In vitro Assessment of Metabolic Stability in Suspension Cryopreserved Hepatocytes

• 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

Equipment

• 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:

• Protocol for Thawing and Use of Plateable and Suspension Cryopreserved Hepatocytes
• Protocol for Counting Primary Hepatocytes using Trypan Blue Exclusion Analysis
• www.lifetechnologies.com/hepatocytes

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 (t_1/2) of the parent compound by regression analysis of the percent parent disappearance vs. time curve.


11. Intrinsic clearance in vitro (Cl_{int in vitro}) can be calculated according to the equation: Cl_{int 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. Cl_{int in vitro} may be scaled to in vivo predictions according to Obach (5) and McGinnity (4).

For questions related to this protocol, contact us at:
Email: hepaticproducts@lifetech.com
Phone: +1 919 237 4500 (Toll)
Phone: +1 866 952 3559 (U.S. Toll-free)

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.