Human hepatic organoids provide a long-term, genetically stable, and well-characterized model system that can recapitulate key hepatic functions and pathophysiology of diseases, as well as provide functional readouts, including CYP3A4 activity, albumin secretion, and urea production. Below, we describe a protocol for assessing cytochrome P450 (CYP) enzymatic activity in human hepatic organoids using liquid chromatography-mass spectrometry (LC-MS) for drug testing.

Materials

• Biosafety cabinet
• Pipet-aid
• Pipettes and tips (P1000, P200, P20, P10, P2)
• For harvesting organoids:
  • 5 mL serological pipettes
  • 15 mL conical tubes
  • 1.5 mL tubes
  • Gentle Cell Dissociation Reagent (GCDR) (Catalog #100-0485)
• Tube racks
• Styrofoam box with ice
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• 37°C water bath
• 24-well plate of healthy hepatic organoid cultures in HepatiCult™ Organoid Growth Medium (Human) (Catalog #100-0385) or HepatiCult™ Organoid Differentiation Medium (Human) (Catalog #100-0383)
• Reagents for CYP reaction and mass spectrometry:
  • Acetonitrile
  • Substrates (e.g. midazolam, tolbutamide, dextromethorphan)
  • Metabolites for standard curve generation
  • CYP reaction buffer (e.g. Krebs-Henseleit Buffer, Hank’s Balanced Salt Solution, culture media)
  • Quench solution (acetonitrile + 0.1% formic acid)
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For information regarding the culture and passaging of human liver organoids using HepatiCult™, refer to the Technical Manual (Document #10000008300).

Protocol

(Optional) Harvest hepatic organoids from Matrigel® domes if required (Day 8 - 13 of differentiation):

1. Gently aspirate culture medium.
   Note: Multiple domes per sample may be required to yield an appropriate amount of material for mass spectrometry analysis from the harvesting step. It is recommended to harvest 6 domes of differentiated organoids in HepatiCult™ Organoid Differentiation Medium (ODM) from a 24-well plate.
2. Add 1 mL of room temperature GCDR to each well of a 24-well plate. You can use this addition to gently detach the domes off the plate.
3. Aspirate detached domes and GCDR with a P1000 pipette and transfer to a 15 mL conical tube.
4. Wash each well with 1 mL GCDR to collect any remaining organoids and add them to the tube.
5. Pipet up and down five times with a 5 mL serological pipet to disrupt the Matrigel® domes and release the organoids.
6. Incubate on ice for 5 minutes.
7. Centrifuge at 300 x g for 5 minutes.
8. Aspirate the supernatant.
   Note: A Matrigel® cushion may be observed on top of the pellet when pooling multiple domes. This should also be carefully aspirated.
9. Gently resuspend the organoids in 1 mL CYP reaction buffer by flicking the tube and swirling with a pipette tip.
   Note: CYP reactions can be performed in a buffer or culture medium.
10. Centrifuge at 300 x g for 5 minutes.
11. Aspirate the supernatant.
12. Resuspend the organoids in a final volume of 450 μL CYP reaction buffer and transfer to a 1.5 mL tube.
    Note: Proliferative organoids in HepatiCult™ Organoid Growth Medium (Human) (OGM) tend to fragment during harvest from Matrigel®. However, the fragments still display CYP activity in a donor-specific manner.

Cytochrome P450 Reaction:

1. Prepare CYP substrates at 1,000X final concentration in acetonitrile.
   Note: 1X substrate reaction concentration recommendations are listed in Table 1.
2. Dilute CYP substrates to 10X in the CYP reaction buffer.
3. Prepare a 1.5 mL tube with 450 μL CYP reaction buffer without organoids as a negative control.
4. Pre-warm organoids in CYP reaction buffer at 37°C for 5 minutes.
   Note: If using organoids without recovery from Matrigel® domes, carefully remove the culture medium and replace it with 450 μL CYP reaction buffer before returning to the incubator to pre-warm.
5. Add 50 μL of 10X CYP substrates to each organoid sample and negative control, and return to 37°C.
6. Collect 50 μL samples of supernatant at desired time points.
7. Immediately terminate the reactions by adding 50 μLof ice-cold quench solution.
   Note: If a different volume of supernatant is collected, add an equal volume of ice-cold quench solution.
8. Vortex samples vigorously.
9. Store samples in airtight vials at -80°C until analysis by LC-MS.

Table 1. Substrate Reaction Concentration Recommendations

LC-MS Sample Preparation:

1. Thaw samples on ice.
2. Centrifuge at ≥ 3,000 x g for 10 minutes at 4°C.
3. Transfer supernatant to mass spectrometry vial or sample plate.
4. Perform analysis.