Liver Cells for ADME & Toxicology Studies

The liver plays a critical role in vertebrate biology. It has many important metabolic functions, including the regulation of glucose and cholesterol metabolism, the production of plasma proteins including clotting factors, and the detoxification of endogenous and exogenous compounds. The liver also produces various hormones involved in insulin regulation, blood pressure, and blood lipid levels. Because of the many physiological processes that depend on the liver, a fundamental understanding of liver biology and the ability to address these at the benchtop is essential for researchers involved in creating new, life-saving medicines.

The liver is composed of five major cell types, and the hepatocyte is the most abundant cell type. Hepatocytes comprise approximately 70% of the liver cell population and are responsible for most metabolic and hormonal processes. The other four cell types, collectively known as hepatic non-parenchymal cells, consist of resident macrophages called Kupffer cells, stellate cells, liver sinusoidal endothelial cells, and cholangiocytes. These cells serve to support the liver structure, transport molecules in and out, and communicate with the immune system.

One of the greatest challenges in drug development is the prediction of the safety and the metabolic fate of a new drug before it enters human clinical trials. Because the liver is the major site of metabolism and detoxification, in vitro cell systems that mimic the liver are significantly useful for predicting the consequences of drugs administered to humans in the clinic. The ability to isolate highly pure hepatocyte populations from non-transplantable, donated human livers to mimic the human liver environment has therefore become an integral part of the drug development pipeline.

Figure 2 is a schematic that shows the procedure for the isolation of primary human hepatocytes, and their cellular morphology in suspension and on plates. The functionality of primary hepatocytes in culture is very similar to that of in vivo hepatocytes, as indicated by albumin production, urea production, and a variety of metabolic enzyme activities.

Hepatocyte isolations also in the ability to obtain a mixed population of all the other major cell types from the liver. From this mixture, enrichment of Kupffer, stellate, and endothelial cells can be performed to enable the development of complex tissue-like models of the liver in vitro. The following sections detail some of the major applications for hepatocytes and other liver cells to support new and improved drug development efforts.

Plated Metabolism Assays

The most common application of plated hepatocytes for in vitro drug metabolism and pharmacokinetics (DMPK) studies is to determine the potential of drug-drug interactions via induction of the gene expression of various CYP450 enzymes. The amount and activity of these enzymes are known to increase when cells are exposed to certain chemical substances. However, an increase in the activity of a particular P450 enzyme might also affect a different drug in a patient's regime, leading to a drug-drug interaction (DDI) that could impact the efficacy and safety of more than one drug. Regulatory agencies require an analysis of the DDI potential in hepatocytes for CYP3A4, CYP1A2, and CYP2B6, which indicates whether a clinical DDI study is required. Our Cryopreserved Human Hepatocytes, Interaction qualified, (HUCPI) are pre-characterized to exhibit enzyme induction levels recommended by the FDA for evaluation of DDI.

A second major application of plated hepatocytes is the determination of the inhibition of the transporter-mediated efflux of a substance by a particular drug. Our Interaction Qualified Hepatocytes (HUCPI) are therefore also qualified for transporter function.  By characterizing for both enzyme induction and transporter function, we enable use of the same donor hepatocyte batch for different types of DDI studies.

Finally, the pharmaceutical discovery of drugs that are more slowly metabolized has become increasingly desirable. Slower drug metabolism translates into longer-lasting efficacious levels of a drug, so a patient can take the drug less frequently, which can increase compliance and decrease production costs. However, suspension hepatocytes are not viable or metabolically active long enough to allow the accurate measurement of the basic metabolic properties of such drugs. Since plated hepatocytes maintain their metabolic activity much longer than cells in suspension, researchers use them for assays that have a duration of four hours or longer. We qualify and report basal metabolism and clearance rates for several P450 enzymes in both of our plateable hepatocyte products, HUCPG and HUCPI.  

¹ Clearance refers to the rate at which a drug is cleared from the system.

Suspension Metabolism Assay

Metabolism of drugs can be measured in hepatocytes while cells are still in suspension, either directly after isolation or after recovery of cryopreserved hepatocytes. Suspension metabolism assays include: metabolic profiling, metabolite identification, and metabolic stability. Suspension hepatocytes are also used to determine drug uptake and kinetics. A common approach known as the oil-spin technique measures radio-labeled drug concentrations inside the cell following a very short incubation period. These routine metabolism and uptake assays using hepatocytes in suspension are typically applied to large numbers of molecules early in development to help prioritize chemicals for further development.

Use the table below to determine what formats of hepatocytes you might need for your applications.

*Check individual CoAs to learn the number of donors pooled in each batch.

Cell Toxicity Models

Liver toxicity is a major cause of clinical trial failures and has played a significant role in the withdrawl 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.  

Building Complex Liver Toxicity Models

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 models (9).

Humanized Mouse Models

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 (HUCPG).

Request more information to learn how primary hepatocytes can inform your toxicity testing and research.

Hepatitis

Many varieties of the hepatitis virus cause inflammation of the liver, which can lead to severe illness or even death when left untreated. Millions of people are infected with hepatitis viruses worldwide and it remains a leading cause of hepatocellular carcinomas, especially in individuals born in the U.S.A. between 1945 and 1965. While cures have been developed for the deadliest variant, Hepatitis C (HCV), reinfection can occur because HCV is very prone to mutation. This tendency to mutate has also hindered the development of a HCV vaccine. While an effective vaccine is available for the more common Hepatitis B (HBV) virus, it still infects millions annually and causes illnesses ranging from flu-like symptoms to liver failure. HBV is a DNA virus that utilizes the nuclear machinery of the hepatocyte to replicate. Infection and subsequent replication can be modeled in primary human hepatocytes in culture, and has been used to develop large scale screening protocols for new drugs that attack the progression of the virions to the closed circular DNA, which eventually leads to inflammation and other symptoms. So far, HBV has eluded efforts to develop curative drugs. (4) For hepatitis studies in vitro, our hepatocytes prequalified to be plateable for 5 days in culture are ideal. These include the General Purpose (HUCPG), Interaction Qualified (HUCPI).

Malaria

Nearly half of the world’s population lives in regions where inhabitants are at risk for malaria infection and more than 500,000 people worldwide die from the disease each year. Malaria is caused by a Plasmodium parasite, which has a life cycle that includes the human liver; specifically, the infection of hepatocytes. As shown in Figure 4, Plasmodium sporozoites undergo a major replication event in the hepatocyte before entering the next stage of the life cycle in the blood. Once the parasite is in the blood, the infected person begins to experience the symptoms associated with malaria; alternating chills and fever, fatigue, and others. The development of clinical interventions for the liver stage of the Plasmodium life cycle has become of interest, not only to prevent malaria symptoms, but also the spread of the parasite back to the mosquito, through the blood. For hepatitis studies in vitro, our hepatocytes prequalified to be plateable for 5 days in culture are ideal. These include the General Purpose (HUCPG), Induction Qualified (HUCPI).

The challenge of using hepatocytes for research on Plasmodium infection has been that the hepatocytes must survive for several weeks in culture. Recent work has shown that co-cultures of cryopreserved hepatocytes with liver Kupffer cells (i.e., the resident macrophage in the liver) (HLKC) can successfully mimic the liver stage of the Plasmodium life cycle. (11).

Non-alcoholic steatohepatitis (NASH) models

Non-alcoholic steatohepatitis (NASH) is a disease of the liver characterized by the infiltration of inflammatory cells, increased lipidosis in the hepatocytes, and early stages of collagen deposition leading to fibrosis. NASH is a progression of non-alcoholic fatty liver disease (NAFLD), which is associated with obesity and diabetes. The incidence of NAFLD is thought to be as high as 30% of the population and continues to rise with the co-incident rise in obesity.

The interest in developing cell-culture models for NASH is very high. Currently, a non-invasive diagnostic is not available for NASH and effective treatments are still in the early research and development stages. However, the onset of fibrosis, which is characterized by increased collagen deposition, is a hallmark pathological feature of NASH. Cell models that recapitulate the features of NASH contain both hepatocytes and stellate cells in co-culture or in 3D models. (12) For NASH studies in vitro, our hepatocytes prequalified to be plateable for 5 days in culture are ideal. These include the General Purpose (HUCPG), Interaction Qualified (HUCPI). Researchers may also be interested in low-passage Stellate cells (HUCLS), Kupffer cells (HLKC), and liver endothelial cells (HLECP2) for building physiologically relevant 3D culture models to mimic the progression of NASH in vitro.

Use the table to determine what format of hepatocytes is best for your disease modeling efforts.

Getting Started with Hepatocytes in 3D: Verified for Spheroids^TM Human Hepatocytes 

Making Liver Tissue-like Structures Using RAFT^TM System

The RAFT^TM System allows for the creation of tissue-like structures. The 3D matrix of the type 1 collagen-based RAFT^TM Culture provides a more natural cell culture environment and therefore a potentially superior model for in vitro screening. In a recent study, we compare cell viability and cell morphology of rat and human hepatocytes, and the maintenance of Cytochrome P450 (CYP) activity in human hepatocytes grown in the traditional Sandwich Model with that of cells cultured in the 3D RAFT^TM System. Our results show that the RAFT^TM 3D System represents a robust model for the long-term maintenance of liver-specific functions. View our recent work describing how to use hepatocytes and RAFT^TM System together to generate a long-term culture system.  

Add Physiological Fluid Flow to Liver Cultures Using Quasi Vivo^TM System

Another method shown to improve hepatocyte health and metabolic stability involves using fluidic flow systems, such as the Quasi Vivo^TM System, where cells are cultured as a monolayer in chambers with media continuously flowing over them. While fluidic flow systems may not be suitable for high-throughput needs, they can stabilize metabolic activity for more than 14 days. In addition, these systems allow for long-term culture of hepatocytes without the need for frequent media changes, improving the chances of detecting the bioavailability of a metabolite.

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.

Request additional information to learn how to incorporate more advanced models into your liver research.

Drug-drug interactions can have a significant impact on drug efficacy and patient safety. As such, regulators have made the prediction of drug-drug interactions a requisite in the development of new drug candidates, which has to be supplied as part of the submission of the registration dossier. In vitro identification and measurement of the contribution of major cytochrome P450 enzymes involved in human metabolism of a new drug candidate (CYP phenotyping), helps predict the impact of co-administered drugs on the pharmacokinetics of the new chemical entity.

Traditionally, these studies were conducted using one of three approaches – correlation analysis, antibody or chemical inhibition, and metabolism by recombinant human enzymes. However, each of these approaches has advantages and disadvantages, which is why a combination is recommended by the FDA and EMA to reliably identify the CYPs involved in the metabolism of a compound. For example, correlation analysis doesn’t provide any quantitative measurement of the contribution of each CYP to the metabolism of a drug, while models of recombinant CYP450 enzymes are not fully representative of the complete liver enzyme profile. Additionally, many chemical and antibody inhibitors actually lack sufficient specificity, resulting in little confidence in the results obtained.

To help you overcome these challenges, we provide Silensomes^TM HLM. Silensomes^TM are validated human-pooled liver microsomes (HLMs) that are chemically and irreversibly inactivated for one specific CYP using mechanism-based inhibitors. Silensomes^TM HLM provide you with a simplified, fully characterized pre-made solution that is ready for CYP phenotyping, resulting in more consistent and reliable results.

For more information on the use of Silensomes^TM HLM, including case studies, visit our product page.

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