Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

NOTE: The general workflow for the metabolic activity assay is outlined in Figure 2. Each step of this workflow is subsequently detailed in the next paragraphs. Detailed tables of reagents and equipment are given in the Table of Materials.

Figure 2: Workflow for the Metabolic Probe Assay. The cells are seeded and grown to adequate density. A CYP induction step might be required depending on the enzyme activity assayed. The probe substrate and inhibitor is added to the cell culture. After an incubation period, the medium is collected for analysis and the cells are lysed for protein quantification. The medium is processed for analysis of the metabolic product of the probe substrate by mass spectrometry or luminometry. Please click here to view a larger version of this figure.

1. Cell Culture

Figure 3: Light Transmission Micrography of a Hepatic Cell Line Culture. The cell line described in this protocol⁵ typically differentiate with a 50% split between cholangiocytes and hepatocytes in culture (100x). Scale bar = 100 μm. Please click here to view a larger version of this figure.

1. Hepatic cell line culture
   NOTE: A commercially available hepatic cell line was used as an example of metabolically competent cells. The cell line was derived from a hepatocarcinoma caused by viral infection⁷. At confluency, this cell line differentiates into cholangiocyte- and hepatocyte-like cells (Figure 3) and further details can be found in Guillouzo⁷. The inclusion of 2% DMSO to the culture medium supports the differentiation of this cell line and expression of a variety of CYPs.
   1. Remove the cryogenic vials with cells from the liquid nitrogen and place on dry ice for transport. Transfer the cryovial to a water bath pre-warmed to 37 °C for 1-2 min, and sterilize the outside of the vial with 70% ethanol before placing in a laminar flow hood.
   2. Whilst working under sterile conditions, carefully open the cryogenic vial to release any excess pressure and pipette the contents of the cryogenic vial into a 10 mL tube filled with 9 mL of pre-warmed Williams' E medium at 37 °C.
   3. Centrifuge the solution for 10 min at 400 x g at room temperature, discard the supernatant, and resuspend the cells in 5 mL of proprietary thaw medium pre-warmed at 37 °C, supplemented with glutamine or a glutamine supplement (2 mM final concentration) (Williams' E + glutamine supplement + thaw supplements).
   4. Count viable cells using a 500 μL aliquot with a cell counter or any other suitable cell counting technique.
   5. Seed the cells in a 24-well collagen-coated plate at a density of 0.6 x 10⁶ viable cells/well in a final volume of 0.5 mL of thaw medium.
   6. Place the plate(s) in an incubator at 37 °C with a 5%/95% CO₂/air and 100% relative humidity.
   7. 24 h after seeding the cells, replace the medium with 0.5 mL of pre-warmed maintenance/metabolism working medium (Williams' E + glutamine supplement + metabolism supplement (this medium already contains DMSO)). Change the medium every two days.
   8. After a few days in culture the cells form a sub-confluent trabecular structure (Figure 3). The optimum metabolic activity is typically observed between day 7 and 10 and is lowest at day 4.

Figure 4: Cross Section of Alcian Blue Stained Airway Cells Grown at the Air-liquid Interface⁸.
Ciliated, mucus producing and basal cells are clearly visible. Scale bar = 250 μm.

2. Differentiated airway epithelium cultures
   NOTE: The protocol is shown with a commercially available fully differentiated primary airway epithelium grown at the air-liquid interface on an insert⁸ (Figure 4). The tissue culture retains a mucocilliary phenotype and cell polarity⁸. Cells can be of nasal or bronchial origin. Here, use human nasal cells. The cells are delivered on a culture plate format with 24 inserts at the air-liquid interface. To facilitate transport and avoid medium spillage the inserts are shipped with the basal section embedded in an agarose matrix mixed with culture medium.
   1. Upon delivery of the cells, sterilize the plates containing the 24 inserts with a 70% ethanol solution before placing in a clean laminar flow hood.
   2. Prepare a sterile 24-well plate by adding pre-warmed 700 μL proprietary culture medium.
   3. Using sterile tweezers transfer the cell inserts to the new plate pre-loaded with medium by gently dislodging the insert from the agarose matrix.
   4. Place the plate(s) in an incubator at 37 °C with a 5% / 95% CO₂/air and 100% relative humidity.
   5. Every 2 days transfer the inserts to a new sterile 24-well plate pre-loaded with 700 μL warm proprietary airway cell culture medium.
   6. Assess the integrity of the airway tissue using methods, such as trans-epithelial resistance (TEER), or cilia beat frequency (CBF) using a microscope fitted with a live video imaging system⁹. The measurement of TEER and CBF is described in Kuehn¹⁰.

2. CYP Activity Quantification in Cell Cultures

NOTE: As a general rule, solvents such as DMSO and methanol can be used to prepare stock solutions for the different metabolic probes and inhibitors presented in this paper. However, it is recommended to keep the final DMSO concentration in the culture medium to a minimum (1% or lower) since high concentrations can inhibit metabolic enzymes. When possible, it is also recommended to use phenol red free and serum free medium during the incubation with metabolic probes since phenol red can interfere with CYPs and Phase II enzyme activity and serum component such as albumin can decrease the probe availability. It is not always possible to strictly adhere to these guidelines. For instance, airway cell culture medium is proprietary and contains phenol red. Proprietary hepatic cell line metabolism medium⁷ already contains DMSO above 1% as it is required for the cells to adopt the hepatocyte-like phenotype.

NOTE: Finally, here, the term specific probe/inhibitor is used but this terminology has to be considered with caution. Indeed, it is very rare for a metabolic probe to be an enzyme exclusive substrate. The probes described here, however, have a high affinity for their enzyme target and therefore are regarded as reliable markers of activity.

1. Metabolic probes, inducers, and inhibitors
   1. Probes using Mass Spectrometry based methods
      NOTE: The probes and inhibitors described in this section are suitable to test the activity of CYP2As¹¹, CYP1A2^12,13, CYP2B6^7,14, CYP2F1^15,16, and CYP2E1¹⁷ by UPLC-mass spectrometry. The probes, inhibitors and corresponding CYP are described in Table 1 with corresponding references. Table 1 also lists the metabolic product of the CYP activity which is quantified by mass spectrometry. All work involving tissue culture and solutions for use in tissue culture is to be carried out in a sterile tissue culture hood. A stock solution can be prepared in DMSO for all the probe substrates.
      1. Prior to the experiment prepare two series of cell inserts or wells, use one series for the inhibition experiment, the other for the metabolic probe only. Prepare a third series of cells as a medium blank control. They can be on separate plates or in the same plates with a minimum of three replicates. An example of plate layout is presented in Figure 5.
      2. On the day of the experiment, replace the cell culture medium with 450 μL of fresh medium.
      3. Prepare in a sterile hood a 10x concentration of the following solutions using the cell culture medium. Mix 10x CYP probe, 10x CYP inhibitor, and 10x CYP inhibitor plus 10x probe.
         NOTE: Master stock solutions of inhibitor and probe can be prepared in 100% DMSO (e.g., 1,000x stocks) and further diluted in media to obtain a 10x solution in order to avoid a final DMSO concentration in excess of 1% with the cell culture. The final 1x concentrations that are aimed for the incubation with the cells are shown in Table 1.
      4. Filter the different 10x probe solution with a 0.2 μm syringe filter.
      5. Add 50 μL of medium containing the 10x CYP inhibitor to the series of cells prepared for the inhibition experiment and pre-incubate for 30 min.
      6. Replace the medium of cells in the inhibition series with 450 μL of fresh medium and add 50 μL of medium containing both 10x CYP inhibitor and the 10x probe substrate.
      7. Add to the other cells 50 μL of medium with the 10x probe substrate and add an equivalent amount of vehicle diluted in medium (e.g., DMSO) to the blank cell controls as shown in Figure 5.
      8. Incubate the cells for 0 – 5 min (time 0, background control) and 16 h in the cell incubator.
         NOTE: This incubation time was used successfully with the airway cells and hepatic cell line; however fresh primary hepatocytes have a high metabolic competency, pending the quality of the cell preparation and genetic makeup of donor, hence shorter times (e.g., 2-4 h) might be suitable. It is always recommended to perform a time course experiment when testing different cell lines or cell types.
      9. At the end of the incubation period, collect the medium in separate labelled tubes for quantification of the metabolic product of the probes. Freeze the medium for future processing.
      10. Lyse the cells for protein quantification.
          NOTE: Any standard protein quantification kit and protocol is suitable for this step. BCA is one suitable option. This step is optional if the cells have to be used in another assay.
   2. Medium treatment
      NOTE: The medium collected from the incubation stage contains the parent probe chemical as well as its metabolic products. To measure a specific CYP activity only the product of the reaction catalyzed by the CYP of interest should be quantified. Phase I metabolism products are often processed in tandem with phase II metabolism (conjugation) and therefore cannot be detected as such. Thus in order to obtain the most accurate quantification of a CYP activity, a deconjugation step is required to remove any Phase II metabolism modification. Since conjugation of Phase I metabolites often involves glucuronidation or sulfation, deconjugation is achieved by treatment of the samples by glucuronidases and sulfatases¹⁸. Glucuronidase and sulfatase activity is pH dependent with highest glucuronidase activity being achieved at pH 4.5-5.0 and sulfatase activity being highest at pH 6.0-6.5. Therefore, this step requires pH adjustment. In the method described here, we measure the pH using pH strips for practical reasons since most pH meter probes do not fit in a 1.5 mL microcentrifuge tube. Commercially available pH strips can be found with a resolution as low as 0.3 to 0.2 pH units.
      1. Transfer 250 μL of incubation medium to a 1.5 mL microtube and add 4-methylumbelliferone internal standard stock solution to reach a final concentration of 100 nM.
      2. Adjust the pH of the medium to 4.5-5.0 by adding 1 M HCl. Add 1 μL of HCl at a time and verify pH by pipetting 2 μL of medium onto a pH strip. Reaching a pH of 5.0 would typically require no more than 5-10 μL of 1 M HCl.
      3. Add 150 units (1.8 μL of an 85,000 unit/mL stock) of β-glucuronidase/arylsulfatase from Helix pomatia and incubate for 1 h at 37 °C. Stop the reaction by adding 250 μL of cold methanol (-20 °C). Store the samples at -20 °C or process further.
      4. Evaporate the solvent using a vacuum centrifuge at 45 °C. Reconstitute the samples with 500 μL of a 30% acetonitrile:water solution. The samples are ready for mass spectrometry analysis and can be stored at -20 °C in amber glass vials.
   3. Mass Spectrometry based quantification
      NOTE: All the UPLC and mass spectrometer settings are detailed in the dedicated Supplemental Table 1. For a quantitative analysis, establishing a calibration curve is required. The calibration curve is obtained by running a known dilution range of the compound to be quantified. In this case, the calibration curves were generated over 10 different concentrations (Table 2), at least 6 of which should be within 20% of the linear range¹⁹. Lowest level of detection is determined as the lowest concentration which gives a signal greater than, or equal to 3x the average background signal. The lowest level of quantification is determined as the sample of lowest concentration which gives a signal greater, or equal to 10x the average background signal both of which are determined over at least 3 independent runs with triplicate samples.
      1. Prepare a serial dilution of a synthetic metabolite in the tissue culture medium according to Table 2, including the internal standard. A final volume of 250 μL per standard dilution is adequate .
      2. Evaporate the serial dilutions of standard as described in step 2.1.2.4 and resuspend in a volume of 30% acetonitrile:water equivalent to the volume of media prior evaporation (e.g., 250 μL 30% acetonitrile:water if the starting standard solution was 250 μL of medium).
      3. Add 250 μL of each serial dilution standard to an amber UPLC glass vial for injection in the UPLC-MS autosampler.
      4. Add the test samples (250 μL) to amber UPLC vials and place them in the UPLC autosampler.
      5. Randomize all the vials.
         NOTE: The mass spectrometer configuration we used is a hybrid triple quadrupole/linear ion trap mass spectrometer coupled to a UPLC detailed in the Supplemental Table 1. Other platforms would have different software and configuration but would follow similar steps to perform the quantification.
      6. Run the UPLC-MS using the settings detailed in Supplemental Table 1 depending on the specific probe to quantify.
      7. Use the "Quantitate – Build Quantitation method" tool in mass spectrometer quantification tools (Table of Materials) to generate the calibration curve from the acquired synthetic standard and internal standard.
      8. Use the "Quantitation Wizard" to select the sample batch and samples to quantify and execute the "Quantitation Method". The mass spectrometer quantification software displays a spreadsheet with the quantitation values of the target compound in each test sample.
   4. Probes using Luminescence based methods
      NOTE: Luminogenic probes to measure a range of CYP activities are commercially available; however, not all are suitable for cell based assays. Here, a protocol is presented to measure the activity of CYP1A1/CYP1B1. CYP1A1/1B1 was selected to illustrate how to assay the activity of inducible CYPs and to give an example of how luminogenic probes can be used as an alternative to mass spectrometry-based methods. Table 3 summarizes the concentrations and incubation time we used for the airway cells and the hepatic cell line; however, those might have to be adapted for other cell types. Other luminogenic probes are commercially available and can be used as substrates for a variety of CYPs.
      1. Prepare sufficient cells to include a non-induced control, an induced test, and an induced + inhibitor test. An example of a plate layout is shown in Figure 5B.
      2. To induce CYP1A1/1B1, incubate the cells with a final concentration of 10 nM TCDD in medium for a period of 72 h (shorter periods of 24 to 48 h may also be suitable).
         CAUTION: TCDD is a carcinogen and teratogen. Wear adequate protective equipment, review the supplier MSDS sheet and perform a risk assessment before use.
      3. After this period, remove the medium, rinse the wells with PBS 3 times, and add some fresh medium.
      4. Prior to incubating the cells with the luminogenic reporter probe, pre-incubate the designated subset of induced cells for 30 min with a final concentration of 10 μM CYP1A1/1B1 inhibitor, α-naphthoflavone, in medium.
      5. Add the luminogenic probe LUC-CEE (Luciferin 6'-chloroethyl ether) at a final concentration of 50 μM to the non-induced, induced, and induced + inhibitor cells.
      6. Incubate for 4 h in a cell culture incubator. At the end of the incubation time collect the medium for quantification of the luminogenic probe.
      7. Transfer 25 μL of culture medium to a white opaque 96 well plate and add 25 μL of luciferin detection reagent supplied by the manufacturer. Incubate for 20 min at room temperature. Read the plate immediately using a luminometer.
      8. Rinse the cells with PBS and lyse for total protein quantification.
         NOTE: This step is optional if live cells are retained for future experiments. It is important to note however that inducers such as TCDD are toxic and will damage the cells.

Table 1: List of Metabolic Probes, Products, and Inhibitors with their Corresponding Enzyme. Molecular weight and recommended concentrations used with the airway and hepatic cells are also indicated. Please click here to view a larger version of this figure.

Figure 5: Suggested 24-well Plate Layout to Perform a CYP Assay. (A) The recommended layout for CYPs constitutively expressed includes a series of wells for the blank medium, the metabolic probe, and the metabolic probe plus inhibitor. This layout can be multiplied by the number of time points that are being tested. (B) The example layout for inducible CYPs includes a series of well for a medium blank, a metabolic probe only control, induced cells (e.g., with TCDD) incubated with the probes and incubated with the probes and an inhibitor.

Table 2: Dilution of Synthetic Standards used for the Calibration Curves and Respective LODs and LOQs. The dilution range to use can vary based on the mass spectrometry platform and sensitivity. Here, we used a hybrid triple quadrupole/linear ion trap mass spectrometer linked to a UPLC. Please click here to view a larger version of this figure.

Table 3: Luminogenic Probe, Inducer and Inhibitor for CYP1A1/1B1 Assay. Please click here to view a larger version of this figure.