HepatoMune® Technology

The involvement of inflammation and its underlying mechanisms are becoming increasingly important in the early detection of drug-induced hepatotoxicity. At present there is a clear, unmet need for an in vitro model of basal and inflamed human liver that is capable of predicting clinical outcomes. HepatoMune tri-cultures are HepatoPac® co-cultures (micropatterned primary hepatocytes and fibroblasts) supplemented with primary Kupffer cells that represent a highly functional miniature human liver tissue that models inflammation-mediated hepatotoxicity within an industry-standard multi-well format. The HepatoMune tri-cultures are viable for at least 10 days while maintaining both liver cell and Kupffer cell functionality.

HepatoMune® tri-cultures enable:

  • Evaluation of inflammation-mediated hepatotoxicity
  • Modeling healthy liver physiology and inflammation
  • Retention of long-term cultures (10 days) for testing hepatotoxicity of compounds
  • Monitoring of protein therapeutic-drug interactions
  • Determination of mechanistic information

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HepatoMune Creation

To engineer HepatoMune, hepatocytes are organized into colonies of prescribed, empirically-optimized dimensions using microfabrication tools and subsequently surrounded by supportive non-parenchymal cells. This combination is then augmented with primary, species-matched Kupffer cells at a precise hepatocyte:Kupffer cell ratio of 1:0.4. The presence of functional Kupffer cells in HepatoMune tri-cultures is then confirmed via pHrodo-S. aureus phagocytosis and CD68 staining.  The resulting HepatoMune tri-culture represents a liver microenvironment that mimics an inflamed liver state. HepatoMune hepatocytes retain in vivo-like morphology, express liver-specific genes, metabolize compounds using active Phase I/II drug metabolism enzymes, secrete diverse liver-specific products, and exhibit transporter activity (Phase III).  Addition of the primary Kupffer cells does not affect the functionality of the hepatocytes as determined by CYP3A4 activity and urea synthesis throughout a 10 day culture.

Hepatomune Tri Culture
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HepatoMune tri-culture were created using patented microfabrication tools and consist of primary hepatocytes arranged in optimized domains and surrounded by stromal fibroblasts. Cryopreserved primary Kupffer cells are added on day 7 of culture. pHrodo and CD68 staining illustrates the presence of functional Kupffer cells on day 10 of culture.

Hepatomune Primary Human Kupffer
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Addition of primary human Kupffer cells does not significantly affect functionality of the primary hepatocytes as determined by Urea synthesis or Cyp3A4 activity.

 

HepatoMune Validation

The model has been thoroughly vetted in order to ensure that it will demonstrate expected responses without compromising hepatocyte function. The role of cytokine exposure was examined in HepatoMune cultures in response to non-cytotoxic concentrations of exogenous cytokines (IL-2, IL1β, IL-6 and TNF-alpha), and indicate that the inflammatory nature of the Kupffer cells within the tri-cultures is not inherently detrimental to hepatocyte viability. In examining suppression of hepatic P450 enzymes, both IL-6 and IL-1β caused CYP3A4 inhibition as expected. The observed inhibition of CYP3A4 is important in the context of understanding compromised drug clearance, which can be associated with P450 downregulation in the liver.

HepatoMune Validation
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Kupffer cell augmentation does not alter hepatocyte viability, as measured by ATP levels, following cytokine exposure over a 4 day dosing cycle. Addition of IL-6 suppresses CYP3A4 activity in HepatoMune, while addition of IL-1β to HepatoMune produces a Kupffer cell-dependent decrease in CYP3A4 activity.

A Better Model of an Inflamed Liver Environment

Liver inflammation involves the early release of multiple pro-inflammatory factors, reactive oxygen species and chemokines by specialized macrophages, known as Kupffer cells. This inflammatory response occurs alongside the downregulation of major liver-specific metabolizing enzymes (ie., CYP450), which can alter the liver's metabolism of foreign biologics, such as therapeutic antibodies or xenobiotics or cause hepatotoxicity.  

In order to effectively understand the mechanism behind inflammatory or infectious disease drug-induced liver injury, in vitro models must exhibit all of the complex pharmacodynamic interactions and processes that occur during the inflamed liver state, including cell signaling events between parenchymal and non-parenchymal cells.  This is difficult to achieve in current in vitro models given their short life and declining activity.

HepatoMune provides a physiologically-relevant in vitro model that allows for the evaluation of inflammation-mediated drug-induced liver injury.  Because it is a metabolically stable and long-lived tri-culture, it provides a platform for the evaluation of mechanistic pathways and time-dependent aspects of inflammation-based hepatotoxicity.  Ultimately, understanding such pathways and their role in altering DILI will aid in the development of non-hepatotoxic drug candidates.

Increased toxic responses to hepatotoxic compounds (e.g. trovafloxacin, chlozapine) have been associated with interactions between these compounds and inflammatory or infectious disease. We have demonstrated a proof-of-concept study using known immune-mediated hepatotoxins to detect inflammation-based toxicity in the HepatoMune platform. We monitored the release of endogenous inflammatory cytokines upon Kupffer cell activation by toxic agents, which can accompany liver injury. We found that activated Kupffer cells rendered trovafloxacin (TVX), but not its non-toxic analog, levofloxacin (LVX), hepatotoxic by a TNF-α-mediated mechanism, reflecting a response seen in animal models (Shaw et al., 2009).  These data validate the specificity of our HepatoMune platform and impact our understanding of the injury potential of drug-cytokine combinations during inflammation and disease.

Hepatomune Trovafloxacin
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Trovafloxacin (TVX) toxicity is potentiated in LPS-treated Human HepatoMune.   LPS stimulation of human HepatoMune cultures causes robust secretion of TNF-α and IL-6. LPS-stimulation of HepatoMune cultures exacerbated TVX-induced toxicity, marked by the leftward shift in the ATP dose-response curves. Levofloxacin was not toxic showing no changes in cellular ATP content upon LPS stimulation.  Simultaneous treatment with TNF-α and TVX exacerbates TVX toxicity in the HepatoPac co-culture model in a manner similar to that observed in LPS-TVX treatment of HepatoMune tri-cultures.

Technical Resources

Type Title
Application Note HepatoMune: A Bioengineered Micro-liver Platform for the Study of Inflammatory Responses in Toxicology Investigation
Poster A Micropatterned Culture with Primary Hepatocytes and Kupffer Macrophages for Studying Inflammation-Drug Interactions.
Product Brochure HepatoMune™ Modeling of Inflammation-mediated Hepatotoxicity Brief Overview of the HepatoMune Tri-culture In Vitro Model.