An Intestine/Liver Microphysiological System for Drug Pharmacokinetic and Toxicological Assessment

1. Production of tissue equivalents for cultivation in the 2-OC

1. Small intestine barrier equivalent production
   1. Maintain Caco-2 and HT-29 cells using the intestine equivalent medium: DMEM supplemented with 10% FBS, 1% penicillin and streptomycin, and 1% non-essential amino acids, which is named as “DMEM S” in this manuscript.
   2. Remove the medium, wash twice with 1x DPBS and add 8 mL of 0.25% Trypsin/EDTA to dissociate Caco-2 cells grown in cell culture flasks (175 cm²). Incubate for 5 min at 37 °C and stop the reaction by adding at least the double volume of trypsin inhibitor. Perform the same procedure for HT-29 cells, adjusting the reagent volumes since a smaller quantity of these cells is needed and they are maintained in smaller flasks (75 cm²).
   3. Centrifuge at 250 x g for 5 min, remove the supernatant from both tubes, and resuspend the cell pellets in 10 mL of DMEM S. Count cells, assuring a cell viability higher than 80%. Aseptically integrate cell culture inserts in a 24-well plate previously filled with 400 µL of DMEM S per well in the basolateral side (which represents the human bloodstream).
   4. Co-cultivate Caco-2 and HT-29 cells at a ratio of 9:1²¹. Use 2.25 x 10⁵ Caco-2 and 2.5 x 10⁴ HT-29 cells to each intestine equivalent in a final volume of 200 µL of DMEM S. Adjust cell numbers and volume according to the desired number of organoids. Mix carefully.
   5. Pipette 200 µL of cell solution into each insert’s apical side (which represents the human intestinal lumen side), seeding 250,000 cells per insert. Co-cultivate the cells in the inserts for three weeks²². Change the medium at least three times a week, aspirating it from both the apical and basolateral sides with a sterile Pasteur pipette, taking care not to damage the intact cell barrier.
      NOTE: Proceed with the aspiration on the apical side, so as not to touch the cell barrier (aspirate by supporting the Pasteur pipette on the plastic rim of the cell insert).
   6. Check the tight monolayer formation by measuring the TEER (transepithelial electrical resistance) every three days using a voltmeter²³, according to the manufacturer's instructions.
      1. Perform a blank, measuring the resistance across a cell culture insert without cells, but with the same cell medium and at the same cell plate.
      2. Calculate tissue resistance by subtracting the blank resistance from the tissue-equivalent resistance, and multiply by the effective surface area of the filter membrane (0.6 cm²). A good intestine barrier resistance is in a range of 150 to 400 Ω∙cm².
         NOTE: After 21 days the cells must be fully differentiated and the intestinal barrier formed, so the intestine equivalents are ready to be integrated into MPS.
2. Liver equivalents production
   1. Maintain HepaRG cells using the liver equivalent medium, which is William’s Medium E supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin, 5 µg/mL human insulin and 5 x 10^-5 M hydrocortisone, and is named “Williams E S” in this manuscript. Renew HepaRG media every 2-3 days and maintain cell culture for two weeks to initiate the differentiation in hepatocytes and cholangiocytes.
   2. After the first two weeks, add 2% DMSO to HepaRG’s medium for an additional two weeks to complete the cell differentiation^24,25. Grow HHSTeC in Stellate Cell Media (SteC CM), using poly-L-lysine-coated cell culture flasks, changing media every two or three days.
   3. Remove the medium, wash twice with 1x DPBS and add 8 mL of 0.05% Trypsin/EDTA, to dissociate HepaRG cells grown in cell culture flasks (175 cm²). Incubate for 5 to 10 min at 37 °C and stop the reaction by adding at least double the volume of trypsin inhibitor. Perform the same for HHSTeC, adapting the reagent volume since a smaller quantity of these cells are needed and they can be maintained in smaller flasks (75 cm²).
   4. Centrifuge both at 250 x g for 5 min, remove the supernatant and resuspend the cell pellets in Williams E S medium. Count cells, assuring a cell viability higher than 80%.
   5. Generate the liver spheroids combining HepaRG and HHSTeC cells at a ratio of 24:1, respectively, in Williams E S medium¹⁶. Add 4.8 x 10⁴ differentiated HepaRG and 0.2 x 10⁴ HHSTeC to compose each liver spheroid of 50,000 cells, in a volume of 80 µL. Adjust cell numbers and volume according to the desired number of spheroids. Mix carefully.
   6. Using a multichannel pipette, dispense 80 µL of the combined cell pool in each well of 384 spheroid microplates, which has round well-bottom geometry.
      NOTE: After four days, spheroids of about 300 µm are formed.
   7. Using wide-bore tips, transfer the liver spheroids to ultra-low-attachment 6 well plates, which allows the required “one-by-one” counting.

2. Integration of intestine and liver equivalents in a 2-OC MPS

1. Intestine 2-OC MPS assembly for absorption assay
   1. Pipette 500 µL of DMEM S into the larger compartment of 2-OC and 300 µL in the smaller one. Aspirate the basolateral and apical media of each intestinal barrier equivalent in the 24 well plates. Using sterile forceps, integrate one insert per 2-OC circuit, specifically into the larger compartment. Apply 200 µL of the intestine medium at the apical side.
      NOTE: Avoid the formation of bubbles when integrating the organoids into the MPS.
   2. Connect the MPS to the control unit, which must be connected to a pressurized air supply. Set the parameters: a pressure of, approximately, ±300 bar and a pumping frequency of 0.3 Hz. Start the flow 24 h before the test substance administration. The next day, perform the APAP treatment.
2. Liver 2-OC MPS assembly for metabolism assay
   1. Pipette 650 µL of Williams E S into the large compartment and 350 µL into the smaller compartment, which will receive the spheroids. In the ultra-low-attachment 6 well plates, count the spheroids using wide-bore tips. Each liver equivalent is composed of twenty spheroids²⁶. Integrate twenty liver equivalents per circuit, using wide-bore tips, which permits the transfer of organoids only, into the smaller compartment of a 2-OC.
   2. Connect the MPS to the control unit, which must be connected to a pressurized air supply. Set the parameters: a pressure of, approximately, ±300 bar and a pumping frequency of 0.3 Hz. Start the flow 24 h before the test substance administration. The next day, perform the APAP treatment.
3. Intestine/Liver 2-OC MPS assembly for absorption and metabolism assay
   1. Combine the two media (intestine and liver) in a 1:4 proportion, which means 200 µL of DMEM S in the intestine apical side and 800 µL of Williams E S in the basolateral side. Integrate intestine and liver equivalents, simultaneously, in the 2-OC.
   2. Connect the MPS to the control unit, which must be connected to a pressurized air supply. Set the parameters: a pressure of, approximately, ±300 bar and a pumping frequency of 0.3 Hz. Start the flow 24 h before the test substance administration. The next day, perform the APAP treatment.
      NOTE: For all experiments, perform each time point in triplicate, which means three separated 2-OC circuits (i.e., 1 and ½ 2-OC devices). The total volume of each 2-OC circuits is 1 mL.

3. Acetaminophen (APAP) preparation

1. Prepare the APAP stock solution, dissolving APAP in absolute ethanol. On the day of the experiment, dilute APAP in the respective medium (APAP solution), to a concentration of 12 µM for “oral administration” and 2 µM for “intravenous administration”.
2. Ensure that the final concentration of ethanol in the vehicle control and treatment solution is 0.5% for both administrations. For the positive control (100 mM APAP), the ethanol concentration is 2%.

4. Test substance administration and media sampling

1. APAP “oral” administration and media sampling
   1. Aspirate the basolateral and apical media of each intestinal barrier equivalent in the 2-OC. Pipette 500 µL of the appropriate culture medium into the large compartment at the organoid basolateral side and 300 µL into the small compartment.
   2. Check for bubbles and proceed with the intestinal barrier equivalent treatment with the test substance in the apical side, emulating oral administration. Emulate APAP “oral” administration by adding 200 µL of a 12 µM APAP solution on the apical side of intestinal culture inserts, which represents the intestinal “lumen side” (Figure 1B). Connect the MPS to the control unit.
   3. Collect the total volume from apical and from basolateral sides at the following time points: 0 h, 5 min, 15 min, 30 min, 1 h, 3 h, 6 h, 12 h, and 24 h^15,27. Perform all experiments in triplicate, at static and dynamic conditions, and collect each sample, of each triplicate, in a separate microtube. Analyze the samples using HPLC/UV.
      NOTE: Separate apical and basolateral samples.
2. APAP “intravenous” administration and media sampling
   1. Emulate the “intravenous” route by administering 2 µM APAP solution directly into the liver compartment. Aspirate all 2-OC medium content. Pipette 650 µL of Williams E S containing the test substance into the large compartment and 350 µL of the same media into the smaller compartment which contains the 20 spheroids. Collect all volumes at the following time points: 0 h, 30 min, 1 h, 2 h, 3 h, 6 h, 12 h, and 24 h^27,28.
   2. Perform all experiments in triplicate, at static and dynamic conditions. Collect each sample, of each triplicate, in a separate microtube. Analyze the samples using HPLC/UV.

5. Instrumentation and chromatographic conditions

1. HPLC analysis
   1. Set all relevant parameters for the HPLC analysis according to Table 1.
   2. Filter the mobile phase through a 0.45 µm membrane filter under vacuum. Filter the samples through a 0.22 µm pore size PVDF syringe filter (diameter 13 mm) and store them in a vial. Start the measurement.
2. Stock solutions, calibration standards, and quality control (QC) samples
   1. Prepare 10 mM of APAP stock solutions in ammonium acetate buffer (100 mM, pH 6.8) and further dilute with DMEM S and Williams E S cell culture media diluted with ammonium acetate buffer (1:1, v/v) to achieve the working solutions ranging from 0.25 to 100.00 µM.
   2. Include a set of calibration samples in triplicate as well as quality control samples at four levels in triplicate. Prepare these standards by serial dilution.
   3. Create calibration curves of APAP peak areas versus APAP nominal standard concentrations. Determine the linear regression fit for each calibration curve. Evaluate the goodness-of-fit of various calibration models by visual inspection, correlation coefficient, intra- and inter-run accuracy and precision values.
   4. Inject blank samples of DMEM S and Williams E S media diluted in ammonium acetate buffer (1:1, v/v) in sextuplicate. Prepare triplicates of quality control samples in DMEM S and Williams E S media diluted with ammonium acetate buffer (1:1, v/v) for the APAP concentrations of 0.50 (LOQ), 4.50, 45.00 and 90.00 µM.
   5. Ensure that the quality control samples are prepared from a new stock solution, different from that used to generate a standard curve. Use quality control samples to investigate intra- and inter-run variations.
3. Validation procedures
   1. Perform the bioanalytical method validation following the previously reported procedures^29,30. Carry out the chromatographic runs on five or six separate occasions, considering Williams E S and DMEM S cell culture media, respectively.
   2. Ensure that calibration points ranging from 0.25 to 100.00 µM of APAP, in DMEM S or Williams E S cell culture media diluted in ammonium acetate buffer (1:1, v/v), are plotted based on the peak-areas of APAP (axis y) against the respective nominal concentrations (axis x). Compare the slopes of these standard calibration curves with slopes of calibration curve prepared in ammonium acetate buffer. Make sure that all calibration curves have a correlation value of at least 0.998.
   3. Determine the precision and accuracy (intra and inter-run) for the analyte in the surrogate matrix using replicates at four different levels LLOQ, low, middle, and high-quality control on five or six different days. Perform intra-run precision and accuracy measurements on the same day in DMEM S or Williams E S cell culture media diluted in ammonium acetate buffer (1:1, v/v) containing 0.50, 4.50, 45.00 and 90.00 µM APAP concentrations (n= 3).
   4. Evaluate each set of quality control samples containing the APAP concentrations from recently obtained calibration curves. Test the selectivity of the assays by the degree of separation of the compound of interest and possible other chromatographic peaks caused by interfering components.
4. Lower limit of quantification (LLOQ) and limit of detection (LOD)
   1. Determine the lower limit of quantification (LLOQ) based on the standard deviation of the response and the slope approach. Calculate using the formula 10α/S, where α is the standard deviation of y-intercept and S is the slope of straight line obtained by plotting calibration curves^29,30. Estimate the limit of detection (LOD) taking into consideration 3.3 times the standard deviation of the blank, divided by the slope of the calibration curve^29,30.

6. Tissue equivalents viability/functionality

1. MTT
   1. Perform an MTT assay to assess organoid viability in all time points of the MPS assay. As the negative control, use cell media plus vehicle. As the positive control, treat the organoids with 100 mM APAP and 1% NaOH diluted in cell medium.
   2. Transfer the 20 spheroids of each replicate for individual wells in a 96 well plate, and the cell culture inserts, containing the intestine equivalents, to 24 well cell plates, placing one intestine equivalent per well. Wash the tissue equivalents three times with 1x DPBS.
   3. Add 300 µL of a 1 mg/mL MTT solution, diluted in the respective cell medium, per well. Incubate the plates for 3 h at standard cell culture conditions.
   4. Remove the MTT solution from each well carefully by pipetting. Extract MTT formazan from the intestine and liver equivalents using 200 µL of isopropanol per well overnight at 4 °C.
      NOTE: Seal the lid to prevent evaporation.
   5. Transfer 200 µL of each supernatant to the respective pre-identified well in a 96 well micro test plate. Use isopropanol as the blank.
   6. Read the formazan absorbance in a plate reader at 570 nm. Calculate the relative ability of the cells to reduce MTT (%) using the average optical density of each time point, compared to the negative control, considered as 100% cell viability.
2. Cytochemistry/Histology
   1. Fix the intestine and liver equivalents, for 25 min at room temperature, using 4% (w/v) paraformaldehyde in 0.1 M phosphate-saline buffer, pH 7.4. Wash the organoids 5 times in PBS buffer for 10 minutes each time. Stain the intestinal and the liver equivalents with tetramethylrhodamine isothiocyanate-phalloidin or Alexa Fluor 647 phalloidin, 1:50 in PBS³¹.
   2. Transfer them to OCT freezing medium for a few minutes to acclimate at RT before transferring them to liquid nitrogen until the complete freezing. Perform liver spheroids cryosections about 10-12 µm thick, using a cryostat.
   3. Mount the tissues sections in mounting medium with DAPI. Examine them by confocal fluorescence microscopy.
   4. Freeze the organoids after fixation to perform hematoxylin & eosin staining according to the established protocols. Mount the slides with mounting medium after slicing the tissue as described above and take histological images using an optical microscope.
3. High content analysis
   1. Mitochondrial and nuclear staining of the cells
      1. Reconstitute the lyophilized powder in DMSO to make a 1 mM mitochondrial staining stock solution (e.g., MitoTracker Deep Red FM). Store aliquoted stock solution at -20 °C protected from light. Dilute the 1 mM mitochondrial staining stock solution to the final concentration (200 nM) in prewarmed (37 °C) tissue culture medium without serum.
      2. Remove the cell culture media. Add the mitochondrial staining solution to completely cover the sample and incubate cells for 15-45 min at 37 °C in a humidified atmosphere with 5% CO₂.
      3. Carefully remove the mitochondrial staining working solution and replace it with 2-4% paraformaldehyde fixative in PBS for 15 minutes at room temperature.
      4. Rinse the fixed cells gently with PBS for 5 minutes. Repeat the washing process twice.
      5. Prepare a 10 mg/mL (16.23 M) nucleic acid staining stock solution by dissolving 100 mg of Hoechst 33342 dye in 10 mL of ultrapure water.
         NOTE: The stock solution should be aliquoted and stored protected from light at -20 °C.
      6. Prepare a 0.2-2.0 µg/mL nucleic acid staining working solution in PBS and incubate the fixated cells with nucleic acid staining working solution for 10 minutes at room temperature.
      7. Remove the nucleic acid staining working solution and rinse the cells gently with PBS for 5 minutes three times. Cells should be kept in PBS at 4 °C, protected from light.
   2. Mitochondrial and nuclear staining analysis
      1. Analyze cells using a fluorescence microscope with filter sets appropriate for the nucleic acid stain (λEx/ λEm: 361/497 nm) and the mitochondrial stain (λEx/ λEm: 644/665 nm). Find cells by nucleic acid positive staining and quantify cell number. Quantify mitochondrial stain fluorescence intensity in mitochondria.
4. Morphometric measurements (spheroids calculations) in ImageJ
   1. Export High Content Analysis (HCA) images as *.flex files from the Columbus software. Import .flex files as a grayscale in ImageJ using Bio-Formats plugin³²: File > Import > Bio-Formats.
   2. In the Import Options window, select Hyperstack viewing and enable Split channels under Split into separate windows. This option will allow the access of all files in a particular channel (e.g., DAPI, mitotracker, etc.). Do not select Use virtual stack under Memory management.
      NOTE: It is preferable to use DAPI channel as “dust” in images as culture medium is reduced in UV wavelength (e.g., 405 nm).
   3. Adjust pixel size (Analyze > Set Scale) if it was not loaded according to embedded values in the .flex file. Apply a Gaussian Blur filter to remove the excess of noise and avoid irregularities in shape contour. Process > Filters > Gaussian Blur. A high value of Sigma (radius) between 2.0 and 3.0 is ideal for most cases. If the stack has several images, apply to all of them (select Yes in Process Stack window).
   4. Generate a binary image to separate background and organoids (objects) using a threshold. Click Image > Adjust > Threshold. Use the red mask to adjust the values according to the intensity of the image, to fit the organoid shape, keeping the morphology intact. Disable Dark background if the image has a white background. Click Apply.
   5. In the Convert Stack to Binary window, choose the Threshold method. Usually, Default or Triangle are preferred in this kind of image processing. Keep the Background as Dark. Select Calculate threshold for each image if there are several images in the stack.
   6. Select Process > Binary > Fill holes. Optionally, remove holes from the background. In Process > Binary > Options, select Black background and execute Fill Holes again. Disable Black background option before proceeding to the next step.
   7. Separate objects. For organoids, the watershed method is a good choice. Click Process > Binary > Watershed. Execute the shape analysis.
      1. Select Analyze > Set Measurements. Several options are available (details in https://imagej.nih.gov/ij/docs/guide/146-30.html). For organoids, select Area, Mean gray value, Min & max gray value and Shape descriptors. Optionally, select Display label to identify objects in the image and Scientific notation. Click OK.
      2. Select Analysis > Analyze Particles. Choose the Size and Circularity limits. Keep 0-infinity and 0.00-1.00, respectively to measure all objects in the image. In Show, choose Outlines so the objects will be identified. Enable Display results to output results; Exclude on edges to exclude objects touching borders; Include holes so eventual interior holes in the objects are considered as part of the main shape.
   8. Repeat Shape Analysis for every image stack and the results will be appended in a single table. Export results table in File > Save As… as Comma Separated Values (.csv) file.
5. Real-Time PCR
   1. Extract RNA from tissue equivalents using a monophasic solution of phenol and guanidine isothiocyanate, following manufacturer’s instructions.
   2. Perform the cDNA synthesis by reverse transcription of 1 – 2 µg of total RNA.
   3. Amplify all targets using gene-specific primers (Table 5) to perform real time quantitative PCR. Each qRT-PCR contains 30 ng of reverse-transcribed RNA and 100 nM of each primer.
   4. Follow PCR conditions: 50 °C for 3 minutes (1 cycle); 95 °C for 5 minutes (1 cycle); 95 °C for 30 seconds, 59 °C for 45 seconds and 72 °C for 45 seconds (35 – 40 cycles).
6. CYP assay
   1. Follow section 2.2 for liver 2-OC assembly. The experimental groups are No-cell control, APAP 2 µM treatments for 12 h, 24 h, and vehicle control. Follow sections 3.3 and 4.2 for APAP 2 µM preparation and treatment.
      NOTE: To ensure that all the samples will be ready at the same time for CYP assay, start the 12 h treatment 12 hours after starting the 24 h treatment. Treat the no-cell control of CYP activity with 0.5% ethanol solution, as well as the vehicle control.
   2. Thaw 3 mM luminogenic substrate stock solution at room temperature and make a 1:1000 dilution in William’s E S. Protect from light.
   3. Collect spheroids and transfer each experimental group to a well of a 96-well plate. Remove the medium, wash twice with 100 µL of 1x DPBS and add 80 µL of 3 µM substrate solution per well. Keep the no-cell control in William’s E S medium. Save a well without spheroids or substrate solution for a background control. Incubate for 30-60 min at 37 °C with 5% CO₂, protected from light.
   4. Equilibrate lyophilized Luciferin Detection Reagent (LDR) using the Reconstitution Buffer with esterase. Mix by swirling or inverting. Store the appropriated volume at room temperature until the next step.
      NOTE: Reconstituted LDR can be stored at room temperature for 24 hours or at 4 °C for 1 week without loss of activity. For long-term storage, store at –20 °C.
   5. Transfer 25 µL of intact spheroids’ supernatant in three different wells of a white opaque 96-well microplate, after incubation. Add 25 µL of LDR per well and homogenize.
   6. Incubate the white plate at room temperature for 20 minutes. Read the luminescence on a luminometer. Do not use a fluorometer.
   7. Calculate net signals by subtracting background luminescence values (no-cell control) from the test compound-treated and untreated (vehicle control) values. Calculate percent change of CYP3A4 activity by dividing the net treated values by the net untreated values and multiplying by 100.
7. Western blotting
   1. Transfer the liver spheroids to an identified 1.5 mL microtube. Remove the medium and wash twice with 100 µL of 1x DPBS.
   2. Lysate the liver spheroids in 100 µL of cell lysis RIPA buffer at 4 °C for 20 min. Centrifuge for 15 min, 4 °C, and 11000 rpm. Transfer the supernatant to another identified 1.5 mL microtube.
   3. Quantify the amount of protein obtained through the Bradford method. Load between 10 and 50 µg of protein from the quantified cell lysate per well of a gradient polyacrylamide gel 3-15% and perform an SDS-PAGE.
   4. Transfer the loaded protein from the gel to a 0.22 µm PVDF membrane through the semi-dry system equipment. Use a transference solution of 50 mM Tris-HCl and 192 mM glycine. Set equipment parameters according to the number of gels to be transferred (1 to 2 at a time).
   5. Block nonspecific interactions on PVDF membrane with a 3-5% skim milk solution in TBS-T buffer: Tris-Buffered Saline (50 mM Tris pH 7.6, 150 mM sodium chloride) supplemented with 0.1% of Tween 20. Keep the membrane under gently constant shaking for 1 hour at room temperature.
   6. Wash with TBS-T under gently constant shaking for 3-5 min at room temperature. Repeat this washing step twice.
   7. Dilute albumin and vinculin primary antibodies to 1:1000 and 1:2000 respectively on TBS-T. Incubate the membrane with primary antibodies overnight at 4 °C, under gently constant shaking.
      NOTE: Always follow the manufacturer’s instructions to dilute antibodies.
   8. Remove the primary antibody and wash membrane 3 times (step 6.7.6). Dilute ECL anti-mouse IgG secondary antibody to 1:5000 on TBS-T. Incubate the membrane with a secondary antibody under gently constant shaking for 2 hours at room temperature.
   9. Remove the secondary antibody and wash the membrane (step 6.7.6). Perform protein detection using ECL Western Blotting Substrate. Expose the autoradiographic films for 30 s to 30 min. Perform the immunoblotting detection in triplicate.