Hepatocytes, the functional parenchymal cells of the liver, are essential for scientists studying absorption, distribution, metabolism, and excretion (ADME), as well as drug metabolism and pharmacokinetics (DMPK). These cells play a crucial role in detoxifying the body and are responsible for metabolizing the majority of xenobiotic compounds.
Image Credit: BioIVT
Understanding how these functions impact a given compound is essential for effective preclinical drug development.
Hepatocytes comprise approximately 80 % of the liver’s volume but only represent around 60 % of the total cell population. Due to their size and other physical properties, hepatocytes can be purified from other liver cell types.
Who uses hepatocytes?
Hepatocytes are widely used across the drug development field, particularly by screening and discovery groups, research and development teams, ADME/DMPK groups, cell and gene therapy teams, and academic researchers.
Their applications range from drug or cell and gene therapy safety and development to pathogen-host interactions and general studies in cell biology or biochemistry.
Advantages of hepatocytes over hepatic subcellular fractions
Primary hepatocytes are regarded as the gold standard for ADME and DMPK studies, as they are considered the most representative in vitro test system. Their enzymatic activities are particularly valuable for drug development, and because these cells remain viable, they maintain functional biochemical pathways that subcellular fractions cannot replicate.
Notably, hepatocytes offer a more comprehensive view because their enzymatic activities are not experimentally enriched, as is the case with microsomes, allowing scientists to better predict in vivo results. Regulatory agencies require hepatocytes in specific assays, such as induction and hepatotoxicity assays, which cannot be performed using subcellular fractions.
Even though hepatocytes are the gold standard for ADME/DMPK work, several factors must be considered when selecting the appropriate test system.
Hepatocytes can become limited from certain donors, are more expensive, and require more refined handling techniques than subcellular fractions. However, BioIVT maintains a steady source of donor tissue for hepatocyte isolation and offers training in the techniques needed for successful hepatocyte experiments, making their use strongly recommended when appropriate.
Selecting the right hepatocytes for a study
It is important to note that not all hepatocyte formats are appropriate for use in all assays. Specific hepatocyte products have inherent characteristics that make them better suited for certain assays.
The major differences between hepatocyte products are outlined here, as well as how these might be useful in a research setting.
Primary versus immortalized hepatocytes
At the most basic level, hepatocytes are classified as either primary or immortalized. The first decision to make is which type to use, so here is a quick comparison between the two.
Primary hepatocytes:
- Are isolated from living tissue.
- Are genetically unmodified.
- Are terminally differentiated.
- Do not proliferate in culture.
- They are representative of the donor from whom they have been isolated.
- Have a finite time in culture.
Immortalized hepatocytes:
- Have undergone spontaneous or purposeful genetic modifications resulting in uncoupled growth characteristics.
- Can resist senescence and cellular death.
- Proliferate in culture.
- Cannot fully replicate phenotypes found in primary hepatocytes (similar to differentiated stem cells).
Immortalized cells are limited in scope because these cells do not represent the full range of phenotypes found in liver tissue and primary hepatic cells.
Despite these limitations, immortalized cells are useful for assays such as lysosomal trapping and induction screening or in experiments focused on cell biology and biochemical pathways.
If you are looking for immortalized cells, there are several cell lines available. BioIVT offers the Fa2N-4 immortalized cell line.
Fresh versus cryopreserved primary hepatocytes
Most studies will benefit from the use of primary hepatocytes, either freshly isolated or cryopreserved. Both cell types are well described in academic literature and have been regarded as acceptable test systems for some time.
Fresh hepatocytes:
- Are never frozen for cryostorage.
- Must be used shortly after isolation.
- Are available in suspension or plated formats.
- They are considered acceptable test systems by regulatory agencies and the wider field.
Cryopreserved hepatocytes:
- Have been frozen to ensure stable enzymatic activity despite long-term storage.
- They can be used for multiple years, as required.
- Are available in a range of different formats.
- They are considered acceptable test systems by regulatory agencies and the wider field.
Freshly isolated cells can be used in suspension or plated format from either human or animal tissue, but experiments must begin almost immediately after the cells are isolated and delivered. Cryopreserved cells, however, offer researchers the flexibility to use them at their convenience.
In addition, cryopreserved hepatocytes allow researchers to select a specific lot based on characterized activities, which are often unknown when using freshly isolated cells.
These hepatocytes are extensively characterized, with lots providing valuable data such as post-thaw viability, yield, phase I and phase II activities, bile canaliculi formation, fold induction, spheroid formation, transporter function, optimal plating density, and micrographs of the cell monolayer.
However, to fully leverage the benefits of cryopreserved hepatocytes, labs need access to ultra-low temperature storage and tissue culture equipment for thawing and handling the cells.
Animal versus human primary hepatocytes
It is also important to consider the species from which hepatocytes are sourced. Using animal primary hepatocytes can be beneficial, as it allows for species comparisons with human test systems. This is also the case with animal subcellular fractions.
Several widely used small animal models, including Cynomolgus (Cyno) monkeys, Sprague-Dawley (SD) rats, CD-1 mice, minipigs, and beagle dogs, are available for comparison with human test systems. Species comparison studies help scientists choose the most suitable small animal models for in vivo research.
While animal hepatocytes are valuable and offer certain advantages over human cells, human hepatocytes remain the most relevant test system for drugs intended for human use. As such, studies such as CYP induction assays must be conducted using primary human hepatocytes.
Individual versus pooled donors
Individual donor hepatocytes are cells sourced from a single donor’s tissue while pooled hepatocytes are comprised of a mixture of different individual tissue donors’ cells in a single vial.
The number of donors in a pool can vary between products, typically ranging from 3 to 200. Pools are useful in experimental design as they minimize, or even eliminate, donor-dependent variability, making the data more reproducible across batches.
Suspension versus plated cultures
The final decision between using suspension or plateable hepatocytes depends on the assays you plan to perform and the specific endpoints you are targeting.
Suspension cultures are suitable for:
- Screening.
- Metabolic stability, metabolite identification, and reaction phenotyping.
- CYP/non-CYP inhibition.
- Uptake assays.
Plated cultures are useful for:
- CYP induction assays.
- Transporter assays, including uptake and efflux assays.
- Metabolic stability, metabolite identification, and reaction phenotyping.
- Inhibition assays.
- Investigating the metabolism of microsomal low turnover compounds.
- Host-pathogen interaction experiments.
- Three-dimensional or enhanced two-dimensional cultures.
- Hepatotoxicity.
- Gene therapy assays.
Suspension hepatocyte lots consist of hepatocytes cultured in suspension that generally do not attach to surfaces or form a confluent monolayer on collagen-coated plates. As they remain suspended in the media, their useful incubation time is limited to 2-4 hours, as their viability and activity decline over time due to anoikis.
In contrast, plateable hepatocyte lots consist of cells that attach to collagen-coated surfaces, flatten, and form a monolayer covering more than 70 % of the culture surface. These cells can be maintained in culture for at least five days. Here are some tips and tricks for working with plated hepatocytes.
Selecting the most appropriate primary hepatocytes
Researchers using cryoplateable hepatocytes should confirm that these hepatocytes form an appropriate monolayer and that this monolayer is sustained throughout the duration of the planned experiment.
Post-thaw viability is less critical for attaching hepatocytes, as only viable cells contribute to monolayer formation. It is also important to ensure that the selected lot can be plated in the intended well format, as not all lots plate efficiently in every format.
When selecting plateable hepatocytes for induction assays, it is advisable to review the data sheets and choose lots with an mRNA fold induction of 6-fold or greater. These should also demonstrate at least a 2-fold induction for specific CYP activity rates.
While hepatocytes with lower induction levels can be used, those inducing at least 6-fold will provide improved sensitivity and are less likely to miss induction.
Not all lots are suitable for every assay, so data sheets offer essential information to help researchers select the most appropriate hepatocyte lots for their experiments, such as the relevant CYP activity levels, yield, uptake, viability, induction, and monolayer formation capacity.
BioIVT’s catalog of hepatocytes and mediums
BioIVT offers a comprehensive portfolio of hepatic test systems, providing a wide range of primary or immortalized cells, cryopreserved or fresh, human or animal, from individual or pooled donors, and in plateable or suspension formats. Hepatocytes can even be plated for customers prior to delivery.
An extensive inventory of individual donor human cryoplateable hepatocytes is available, ideally suited for IND-enabling CYP induction studies. These cryoplateable lots can also be utilized in a diverse array of experimental designs across multiple research fields and drug modalities.
In addition to its extensive inventory, BioIVT offers individual donor human hepatocyte lots that have undergone further characterization and are qualified for extensive transporter activities (T-CERT). It also provides lots certified for spheroid formation (S-CERT).
Pooled hepatocyte products, comprising multiple human donors, are highly suited for use in IND-enabling drug metabolism studies. Using pooled products in these assays is advisable, as it reduces the risk of skewed metabolism data due to donor-dependent variability.
BioIVT offers LIVERPOOL® and CryostaX® — human pooled hepatocytes available in both cryoplateable and cryosuspension formats — all of which are suitable for these types of assays. BioIVT’s pooled hepatocytes are extremely versatile and can be custom-prepared to meet individual experimental needs.
In addition to its traditional hepatic test systems, BioIVT has developed advanced test systems designed to address specific requirements that standard systems may not easily accommodate, such as transporter-certified, spheroid-certified, and HEPATOPAC® long-term cultures.
HEPATOPAC® is a micropatterned, long-term co-culture system ideal for in vitro studies of compounds with low clearance or metabolism rates. It is also well-suited for investigating the effects of chronic or extended exposure to compounds.
Models leveraging HEPATOPAC® technology provide critical data that cannot be obtained from models using plated, suspension, or sandwich-culture hepatocytes. The micropatterned architecture of HEPATOPAC® cultures also enhances consistency and reproducibility compared to random hepatocyte co-cultures.
HEPATOMUNE® cultures offer researchers a highly functional in vitro liver platform ideal for modeling inflammation-mediated hepatic phenotypes.
Using a proprietary fabrication method, human hepatocyte 'islands' are cultured with stromal and Kupffer cells in a precise ratio, mimicking the liver’s physiological microenvironment. This configuration enables the study of innate immune responses to hepatic phenotypes and cytokine-mediated drug-induced liver injury (DILI).
HEPATOMUNE® cultures remain viable for at least 10 days, providing a robust in vitro model for studying hepatic inflammation and responses to various experimental conditions.
BioIVT offers a comprehensive range of hepatocyte test systems.
Human hepatocyte products
- Cryoplateable hepatocytes
- LIVERPOOL® pooled cryoplateable hepatocytes
- LIVERPOOL® pooled cryosuspension hepatocytes
- CryostaX® individual and pooled plateable hepatocytes
- CryostaX® individual and pooled hepatocytes for suspension
- TRANSPORTER CERTIFIED® hepatocytes
- SPHEROID CERTIFIED™ hepatocytes
- HEPATOPAC® long-term co-cultures
- HEPATOMUNE® long-term tri-cultures
- Cryosuspension hepatocytes
- Genotyped hepatocytes
- Fresh hepatocytes
Animal hepatocyte products
- Cryoplateable rat, mouse, monkey, dog, and minipig hepatocytes
- Cryosuspension rat, mouse, monkey, dog, guinea pig, rabbit, and minipig hepatocytes
- CryostaX® animal hepatocytes
- HEPTATOPAC® long-term cultures
- Fresh rat, mouse, monkey, and dog hepatocytes
BioIVT produces medium that has been specifically formulated to enable incubating, thawing, plating, spheroid formation, and maintenance of the company’s hepatocytes in culture.
INVITROGRO™ hepatocyte media is antibiotic-free, which extends its shelf life. The company's TORPEDO™ Antibiotic Mix is provided separately, allowing customers to add the antibiotic and supplement mix to INVITROGRO™ hepatocyte media as needed.
A variety of CryostaX® media is available to ensure optimal performance of hepatocytes in the CryostaX® format. Additionally, ACCULIVER kits are specifically designed for use with TRANSPORTER CERTIFIED® hepatocytes.
Human and animal tissue procurement
BioIVT’s extensive range of tissues is sourced from accredited and licensed providers, with full, verifiable consent given for their use in research. Human tissue is only accepted when it cannot be used to save lives, and all accepted tissues are tested negative for major animal or human pathogens.
BioIVT recommends using appropriate PPE when working with reagents. Official statements detailing how BioIVT obtains its tissues are available and can be provided to researchers to meet regulatory or ethical requirements, including those of academic journals.
Acknowledgments
Produced from materials originally authored by Dr. Chris Bohl from BioIVT.
About BioIVT
BioIVT, formerly BioreclamationIVT, is a leading global provider of high-quality biological specimens and value-added services. We specialize in control and disease state samples including human and animal tissues, cell products, blood and other biofluids. Our unmatched portfolio of clinical specimens directly supports precision medicine research and the effort to improve patient outcomes by coupling comprehensive clinical data with donor samples.
Our Research Services team works collaboratively with clients to provide in vitro hepatic modeling solutions. And as the world’s premier supplier of ADME-Tox model systems, including hepatocytes and subcellular fractions, BioIVT enables scientists to better understand the pharmacokinetics and drug metabolism of newly discovered compounds and the effects on disease processes. By combining our technical expertise, exceptional customer service, and unparalleled access to biological specimens, BioIVT serves the research community as a trusted partner in ELEVATING SCIENCE®.
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