Key words

1 Introduction

Metabolism is a key function of liver , which is carried out by drug-metabolizing enzymes, such as cytochromes P450. Over the years, several in vitro cell models have been developed to mimic liver function, but many lack drug-metabolizing enzyme activities, which is true, for example, with HepG2 cells [1]. Other models, such as primary human hepatocytes and HepaRG cells, express drug-metabolizing enzyme activities, but are expensive, scarce, or require higher levels of maintenance [2]. Therefore, a cost-effective, metabolically competent, and robust in vitro cell model is needed for medium to high-throughput screening purposes.

Human hepatoma HuH-7 cells were derived from a Japanese male with well differentiated hepatocellular carcinoma in 1982 [3]. Studies have shown that HuH-7 cells could be induced by DMSO and differentiate into primary human hepatocyte-like cells [4, 5]. The DMSO-treated HuH-7 cells resemble primary human hepatocyte characteristics, such as, polygonal shape and binucleated cells (Fig. 1). Furthermore, DMSO-treated HuH-7 cells express the functional CYP 3A4 enzyme, the most abundant liver enzyme which mediates the metabolism of more than 50 % of all marketed drugs [5]. Inhibition of CYP3A4 activity could cause drug–drug or food–drug interactions, which may lead to adverse effects [6].

Fig. 1
figure 1

Phase contrast photomicrographs of DMSO -treated HuH-7 cells with arrows pointing to binucleated cells . Bar represents 100 μm

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This chapter describes a protocol to establish DMSO-treated HuH-7 cells for measuring CYP3A4 inhibition and hepatotoxicity . A general timeline for developing DMSO-treated HuH-7 cell model is illustrated in Fig. 2. The cells are passaged for two or three times after thawing, then seeded in a flask for confluence and DMSO treatment. Finally, the cells are assayed for both reversible and time-dependent CYP3A4 inhibition, as well as tested for hepatotoxicity using control compounds.

Fig. 2
figure 2

Development of DMSO -treated HuH-7 cells. Timeline begins two to three passages after initial thawing of cells

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2 Materials

2.1 Equipment

  1. 1.

    BMG FLUOstar Omega multimode microplate reader.

  2. 2.

    New Brunswick Galaxy 48R CO2 incubator.

  3. 3.

    Inverted microscope.

  4. 4.

    Countess, automated cell counter; counting slides; Trypan blue stain 0.4 %.

  5. 5.

    96-well, black, clear flat bottom, tissue culture-treated polystyrene microplates, with lids, sterile.

  6. 6.

    96-well, white, opaque flat bottom, untreated polystyrene microplates, nonsterile.

  7. 7.

    Vacuum filter/storage bottle system, 0.22 μm pore, cellulose acetate membrane filter, sterile.

  8. 8.

    8-channel pipette and tips; multichannel reservoir.

2.2 Cells and Culture Medium

  1. 1.

    Human hepatoma HuH-7 cells, Health Science Research Resources Bank, Japan Health Sciences Foundation.

  2. 2.

    Dulbecco’s Minimal Essential Medium (DMEM), low glucose, GlutaMAX Supplement, pyruvate.

  3. 3.

    100× MEM nonessential amino acids and 1 M HEPES.

  4. 4.

    Fetal bovine serum, Atlanta Biologicals Premium Select.

2.3 Reagents and Solutions

  1. 1.

    Rat tail collagen type I.

  2. 2.

    Glacial acetic acid.

  3. 3.

    Dulbecco’s phosphate-buffered saline (DPBS, no calcium, no magnesium) and Versene (0.02 % EDTA).

  4. 4.

    Trypsin–EDTA (0.5 %), without phenol red.

  5. 5.

    Promega P450-Glo CYP3A4 (luciferin-IPA substrate) and CellTiter-Glo assays; beetle luciferin, potassium salt.

  6. 6.

    DMSO, ketoconazole , troleandomycin , salicylamide , nitrofurantoin , and nefazodone hydrochloride .

3 Methods

3.1 Cell Culture

  1. 1.

    Coat tissue culture surface using rat tail collagen type I (Table 1). Dissolve collagen in 0.02 N acetic acid to make a 100 μg/mL solution. Coat tissue culture surfaces at 10 μg/cm2. Incubate coated vessels at room temperature for at least 2 h and rinse with DPBS. Use fresh or air dry and store at 2–8 °C for up to 1 month.

    Table 1 Collagen coating
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  2. 2.

    Prepare basal medium (Table 2) and 1 % DMSO supplemented basal medium. Measure and mix all components under sterile conditions, followed by filter sterilization using a 0.22 μM filter. Medium is stable for 1 month if stored at 2–8 °C. 1 % DMSO supplemented basal medium is prepared fresh for medium change after cells reach confluence.

    Table 2 Basal medium
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  3. 3.

    Thaw and recover HuH-7 cells into T25 or T75 flasks using basal medium. Passage cells two or three times when reach 85–90 % confluence.

  4. 4.

    When desired cell amount is reached, remove the basal medium from flask using aspiration and rinse twice with Versene solution.

  5. 5.

    Add 0.025 % trypsin–EDTA reagent (prepared using 0.5 % trypsin–EDTA and DPBS) and rinse the flask quickly. Remove extra trypsin reagent and leave 1 mL in the flask. Incubate the flask (37 °C) until cell detachment (3–5 min).

  6. 6.

    Use a microscope to observe. When cells are completely detached, add 3–4 mL prewarmed (37 °C) basal medium to stop trypsinization. Failure to add medium promptly can result in over-trypsinization and significant cell death.

  7. 7.

    Determine the viable cell concentration (cells/mL) using a cell counter. Adjust cell concentration using basal medium as needed.

  8. 8.

    Seed HuH-7 cells at 6.3 × 104 cells/cm2 onto collagen coated T25 or T75 flasks using basal medium. Wait 10 min at room temperature before returning to the incubator. Replenish medium twice per week until 100 % confluence, which takes about 3–4 days after seeding.

  9. 9.

    Upon cell confluence, switch basal medium to 1 % DMSO supplemented basal medium (fresh prepared each time). Replenish medium twice per week for 2 weeks using 1 % DMSO supplemented basal medium.

  10. 10.

    After the 2-week DMSO treatment, seed DMSO-treated HuH-7 cells at 3.1 × 105 cells/cm2 onto a collagen coated clear bottom 96-well plate using basal medium. Use plated cells for assays within 48 h after seeding (see Note 1 ).

3.2 CYP3A4 Reversible Inhibition Assay

  1. 1.

    Reagent preparation.

    • Serum-free medium, prepare according to Table 2, leave out fetal bovine serum, and compensate volume with DMEM.

    • Luciferin-IPA substrate, thaw 3 mM stock solution, protect from light.

    • Salicylamide , Phase II conjugation inhibitor, prepare 3 M in DMSO fresh each time.

    • Beetle luciferin, prepare 2 mM stock in H2O, serial dilute using serum-free medium (0.2–80 nM, see Note 2 ).

    • Luciferin detection reagent, equilibrate to room temperature.

    • Ketoconazole , prepare 2 mM stock in DMSO and conduct serial dilution using DMSO (0.01–2 mM).

    • Dissolve luciferin-IPA substrate (3 μM), salicylamine (3 mM) and various concentrations of ketoconazole (0.01–2 μM) using serum-free medium (see Note 3 ) and prewarm to 37 °C. Preparation without ketoconazole serves as the control.

  2. 2.

    CYP3A4 reversible inhibition.

    Wash DMSO-treated HuH-7 cells on 96-well plates with serum-free medium and incubate with ketoconazole (0.01–2 μM) simultaneously with CYP3A4 substrate luciferin-IPA (3 μM, Km value) at 37 °C (100 μL/well) for 30 min. Include beetle luciferin standards (100 μL) in blank wells.

  3. 3.

    Assay detection.

    Terminate reaction by transferring 50 μL of incubation medium from each well to a separate white flat bottom plate with wells containing 50 μL of luciferin detection reagent at room temperature. Mix well and incubate the plate for 20 min, then measure luminescence using a plate reader (Fig. 3a).

    Fig. 3
    figure 3

    (a) CYP3A4 r eversible inhibition by ketoconazole and (b) time-dependent inhibition by troleandomycin in DMSO-t reated HuH-7 cells. Figure reproduced from reference [5]

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  4. 4.

    Calculate IC 50, the half-maximal inhibitory concentration of ketoconazole determined using a nonlinear regression Hill model.

3.3 CYP3A4 Time-Dependent Inhibition Assay

  1. 1.

    Reagent preparation.

    • Serum-free medium, prepare according to Table 2, leave out fetal bovine serum, and compensate volume with DMEM.

    • Luciferin-IPA substrate, thaw 3 mM stock solution, protect from light.

    • Salicylamide , Phase II conjugation inhibitor, prepare 3 M in DMSO fresh each time.

    • Beetle luciferin, prepare 2 mM stock in H2O, serial dilute using serum-free medium (0.2–80 nM, see Note 2 ).

    • Luciferin detection reagent, equilibrate to room temperature.

    • Troleandomycin , prepare 10 mM in DMSO and conduct serial dilution using DMSO (0.2–10 mM). Dissolve each concentration using serum-free medium (5–100 μM) and prewarm to 37 °C. Incubation without troleandomycin serves as the control.

    • Dissolve luciferin-IPA substrate (3 μM) and salicylamide (3 mM) using serum-free medium and prewarm to 37 °C.

  2. 2.

    CYP3A4 time-dependent inhibition: preincubation.

    Wash DMSO-treated HuH-7 cells on 96-well plates with serum-free medium and incubate with troleandomycin (5–100 μM, 100 μL/well) for 0, 15, 30, and 60 min at 37 °C.

  3. 3.

    CYP3A4 time-dependent inhibition: substrate incubation.

    At different time points, wash cells with serum-free medium and incubate with CYP3A4 substrate luciferin-IPA (3 μM, Km value) at 37 °C (100 μL/well) for an additional 30 min. Include beetle luciferin standards (100 μL) in blank wells.

  4. 4.

    Assay detection.

    Terminate reaction by transferring 50 μL of incubation medium from each well to a separate white flat bottom plate with wells containing 50 μL of luciferin detection reagent at room temperature. Mix well and incubate the plate for 20 min, then measure luminescence using a plate reader (Fig. 3b).

  5. 5.

    Calculate Parameters.

    Obtain inactivation rate constant k obs by plotting the natural logarithm of the remaining CYP3A4 activity (%) against preincubation time with troleandomycin [I].

    Calculate kinetic parameters k inact and K I by fitting data to the following equation using a nonlinear regression,

    $$ {k}_{\mathrm{obs}}=\frac{k_{\mathrm{inact}}\left[\mathrm{I}\right]}{K_{\mathrm{I}}+\left[\mathrm{I}\right]} $$

3.4 Hepatotoxicity Assay

  1. 1.

    Reagent preparation.

    Nitrofurantoin and nefazodone, prepare 100 mM in DMSO, protect from light. Conduct serial dilution using basal medium (1–100 μM) on treatment day.

    CellTiter-Glo assay, reconstitute and equilibrate at room temperature on assay day (24 h after treatment day).

  2. 2.

    Treatment.

    Incubate DMSO-treated HuH-7 cells with nitrofurantoin or nefazodone (100 μL/well) for 24 h at 37 °C. Incubation without chemicals serves as the control.

  3. 3.

    Toxicity assay.

    After treatment, equilibrate HuH-7 cells at room temperature for 30 min. Add 100 μL of CellTiter-Glo reagent to each well and mix well. Incubate the plate for additional 10 min, then measure luminescence using a plate reader (Fig. 4).

    Fig. 4
    figure 4

    Cytotoxicity of nitrofurantoin and nefazodone (24 h) in DMSO-t reated HuH-7 cells. Figure reproduced from reference [5]

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  4. 4.

    Calculate EC 50, the half-maximal cytotoxic concentration of hepatotoxicants determined using a nonlinear regression Hill model.

4 Notes

  1. 1.

    DMSO-treated HuH-7 cells maintain CYP3A4 activity in basal medium for at least 48 h after plating.

  2. 2.

    Add 100 μL/well of beetle luciferin standards (0.2–80 nM) to blank wells on the 96-well plate, proceed together with samples for inhibition studies and luminescence detection. When calculate enzyme activity, use beetle luciferin standard curve range 0.1–40 nM.

  3. 3.

    Prepare luciferin-IPA and salicylamide in serum-free medium, then dissolve various concentrations of ketoconazole using a multichannel reservoir.