
By Chris Bohl, PhD, Senior Manager of Products Technical Support, BioIVT
Combing through journal articles to figure out the right materials for your assays is a time-consuming process, so BioIVT has compiled an overview for some of the major options with general guidelines that you can follow to assist your selection.
Hepatocytes
Primary hepatocytes are often referred to as the “gold standard” for in vitro hepatic ADMET (absorption, distribution, metabolism, excretion and toxicity) assays. The liver is one of the main sites where drugs are metabolized. Primary hepatocytes are a good model because, in context of biotransformation, most drug metabolizing enzymes are abundantly found in this particular cell type in the liver. The majority of hepatocyte-specific cellular function can be maintained in culture, which makes them the best model for replicating liver function in vitro, e.g. xenobiotic metabolism, hepatotoxicity, cellular and gene therapy, and host-pathogen interactions. In drug development, hepatocytes are best suited for metabolism, clearance, transport, hepatotoxicity, and drug-drug interaction assays, allowing them to serve as a cost-effective alternative to in vivo testing.
There are various test system formats that are best suited for each assay. Researchers looking for primary hepatocytes are offered many options including species, cryopreserved or fresh, plateable or suspension, and individual or pooled—so how to choose?
Cryopreserved vs fresh
Cryopreserved hepatocytes are the most common and easy to work with. The cryopreservation process enables long-term storage while conserving function, giving researchers access to the same lot to provide consistency in results from tests performed at different times. On the other hand, using fresh hepatocytes necessitates immediate use and no opportunity to use cells from the same isolation in later assays.
Upon thawing, cryopreserved hepatocytes are shown to maintain clinically significant activity levels; however, fresh hepatocytes typically have higher activities, as cryopreservation leads to some cell damage, and are generally more responsive in enzyme induction assays.
Plateable vs suspension
Cryoplateable hepatocytes are the closest alternative to fresh hepatocytes and are used most often in confluent monocultures with an optional overlay of extracellular matrix, which best replicates in vivo conditions. This format allows drug developers to study hepatobiliary drug transport, metabolism, drug-drug interactions, drug transport into and out of the cells, and hepatotoxicity.
Plateable hepatocytes can be used in multiple configurations: monoculture, co-culture, tri-culture, in either 2D or 3D formats. Monocultures are typically best suited for observing activity of enzymes abundant in hepatocytes, while co-cultures can be used to model hepatocyte interactions with other cell types. The addition of other cell types can be used to keep the cells viable and functional for longer or can be used to enhance drug toxicity screening.
BioIVT qualifies plateable hepatocytes by their ability to maintain confluent monolayer for at least five days (critical for cytochrome P450 [CYP] induction studies, metabolism and hepatotoxicity assays), and human lots can be further characterized for uptake and efflux transporter activities, CYP, and relevant non-CYP enzyme activities.
Hepatocytes in suspension are used primarily for compound stability, metabolite formation, extended incubations using the relay method, inhibition studies, uptake, and gene expression studies. Suspension lots are best suited for short-term studies (typically 2-4 hours) due to decreases in viability and metabolic activity over time.
Individual or pooled
You also have the option to select individual donors or pooled lots for both suspension and plateable formats. Single donor lots are necessary for some definitive preclinical studies, such as induction studies. While pooled donor hepatocytes can be used for induction screening, they aren’t typically used for definitive induction studies where data is used to apply for an IND.
Pooled lots provide consistency and wider representation due to larger sample size. Pooled hepatocytes can be used for the majority of the other assays that individual donor hepatocytes are used for with fewer concerns regarding inherent donor variability. Human hepatocyte pools can also be customized for the number of donors and phenotype criteria (race, age, gender, BMI, diabetic, alcohol and drug use, etc.).
Subcellular fractions
Subcellular fractions are widely used as a test system in early stages of drug development for metabolism and screening studies. Fractions can be obtained by successive centrifugation from any tissue and are very easy to work with because incubations require none of the time, extensive training, and resources of cell cultures. An additional benefit is their suitability for full automation, allowing data to be generated quickly. Subcellular fractions include matrices appropriate for use in metabolic stability, metabolite ID, enzyme inhibition, reaction phenotyping, evaluating species differences, and metabolite formation.
Microsomes
Human liver microsomes are the most widely used in vitro model for drug metabolism predictions, including interaction with major drug-metabolizing enzymes. Microsomes are subcellular fractions containing high specific activity of membrane-bound phase I metabolic enzymes. High concentrations of these enzymes, including CYPs and UGTs, make microsomes an ideal test system for species comparisons, metabolic stability, inhibition, and reaction phenotyping studies during discovery and lead optimization. Due to the small size of microsomes, a large donor pool can be used in an assay which can be instrumental to represent enzymatic activity levels expressed in the general population, and ease of use makes them ideal for high-throughput assays.
S9
S9 fractions contain both microsomal and cytosolic enzymes and are more representative of whole cells/tissue than microsomes. Cytosol contains some phase II enzymes not found in microsomes, which may be important to consider if your drug candidate is predicted to be metabolized by these enzymes.
Lysosomes
Purified lysosomes and tritosomes (lysosomes isolated from animals treated with Tyloxapol for improved separation of the organelle from mitochondria) are valuable tools for investigating compound stability of some drugs. Many of the most common enzymes involved in the degradation or catalysis of oligonucleotides, peptides, nanoparticles, antibodies, ADCs, and other biopharmaceuticals are contained in the lysosome. Studies using this test system are helpful in understanding metabolic stability of compounds that are trafficked through the cellular endocytic/lysosomal pathways.
Recombinant enzymes
Recombinant enzymes are the simplest system available to study enzyme interaction. Different isoforms of CYPs and other enzymes are engineered to enable researchers to distinguish a xenobiotic’s effect on activity of individual enzymes. Some enzymes, such as CYP3A4 and CYP3A5, are very similar in structure and are hard to distinguish in LC/MS analysis. Recombinant enzymes, therefore, are most often used to confirm any ambiguity observed in reaction phenotyping studies. In cases of low metabolism, recombinant enzymes can be used at high concentrations to amplify metabolic activity for use in metabolic stability screening and inhibitory potential evaluation.
Additional resources
Further informational resources on ADMET studies and research products, including tables rating test systems for different assays, information on selecting appropriate nonclinical species for toxicology studies, regulatory guidance on studies and test systems, and more are available on BioIVT.com.
About the author
Dr. Chris Bohl received his PhD from the School of Biological Sciences from the University of Nebraska – Lincoln and has been with XenoTech/BioIVT since 2014. XenoTech/BioIVT has been providing ADME research products and services for over 25 years, and Chris utilizes this expertise to provide valuable guidance to scientists seeking to choose the correct test system, troubleshoot difficulties, and much more.