The pharmaceutical and biotechnology industry’s goal is to discover therapeutic agents that are both safe and effective at treating or preventing diseases. Compounds identified as selective and potent in the early drug discovery phase are progressed to preclinical drug development for further evaluation. It is estimated that over 10% of drugs fail in clinical trials due to pharmacokinetic reasons (1), and the US Food and Drug Administration (FDA) guidelines emphasize the identification of metabolic pathways, relevant metabolites and potential drug-drug interactions for new chemical entities (NCEs) where metabolism is the principal route of elimination (2) Consequently, drug metabolism studies are a critical constituent of any drug development program.

The primary site of metabolism for many drugs is the liver. Liver-derived systems such as liver slices, sub-cellular liver fractions, and intact hepatocytes are typically utilized to assess the metabolism of NCEs. Intact hepatocytes contain the cytochrome P450’s (CYPs), other non-P450 enzymes, and phase II enzymes such as sulfo- and glucuronosyltransferases, and thus represent a prime model system for studying drug disposition in vitro (3). NCEs can be screened and rank-ordered according to metabolic half-life estimates or in vitro intrinsic clearance values (Cl int, in vitro) obtained from metabolic stability studies. Moreover, metabolic screening assays enable drug developers to focus on the improvement of compounds through structural activity relationships (SAR) and prevent the progression of labile compounds to more costly in vivo studies. Given that cryopreserved hepatocytes retain enzymatic activities similar to those of fresh hepatocytes and offer convenience to the end user (4), the utility of cryopreserved hepatocytes in efforts to define a drug’s disposition in vitro is advantageous as compared to other model systems.

Important notes

  • Review this protocol to ensure y ou have all the necessary reagents and equipment prior to starting the procedure. Once thawed, cryopreserved hepatocytes must be used immediately and will not maintain viability if refrozen.
  • Use universal safety precautions and appropriate biosafety cabinet when handing primary hepatocytes.


Critical materials and reagents

  • Cryopreserved hepatocytes for suspension use, such as Life Technologies Cat. No. HMCS1S or HMCS2S (check website for additional catalog numbers), enough for 6 x 106 cells per plate
  • Williams’ Medium E, 500 mL, Life Technologies Cat. No. CM6000
  • Hepatocyte Maintenance Supplement Pack (Serum-free), 1 kit, Life Technologies Cat. No. CM4000
  • 12-well non-coated plates (Greiner Bio-One, Cat. No. 665 180 or equivalent).
  • 15-mL conical tubes (1 per compound)
  • Compound stocks: test articles (TA) and positive controls (PC). Suitable positive controls may include:
    • midazolam
    • phenacetin
    • testosterone
    • dextromethorphan
    • (S)-mephenytoin
    •  7-hydroxycoumarin
  • Stop solution


  • 37°C water bath
  • 37°C / 5% CO2 humidified incubator
  • Orbital shaker placed inside incubator




Advanced Preparation

  • Prepare Incubation Medium by combining Hepatocyte Maintenance Supplement Pack (Serum-free) with Williams Medium E per kit instructions, and warm to 37°C in a water bath. At least 5 mL of Incubation Medium will be needed per test article and control.
  • Prepare compound stocks: test articles (TA) and positive control(s) (PC) dissolved in an organic solvent such as methanol or DMSO to desired concentration, such as 1 mM.
  • Prepare hepatocytes immediately prior to assay, diluted to 1 x 106 viable cells/mL in Williams’ Medium E supplemented with Hepatocyte Maintenance
  • Supplement Pack (serum-free). Useful references:


  1. In separate conical tubes, add the test compounds and positive control(s) to warm Incubation Medium to yield the desired working concentration(s). For example, prepare 2 μM by adding 10 μL of 1 mM test article stock solution to 5 mL of Incubation Medium. Note: if DMSO is used as a solvent, the concentration should not exceed 0.1%, with a maximum of 1% in the final Incubation Medium.

  2. Pipette 0.5 mL of Incubation Medium containing the test article or positive control into respective wells of a 12-well non-coated plate. See Figure 1. Note: Final substrate concentration will be 1 μM once step 6 is complete.

  3. Place the plate in the incubator on an orbital shaker to allow the substrates to warm for approximately 5-10 min prior to initiation of the reactions.

  4. For the negative control, boil 1.0 x 106 viable hepatocytes/mL for 5 min to eliminate enzymatic activity. Use enough volume to cover the number of negative controls desired.

  5. Remove the 12-well non-coated plate containing the substrates from the incubator.

  6. Start reactions by adding 0.5 mL of 1.0 x 106 viable cells/mL in each appropriate well of the plate to yield a final cell density of 0.5 x 106 viable cells/mL. Pipette 0.5 mL of the inactivated hepatocytes into the negative control wells.

  7. Return the plate to the orbital shaker in the incubator and adjust the shaker speed to 90-120 rpms.

  8. Remove well contents in 50 μL aliquots at time points 0, 15, 30, 60, 90 and 120 min.

    • Additional time points 180 min and 240 min may be included, but should not be necessary for healthy and metabolically competent hepatocytes to detect high turnover compounds.
    • In abbreviated screening assays where only two time points are used to assess NCE metabolic stability, it is strongly advised that a 0 time point and a secondary time point up to 120 min is selected.

  9. Stop the incubations by addition of sample aliquots (e.g. 50 μL) to tubes containing the appropriate quenching solvent and either freeze at -70°C, or directly extract as per analytical methods.

    Table 1 — Incubation conditions.

    Working substrate concentration Working cell density Amount of working substrate per well Amount of working cells to add Final substrate concentration Final cell density Incubation volume Amount of quenching solvent
    2 μM 1.0 x 106 cells/mL 0.5 mL 0.5 mL 1 μM 0.5 x 106 cells/mL 1.0 mL 50 μL

    Figure 1 — Example of suspension metabolism study design using a 12-well format.

  10. Determine the in vitro half-life (t1/2) of the parent compound by regression analysis of the percent parent disappearance vs. time curve.

  11. Intrinsic clearance in vitro (Clint in vitro) can be calculated according to the equation: Clint in vitro = kV/N, where k = 0.693/t1/2, V = incubation volume (1 mL) and N = number of hepatocytes per well (0.5 x 106 viable cells).

  12. Clint in vitro may be scaled to in vivo predictions according to Obach (5) and McGinnity (4).

Technical support

For questions related to this protocol, contact us at:
Phone: +1 919 237 4500 (Toll)
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  1. Kola I, Landis J (2004) Can the pharmaceutical industry reduce attrition rates? Nat Rev Drug Discov 3: 711–715.

  2. US FDA (2006) Draft Guidance for Industry: Drug Interaction Studies—Study Design, Data Analysis, and Implications for Dosing and Labeling, US Food and Drug Administration Publication.

  3. LeCluyse EL, Alexandre E, Hamilton GA et al. (2004) Isolation and culture of primary human hepatocytes. Methods Mol Bio 290:207–230.

  4. McGinnity DF, Soars MG, Urbanowicz RA, Riley RJ (2004) Evaluation of fresh and cryopreserved hepatocytes as in vitro metabolism tools for the prediction of metabolic clearance. Drug Metab Dispos 32:1247–1253.

  5. Obach RS (1997) Non-specific binding to microsomes: impact on scale-up of in vitro intrinsic clearance to hepatic clearance as assessed through examination of Warfarin, imipramine and propranolol. Drug Metab Dispos 25:1359–1369.