Models for Drug and Chemical Toxicity

Liver toxicity is a major cause of clinical trial failures and has played a significant role in the withdrawal of several drugs from the market. Evaluation of toxicity is performed throughout the drug development pipeline. However, for liver toxicity in particular, the models for detection early in the pipeline which are mostly animal-based fall short of meeting expectations. Increasingly, in vitro human models are being explored as an alternative in or to augment findings in animal models as a means to improve preclinical prediction of liver toxicity.

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Building complex liver toxicity models

The key component of any human in vitro liver toxicity model is primary hepatocytes. Ideally, the hepatocyte culture needs to be robust and have decent longevity in culture to evaluate toxicity over time. Our plateable hepatocytes that are suitable for general applications or for drug drug interaction studies are qualified to maintain healthy monolayers for at least five days in culture systems. Many batches are also qualified to self-assemble into spheroids which can last up to 21 days in culture.

The ability now to create in vitro models that co-culture hepatocytes with Kupffer cells, the liver resident macrophage, can improve the ability to detect these complex toxicities early in development. Other new models such as co-cultures with stellate and liver endothelial cells, in both monolayer and 3D spheroid models, have been shown to increase the predictive power of cell culture models¹.

Hepatocytes and Non-parenchymal Cells - Development and Optimization of a Comprehensive Co-Culture

Tech Note detailing the development of a co-culture protocol for Heps and NPCs Download Document

Hepatocytes in 3D – spheroid culture

Studies have shown that the formation of multicellular hepatocyte spheroids in 3D culture is a promising approach for enhancing liver-specific functions. Hepatocytes cultured in spheroids exhibit polarized cell structures and direct cell-cell contact, and importantly, can maintain steady metabolic function for more than 14 days. This approach also enables the self-assembly of multiple liver cell types, creating a pseudo microtissue that better mimics the in vivo hepatocyte environment.

Lonza tests each batch of our General Purpose and Interaction Qualified hepatocytes (cat. # HUCPG and HUCPI) for capacity to self assemble into spheroid structures for use in longer-term toxicity, metabolism, and disease modeling applications. Refer to our White Paper and Technical Note for detailed evaluations and protocols to incorporate spheroids into your routine cultures.

Development of a long-term primary hepatocyte 3D spheroid model

For use in DILI applications using ViaLight Plus Cytotoxicity BioAssay Kit

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Primary human hepatocyte 3D spheroids co-cultured with non-parenchymal cells

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Build in vivo-relevant liver models by adding non-parenchymals

Additionally, non-parenchymal cells can also be used to better represent normal liver physiology. For example, co-cultures using hepatocytes, Kupffer cells, Stellate cells, and liver endothelial cells are already showing to be powerful tools for modeling the liver in vitro. This is because it is thought that under both normal and pathological conditions, many hepatocyte functions are regulated by substances released from neighboring non-parenchymal cells. These cells play an important role in the modulation of xenobiotic metabolism in the liver and provide more comparable data for diseased and healthy cells.

Kupffer cells are the resident macrophages in the liver and are part of the first line of defense against bacteria and toxins in the gut. Kupffer cells exhibit typical macrophage morphology and can be activated to produce inflammatory cytokines, growth factors and reactive oxygen species.

In vivo, prolonged activation of Kupffers are associated with many diseases of the liver including Non-alcoholic Fatty Liver Disease (NAFLD), Non-alcoholic Steato Hepatitis (NASH), and liver damage associated with certain bioactivated chemicals.

Kupffer cells are isolated and enriched from dissociated human liver tissue and then cryopreserved as passage 0. Characterization of isolated Kupffer cells includes assessing morphology in plated format and evaluating functional responses to LPS stimulation.

Hepatic Stellate cells are a resident cell type of the liver primarily functioning to store retinoids. In response to liver damage, Stellates rapidly lose the stored retinoids and differentiate into a proliferating fibroblast-like cell that begins depositing collagen matrix. This activity causes buildup of collagen in the liver eventually leading to cirrosis.

Stellates can be isolated from disrupted liver tissue, enriched, and placed into cell culture. Isolated and enriched Stellates are provided at passage 1, resulting in Stellates with low activation states.

Endothelial cells are the primary barrier cells that line blood vessels and sinusoids of the liver. Human Liver-derived Endothelial Cells (HLECs) provided by Lonza are isolated and enriched from disrupted liver tissue then cryopreserved for use in a wide range of research models.

HLECs express characteristic markers of endothelial cells including CD32b and vWF as shown by immunofluorescence and maintain appropriate endothelial morphology over limited serial passaging.

Humanized mouse liver

An immunocompromised mouse model can be made to house a liver composed almost entirely of human liver cells. The purpose of creating such a model is to enable human-relevant liver pharmacokinetics and pharmacodynamics studies to be performed in an intact mouse model. These models can be used to re-create human specific liver toxicities in an in vivo environment, but are also used for modeling infectious diseases that impact the human liver. Hepatocytes used for making humanized mouse models should be plateable and of high quality, but don’t necessarily need to be prequalified for drug-drug interaction studies. Lonza therefore recommends Human Cryopreserved Hepatocytes, Plateable, General Purpose cells (cat # HUCPG).

Matching toxicity application needs to hepatocyte culture format

Application	Description	Culture Format	Catalog Numbers
Cell-based toxicity	Short term assays to examine the acute effects of drugs and toxicants on cell health	Plated monolayer cultures	HUCPI HUCPG
		3D/Spheroids	HUCPI HUCPG
		Co-culture	HUCPI HUCPG HUCNP HUCLS-200K HUCLS-1M HLKC-200K HLKC-500K HLECP1
		3D/Spheroid Co-Culture	HUCPI HUCPG HUCNP HUCLS-200K HUCLS-1M HLKC-200K HLKC-500K HLECP1
Mechanistic toxicity	Short and long term assays to determine toxicity mechanisms. Results are used to suggest structure-activity relationships and molecular signatures that indicate risk.	Plated monolayer cultures	HUCPI HUCPG
		3D/Spheroids	HUCPI HUCPG
		Co-culture	HUCPI HUCPG HUCNP HUCLS-200K HUCLS-1M HLKC-200K HLKC-500K HLECP1
		3D/Spheroid Co-Culture	HUCPI HUCPG HUCNP HUCLS-200K HUCLS-1M HLKC-200K HLKC-500K HLECP1
Humanized mouse liver	Human hepatocytes are injected into mice with damaged livers in order to allow the liver to regenerate with human cells		HUCPI HUCPG

References

Yamazaki H, Suemizu H, Mitsui M, Shimizu M, and Guengerich FP. (2016) Combining Chimeric Mice with Humanized Liver, Mass Spectrometry, and Physiologically-Based Pharmacokinetic Modeling in Toxicology. Chemical Research in Toxicology 29(12): 1903-1911.