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Which Hepatocytes Should I Use for What Studies?

A Guide to Hepatocyte Test System Selection for ADME Studies

What are hepatocytes and why are they important?

Hepatocytes are the functional parenchymal cell of the liver. They are important for ADME/DMPK scientists because they’re the cells that carry out the bulk of metabolism for xenobiotic compounds and play a major role in detoxifying the body. Understanding how these functions affect your compound is essential in preclinical drug development.

Hepatocytes make up about 80% of the liver’s volume, but only represent approximately 60% of the total cell population. This basically means that they’re big, and because of their size and other physical properties, we can take advantage of these characteristics to purify them away from other cell types in the liver.

Who Uses Hepatocytes?

A lot of different groups use hepatocytes. This is especially true in the field of drug development with screening/discovery groups, R&D, ADME/DMPK groups, and academics. Hepatocyte uses vary from drug development and safety to pathogen-host interactions to general cell biology/biochemistry.

Why use hepatocytes instead of hepatic subcellular fractions?

Primary hepatocytes are considered the gold standard for ADME/DMPK studies because they are the most representative in vitro test system. They’re the cells with the enzymatic activities that are most interesting and impactful with regards to drug development work. Because they’re still living, they contain functional biochemical pathways that cannot be replicated with subcellular fractions. The activities are also not experimentally enriched like they are with microsomes. This makes them more representative and offers a more comprehensive view for scientists to better predict what could happen in vivo. This is also why regulatory agencies require the use of hepatocytes in certain assays. For instance, induction assays can’t be done in subcellular fractions.

Even though hepatocytes are the gold standard for ADME/DMPK work, there are some things that you need to take into consideration for selecting the appropriate test system. Hepatocytes eventually become limiting from particular donors, cost more, and require more refined handling techniques than subcellular fractions. However, XenoTech has a steady source of donor tissue to isolate hepatocytes and we can train you in the techniques needed for successful hepatocyte experiments, so their use is strongly recommended when appropriate. Read more about when to use hepatocytes vs other test systems.

Selecting the Right Hepatocytes for Your Study

Which test system best suits my needs?

Not all hepatocyte formats are suited for all assays, so we made a flow chart to help you narrow down which test system works best for your experiments. This flow chart is not all-encompassing, but it’s a good start for narrowing down what format you may need:

Hepatocyte Selection Steps Chart

Primary vs Immortalized Hepatocytes

At the most basic level, hepatocytes can be differentiated between primary and immortalized hepatocytes. The first thing that needs to be decided is which to use, so here is a quick comparison between the two.

Primary hepatocytes:

  • Isolated from living tissue
  • Genetically unmodified
  • Terminally differentiated
  • Do not proliferate in culture
  • Are representative of the donor that they’re isolated from
  • Have a finite time in culture

Immortalized hepatocytes:

  • Have undergone purposeful or spontaneous genetic modifications that lead to uncoupled growth characteristics
  • Resist senescence and cellular death
  • Proliferate in culture
  • Can’t fully replicate the phenotypes that are found in primary hepatocytes, much like differentiated stem cells

Since immortalized cells don’t represent the complete array of phenotypes found in liver tissue and primary hepatic cells, they are limited in scope. However, that doesn’t mean that they aren’t useful for certain assays like lysosomal trapping, induction screening, or experiments that are focused on examining cell biology/biochemical pathways. If you need immortalized cells, there are various cell lines to choose from. XenoTech provides the Fa2N-4 cell line as well as cells from the JCRB cell bank.

Fresh vs Cryopreserved Primary Hepatocytes

Most of the time you’ll want to use primary hepatocytes, which are available as freshly isolated or cryopreserved cells. Both options have been accepted test systems for quite some time and are well described in literature.

Fresh hepatocytes:

  • Never frozen for cryostorage
  • Need to be used shortly after isolation from the tissue
  • Suspension or plated formats
  • Accepted test system by the field and regulatory agencies

Cryopreserved hepatocytes:

  • Carefully frozen to maintain stable enzymatic activity during long-term storage
  • Can be used at your discretion for multiple years
  • Available in various formats
  • Accepted test system by the field and regulatory agencies

Freshly isolated cells can be used in suspension or plated format from animal or human tissue, but your experiments have to be ready to go when the cells are isolated and delivered. If you choose cryopreserved cells, you have the convenience to use them whenever it’s best for you and your lab. Cryopreserved hepatocytes also allow you to choose a hepatocyte lot based on characterized activities that aren’t known at the time that the fresh cells are isolated. Cryopreserved hepatocytes are characterized for post-thaw viability, yield, phase I and phase II activities, uptake, fold induction and optimal plating density. We also provide micrographs of the monolayers and, in some cases, we characterize intrinsic clearance rates for select cytochrome P450 enzymes (CYPs). However, your lab will need access to ultra-low temperature storage and other common tissue culture lab equipment to thaw and use them. Watch our thawing demo video or download our protocol (available at the bottom of many of our hepatocyte and media pages) to learn more.

Animal vs Human Primary Hepatocytes

The next thing you need to consider is the species from which the hepatocytes are sourced. Animal primary hepatocytes are useful for the same reason animal subcellular fractions are useful: they allow for species comparisons with human test systems. For instance, Cynomolgus (Cyno) monkey, Sprague-Dawley (SD) rat, CD-1 mouse and beagle dog are all very common small animal models for comparison with human test systems. Species comparison studies help scientists choose the appropriate small animal models for in vivo work and generally have good availability.

Additionally, researchers sometimes use animal hepatocytes simply because they’re less expensive than human. However, while animal hepatocytes are very useful and have some advantages over human, human hepatocytes are still the most relevant test systems for drugs intended for human use, so certain studies like CYP induction assays are required to be carried out using primary human hepatocytes.

Individual vs Pooled Donors

This choice is really only for human hepatocytes, as all small animal model hepatocytes come standard as pools.