New chemical entities (NCEs) with relatively low turnovers have presented a unique challenge in the acquisition of reliable data from short-term (≤ 1-4 hours) metabolism studies conducted in liver microsomes, S9 fractions and suspension cultures of primary hepatocytes. An alternative model that allows for the determination of intrinsic clearance values would be advantageous. Recently, monolayer cultures have been proposed as an effective model system to model the metabolism of low-turnover compounds (1). Because plated cryopreserved human hepatocytes allow for longer duration incubation times (>8 hr), as compared to hepatocyte suspensions, this system allows for more accurate determinations of intrinsic clearance (Clint) values. Inclusion of positive controls recognized as low-turnover compounds is recommended in the study.

Important notes

  • Review this protocol, as well as the protocol, Thawing and Use of Plateable and Suspension Cryopreserved Hepatocytes, to ensure you 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 plated use, such as Life Technologies Cat. No. HMCPMS or HMCPML, enough for ~10 x 106 cells per plate.
  • The reagents in the plating protocol, with the exception of the Geltrex™ overlay, which is not required for this assay.
  • Williams’ Medium E, 500 mL, Life Technologies Cat. No. CM6000
  • Hepatocyte Maintenance Supplement Pack (Serum-free), 1 kit, Life Technologies Cat. No. CM4000
  • 50-mL conical tubes
  • Compound stocks: test articles (TA) and positive controls (PC). Suitable positive controls may include:
    • diazepam
    • imipramine
    • tolbutamide
    • verapamil
  • Stop solution


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


Advance preparation

  • Prepare plated hepatocytes.  Note that a Geltrex™ overlay is not required for this assay.  Incubate the cells for 4–6 hr at 37°C to allow complete attachment before replacing with serum-free incubation medium. Useful references:
  • 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.
  • After hepatocytes have attached, remove the serum-containing plating medium and replace with serum-free incubation medium to wash the hepatocytes.  Return the plates to the humidified incubator. Incubations can be performed immediately after attachment or on the following day.


  1. 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.

  2. In separate conical tubes, add test compounds and positive control(s) to warm incubation medium to yield the desired working concentration(s). For example, prepare 1 µM by adding 20 µL of 1 mM test article stock solution to 20 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.
  3. Remove the wash medium from the hepatocytes and replace with an appropriate volume of incubation medium containing the TA or positive control for the appropriate time point.  Incubate on an orbital shaker for 0, 1, 2, 4, 6, and 8 hr at 80–120 rpm for a 24-well plate.  See diagram below (Figure 1) for plate diagram. Some low-turnover compounds typically require up to 24 hr incubation periods. See diagram below (Figure 2) for an example of an experiment designed in 24-well plate format.

  4. Stop the incubations by addition of the appropriate quenching solvent. Mix briefly and collect samples and either freeze at -70°C, or directly extract as per analytical methods. If plate is to be returned to incubator, aspirate remaining quenching solvent.

  5. Analysis

  6. Analyze parent disappearance (and metabolite formation as desired) by analytical method(s) of choice, typically HPLC or LC-MS/MS.

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

  8. 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 and N = number of hepatocytes per well (Table 1).

  9. Clint in vitro may be scaled to in vivo predictions according to Obach (3) and McGinnity (2).

Figure 1. Example of plated metabolism set-up in a 24-well format up to an eight hour time-point. Numbers in the circles reflect the incubation time (hours) of the solutions in the well.

Figure 2. Example of plated metabolism set-up in a 24-well format up to a 24 hour time-point. Numbers in the circles reflect the incubation time (hours) of the solutions in the well.
Table 1:  Parameters used in intrinsic clearance in vitro (Cl int,in vitro) calculations for human hepatocytes*

The assumption for N is that all hepatocytes adhered to the well with respect to seeding density.
Plate format Seeding density
(106 total cells/mL)
Incubation volume
V (mL)
Hepatocytes per well
N (106 cells)
12-well Either 0.7 / 0.8 / 0.9 1.0 0.7 / 0.8 / 0.9
24-well Either 0.7/0.8/0.9 0.5 0.35/0.4/0.45
48-well Either 0.7/0.8/0.9 0.2 0.14/0.16/0.18
96-well Either 0.7/0.8/0.9 0.1 0.07/0.08/0.09

*Check with your Invitrogen specialist if using animal hepatocytes.  Seeding densities for mouse and monkey are 0.3 to 0.5 x 10 6 and 0.9 to 1.1 x 10 6 total cells/mL, respectively.

Technical support

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  1. Griffin SJ, Houston JB (2005) Prediction of in vitro intrinsic clearance from hepatocytes: comparison of suspensions and monolayer cultures. Drug Metab Dispos 33:115–120.

  2. 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.

  3. 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.
LT134        rev 2-Mar-2011