In vitro Assessment of Induction Potential in Plated Cultures of Cryopreserved Hepatocytes

• 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

1. Prepare plated hepatocytes with  a Geltrex™ matrix overlay following these protocols:
   
   
   • Protocol for thawing and use of plateable and suspension cryopreserved hepatocyte
   • Protocol for counting primary hepatocytes using trypan blue exclusion analysis
     
     
2. The induction protocol can be started 2 hr after overlay completion, or up to 24 hr later

Reagent Preparation

1. 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 (usually 15–20 min).


2. Prepare working concentrations of the inducers as follows:
   
   
   • 2 mM 3-methylcholanthrene (3-MC) in DMSO
   • 200 mM phenobarbital (PB) in DH₂O
   • 10 mM rifampicin (RIF) in DMSO
     
     


3. Prepare the test article (TA) in solvent, such as methanol or DMSO, in multiple concentrations to allow production of a dose-response curve if desired, but minimally 3 concentrations of test article should be assessed spanning the in vivo or predicted C_max exposure levels, such as 0.1X, 1X, and 10X C_max.


4. Prepare working concentrations of the probe substrates as follows:
   
   • 100 mM phenacetin in DMSO
   • 500 mM bupropion in methanol
   • 200 mM testosterone in methanol
   
   Induction Procedure
   


5. In separate conical tubes, dissolve specified amount of inducers into 20 mL, 37°C prepared Incubation Medium (final concentrations are bolded):
   
   
   • 20 μL of 2 mM 3-methylcholanthrene (3-MC)           2 μM
   • 100 μL of 200 mM phenobarbital (PB)                1000 μM
   • 20 μL of 10 mM rifampicin (RIF)                             10 μM
   
   
6. Add 20 µL DMSO to the 1,000 µM PB conical tube so that the percentage of DMSO is equivalent among the treatment groups
   
   
7. Prepare a negative control by adding 20 µL DMSO into 20 mL warm incubation medium.



8. In separate conical tubes, dissolve the test articles in warm incubation medium to yield final inducing concentrations 1,000-fold lower than the respective working concentrations.  For example, 20 µL of a 10 mM test article into 20 mL of incubation medium yields a final concentration of 10 µM.
   
   Note: If test articles are dissolved in a solvent other than DMSO, a negative control with an equivalent organic percentage must be included in the study design for fair comparison.
   
   


9. Remove the hepatocyte plate(s) from the incubator and replace the medium in the appropriate wells with 0.5 mL of the negative control, inducers, or test article solutions, preferably each in triplicate.
   
   Important: Because hepatocytes are susceptible to drying out, aspirate each treatment group separately and replace with the experimental medium before dosing the next set.
   
   
10. Return plate(s) to the incubator for 24 hr.



11. After 24 hr, replace the medium with freshly prepared inducing solutions, repeating steps 5 through 8. Fresh solutions should be prepared daily and applied every 24 hr during the duration of the experiment, which is typically 48 - 72 hours prior to harvest.
    
    Day of Harvest
    


12. On the day of harvest, warm the incubation medium to 37°C, as usual.



13. Remove the induction plate(s) from the incubator.



14. Aspirate the dosing medium from each well, wash and replace with warm incubation medium.  Take precautions to avoid the hepatocytes drying out, as previously mentioned.



15. In separate conical tubes, dissolve 20 µL probe substrates in 20 mL warm incubation medium to yield final incubation concentrations 1,000-fold lower than working concentrations.



16. Remove the wash medium from the hepatocytes and replace with 0.5 mL of the probe substrate solutions prepared in the previous step.  Incubations should be conducted in triplicate with the respective CYP450 treatment group.



17. Incubate on an orbital shaker in the incubator for the time specified in Table 1.  A speed of 120–150 rpm is recommended for a 24-well plate.



18. Stop the incubations at the appropriate time points by collecting and freezing the sample medium, or by removing the sample medium and replacing it with an organic stop solution.  Samples can be stored at -70°C before analysis.



19. If collecting samples for mRNA analysis, see steps 25–27.
    

    Table 1:  Assay conditions for the determination of enzyme activities in plated cryopreserved human hepatocytes
    

    Enzyme	Prototypical inducer	Substrate	Concentration	Incubation time	Marker metabolite
    CYP1A2	3-methylcholanthrene	phenacetin	100 µM	15 min	acetaminophen
    CYP2B6	phenobarbital	bupropion	500 µM	20 min	hydroxybupropion
    CYP3A4/5	rifampicin	testosterone	200 µM	14 min	6β-hydroxytestosterone

    
    Catalytic Activity Determination
    


20. Extract and analyze samples by method of choice, typically HPLC or LC-MS/MS by monitoring the formation of metabolite.



21. CYP activity can be expressed as pmol/min/million cells where pmol is defined as the amount of metabolite formed during the reaction.  The number of hepatocytes per well (N) is related to the seeding density as defined in Table 2.



22. If optional RNA isolations are not performed, cell plates can be sealed and frozen directly for subsequent analyses.
    
    Note: if performing protein determinations to normalize data across wells, keep in mind that the protein is not solely from hepatocytes, due to the presence of detectable protein in the extracellular matrix used to overlay the hepatocyte monolayers.
    


23. The fold-induction enzyme activity is determined by the ratio:
    
    CYP activity(_induced) / CYP activity(_vehicle)



24. The percent adjusted positive control is determined by:
    
    ([CYP activity(_induced) -- CYP activity(_vehicle)]  /   [CYP activity(_{positive control})  -- CYP activity(_vehicle]) x 100


Table 2:  Parameters for human hepatocytes used in catalytic rate calculations. The assumption for N is that all hepatocytes adhered to the well with respect to seeding density.



Plate format	Seeding density (106 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	0.25	0.18/0.20/0.22
96-well	Either 0.7/0.8/0.9	0.125	0.09/0.10/0.11


Sample Collection for mRNA Analysis



25. If mRNA analyses are desired; upon removal of the probe substrates, wash hepatocytes twice with Hank’s Balanced Salt Solution (HBSS).



26. Remove the HBSS from the wells and replace with an appropriate volume of lysis buffer.  For most commercial methodologies, this is 300 µL per well for a 24-well plate.



27. Collect the cell lysates in RNAse-free microcentrifuge tubes, or seal and freeze the entire plate at -70°C for subsequent isolations within 4 weeks.
TOP

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)

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