Introduction to Use of Primary Liver Cells for In Vitro Cell Models
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.
Schematic of multiple liver functions
The liver has many physiological functions including cholesterol and amino acid synthesis as well as detoxification of various small molecules.
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.
Hepatocytes are isolated using a two-step collagenase perfusion protocol. The cellular morphology, structure, and functionality different for suspended and plated cells, and these differences are important for specific applications of hepatocytes.
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.
Application - Drug Metabolism and Drug-Drug Interaction Studies
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.
10-, 20-, and 50-donor single and mixed gender pools of hepatocytes and a proprietary prediction algorithm, DonorPlexTM Hepatocytes go beyond your expectations for a robust and consistent pooled donor hepatocyte product.
*Check individual CoAs to learn the number of donors pooled in each batch.
Applications - Drug and Chemical Toxicity Studies
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 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 stellateand 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.
The culture of human primary hepatocytes as spheroids supports
long-term cell viability and functionality. In this study, we compared
different spheroid culture systems and present optimized culture
Engineering Culture Platforms to Mimic Liver Diseases
Learn from Dr. Salman Khetani, University of Illinois at Chicago, how multiple liver cell-types including hepatocytes, Kupffer, stellate, and endothelial cells can be engineered together to model human liver disease phenotypes in culture.
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).
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).
Malaria parasite life cycle in hepatocytes
A human is infected with malaria when an infected female Anopheles mosquito bites and injects Plasmodium sporozoites into the bloodstream. The sporozoites quickly make their way to the liver, where they infect the hepatocytes. Over the next week or so, the sporozoites multiply asexually in the hepatocytes. No symptoms are apparent at this stage. After they are released from the liver, the merozoites invade the bloodstream where they infect the erythrocytes. Some merozoites replicate asexually in the erythrocytes and are released back into the bloodstream when the erythrocyte ruptures, causing the classical symptoms of malaria. However in other erythrocytes, instead of replicating asexually, the merozoites develop into gametocytes, the sexual form of Plasmodium. The gametocytes are picked up by a mosquito that bites an infected person and undergo a sexual reproduction cycle in the mosquito, producing more sporozoites. The cycle begins again when another human is bitten.
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.
Getting Started with Hepatocytes in 3D: Verified for SpheroidsTM Human Hepatocytes
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.
We now test each batch of our General Purpose and Interaction Qualified hepatocytes (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
Making Liver Tissue-like Structures Using RAFTTM System
The RAFTTM System allows for the creation of tissue-like structures. The 3D matrix of the type 1 collagen-based RAFTTM 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 RAFTTM System. Our results show that the RAFTTM 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 RAFTTM System together to generate a long-term culture system.
Add Physiological Fluid Flow to Liver Cultures Using Quasi VivoTM System
Another method shown to improve hepatocyte health and metabolic stability involves using fluidic flow systems, such as the Quasi VivoTM 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.
The culture of human primary hepatocytes as spheroids supports
long-term cell viability and functionality. In this study, we compared
different spheroid culture systems and present optimized culture
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Application - CYP Phenotyping
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 SilensomesTM HLM. SilensomesTM are validated human-pooled liver microsomes (HLMs) that are chemically and irreversibly inactivated for one specific CYP using mechanism-based inhibitors. SilensomesTM HLM provide you with a simplified, fully characterized pre-made solution that is ready for CYP phenotyping, resulting in more consistent and reliable results.
Dr. Stephen Ferguson, a leading researcher at the National Institute of Environmental Health Sciences (NIEHS), discusses how liver cells assembled in 3D spheroids produce highly differentiated functionality and utility for toxicology screening.