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.

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.

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

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.

Disease Model, Downloadable Publications, Liver Tissue, Webinars

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.

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

3D bioengineered tissues: From advancements in in vitro safety to new horizons in disease modeling

Research aimed at more fully emulating human biology in vitro has rapidly progressed in recent years with advancements in 3D tissue engineering and microphysiological systems. The initial target of such systems has been directed towards drug and chemical safety assessment, with the goal of improving sensitivity and predictive capabilities. Here we discuss recent developments of in vitro organ culture systems, and their future applications in modeling human disease.