Background
Human hepatocytes have become the gold standard for predicting human CYP450 induction in vitro due to inter-species differences (2-6). When freshly isolated human hepatocytes are overlaid with an extracellular matrix component, such as Geltrex™ Reduced Growth Factor Basement Membrane Matrix or Collagen I, Rat Tail, the cells retain the ability to respond to prototypical CYP450 inducers, which reflects what would be observed in vivo in regards to enzyme specificity and potency of enzyme induction (7, 8). In addition, primary cultures of human hepatocytes have the distinct advantage of exhibiting species-specific induction of CYP450 isoforms.
Master regulators of CYP450 induction such as the aryl hydrocarbon receptor (AhR), and the nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR), have been recognized as key mediators of drug-induced changes in the expression of CYP1A, CYP3A, CYP2B and the CYP2C enzymes, as well as of phase II enzymes and transporters (2, 8, 9). Therefore, in vitro induction study designs using selective positive control inducers provide a useful tool to characterize both the induction of CYP450s and the potential receptor pathways that may be involved.
This in situ induction protocol outlines the recommended approaches for using cultures of primary human hepatocytes derived from cryopreserved preparations to assess the potential of test compounds to induce pharmacologically important pathways.
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.
- Induction studies typically assess each inducer, test compound and negative control in triplicate. We suggest using 24-well collagen-coated plates and mapping out your wells in advance for the proper amount of cryopreserved hepatocytes and reagents.
- Consult Tech Support (contact info below) if you are adapting this protocol for use with animal hepatocytes.
- Use universal safety precautions and appropriate biosafety cabinet when handing primary hepatocytes.
Critical materials and reagents
- Cryopreserved plateable hepatocytes prequalified for induction, such as Life Technologies Cat. No. HMCPIS or HMCPIL, enough for ~10 x 106 cells per plate.
- The reagents in the plating protocol, including Geltrex™ Reduced Growth Factor Basement Membrane Matrix, which is 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
- Hank’s Balanced Salt Solution (HBSS)
- Lysis Buffer for RNA isolations
- 50-mL conical tubes
- RNAse-free microcentrifuge tubes
- CYP substrates, such as phenacetin, bupropion and testosterone
- Compound stocks: test articles (TA), vehicle control (e.g. DMSO) and positive controls (PC). Suitable positive controls may include:
- 3-methylcholanthrene (3-MC, a prototypical CYP1A enzyme inducer)
- phenobarbital (PB, a prototypical CYP2B inducer)
- rifampicin (RIF, a prototypical CYP3A enzyme inducer)
Note: Omeprazole may be used as a less selective inducer of CYP1A and CITCO may be used as a selective inducer of CYP2B6
Equipment
- 37°C water bath
- 37°C / 5% CO2 humidified incubator
- Orbital shaker placed inside incubator
- Plates/deep-well blocks for in situ activity harvest
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)