HEPATOMUNE Technology is used to create an in vitro hepatic culture that models inflammation-mediated hepatotoxicity. HEPATOMUNE cultures are tri-cultures created by supplementing HEPATOPAC cultures (i.e. micropatterned primary hepatocytes and fibroblasts) with primary Kupffer cells. The presence of the Kupffer cells results in a culture that mimics highly-functional human liver tissue in inflammatory stress conditions.
HEPATOMUNE cultures are viable for at least ten days in industry-standard multi-well formats.
HEPATOMUNE 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.
Products and Services Using HEPATOMUNE Technology
HEPATOMUNE Technology is available in a ready-to-use Human HEPATOMUNE kit.
Additionally, BioIVT can implement hepatotoxicity studies using HEPATOMUNE Technology on a contract services basis, either as a stand-alone project, or as part of a comprehensive evaluation of ADME Tox properties of compounds.
Process to Create a HEPATOMUNE Culture
To engineer HEPATOMUNE cultures, 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, as shown in the pictures below:
HEPATOMUNE cultures 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, as illustrated in the charts below:
Validation of the HEPATOMUNE Model
The HEPATOMUNE 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, IL-1β, IL-6 and TNF- α), 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.
Data from these studies is shown in the following charts:
Critical Characteristics of a Liver Inflammation Model
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 (i.e., 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 (DILI), 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 cultures provide a physiologically-relevant in vitro model that allows for the evaluation of inflammation-mediated DILI. 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, shown in the following charts, validate the specificity of our HEPATOMUNE platform and impact our understanding of the injury potential of drug-cytokine combinations during inflammation and disease.