorporated in the model. Assessment of miR-122 confirmed early hepatocyte injury in response to TGF-β1 that appeared delayed in the presence of KCs, whereas MTX-induced increases in miR-122 were observed when KCs were incorporated in the model. Although the collagen responses were mild under the conditions tested to mimic early fibrotic injury, a global reduction in cytokines was observed in the KC-modified tissue model following treatment. Furthermore, gene expression profiling suggests KCs have a significant impact on baseline tissue function over time and an important modulatory role dependent on the context of injury. Although the number of differentially expressed genes across treatments was comparable, pathway enrichment suggests distinct, KC- and time-dependent changes in the transcriptome for each agent. As such, the incorporation of KCs and impact on baseline tissue homeostasis may be important in recapitulating temporal dynamics of the fibrogenic response to different agents.

Downloadable Publications, Liver Tissue, Peer-Reviewed, Safety Testing

Optimizing drug discovery by investigative toxicology: Current and future trends

Investigative Toxicology describes the de-risking and mechanistic elucidation of toxicities, supporting early safety decisions in the pharmaceutical industry. Recently, Investigative Toxicology has contributed to a shift in pharmaceutical toxicology, from a descriptive to an evidence-based, mechanistic discipline. This was triggered by high costs and low throughput of Good Laboratory Practice in vivo studies, and increasing demands for adhering to the 3R (Replacement, Reduction and Refinement) principles of animal welfare. Outside the boundaries of regulatory toxicology, Investigative Toxicology has the flexibility to embrace new technologies, enhancing translational steps from in silico, in vitro to in vivo mechanistic understanding to eventually predict human response. One major goal of Investigative Toxicology is improving preclinical decisions, which coincides with the concept of animal-free safety testing. Currently, compounds under preclinical development are being discarded due to the use of inappropriate animal models. Progress in Investigative Toxicology could lead to humanized in vitro test systems and the development of medicines less reliant on animal tests. To advance this field a group of 14 European-based leaders from the pharmaceutical industry founded the Investigative Toxicology Leaders Forum (ITLF), an open, non-exclusive and pre-competitive group that shares knowledge and experience. The ITLF collaborated with the Centre for Alternatives to Animal Testing Europe (CAAT-Europe) to organize an “Investigative Toxicology Think-Tank”, which aimed to enhance the interaction with experts from academia and regulatory bodies in the field. Summarizing the topics and discussion of the workshop, this article highlights Investigative Toxicology’s position by identifying key challenges and perspectives.

Disease Model, Downloadable Publications, Liver Tissue, Posters

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.

Disease Model, Downloadable Publications, Liver Tissue, Posters

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.

Disease Model, Downloadable Publications, Liver Tissue, Posters

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.

Disease Model, Downloadable Publications, Liver Tissue, Posters

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.

Downloadable Publications, Liver Tissue, Posters, Therapeutic Tissue

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.

Disease Model, Downloadable Publications, Liver Tissue, Posters

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.

Downloadable Publications, Liver Tissue, Therapeutic Tissue, Webinars

3D bioprinted therapeutic liver tissue

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.