Three-Dimensional Morphogenesis in Canine Gut-on-a-Chip Using Intestinal Organoids Derived from Inflammatory Bowel Disease Patients

The study was approved and conducted in accordance with the Institutional Animal Care and Use Committee of Washington State University (ASAF# 6993). In this protocol, we utilized a well-established Gut-on-a-Chip microfluidic device made of PDMS which was fabricated in-house² (Figure 1D). Detailed methods of the fabrication of the Gut-on-a-Chip microdevice can be found in previous reports^2,24. This protocol demonstrates a unique integration of intestinal organoids and a microfluidic system (Figure 2).

1. Surface activation of a Gut-on-a-Chip made of PDMS

1. Prepare 1% polyethyleneimine (PEI) solution by adding 1 mL of 50% PEI solution into 49 mL of distilled (DI) water in a 50 mL conical tube. Invert the tube two to three times to mix the solution well, then filter the solution using a 0.2 µm syringe filter.
   NOTE: Keep it stored at 4 °C.
2. Prepare 0.1% glutaraldehyde (GA) solution by adding 100 µL of 50% GA solution into 49.9 mL of DI in a 50 mL conical tube. Invert the tube two to three times to mix the solution well, then filter the solution using a 0.2 µm syringe filter.
   NOTE: Store it at 4 °C and make sure to protect it from exposure to direct light.
3. Place the Gut-on-a-Chip device in a dry oven set at 60 °C and incubate it for a minimum of 30 min to eliminate any remaining moisture.
4. Expose the Gut-on-a-Chip device to UV and Ozone treatment for 60 min using a UV/Ozone generator.
   NOTE: To ensure optimal activation of the PDMS surfaces during treatment, maintain a distance of approximately 3 cm or less between the UV lamp and the device. Avoid overcrowding of devices in the generator to accomplish efficient surface activation.
5. Secure the upper microchannel's inlet, bypass, and outlet tubing by using binder clips to clamp them. Also clamp the inlet tubing for the lower microchannel.
6. Disconnect the outlet tubing for the lower microchannel. Ensure that the bypass tubing of the lower microchannel remains open.
7. Using a P100 micropipette, introduce 100 µL of a 1% PEI solution through the outlet hole of the lower microchannel.
   NOTE: It is recommended to perform this process while the device is still warm from UV/Ozone exposure. Observe the PEI solution flowing through the microchannel and the PEI solution drops to come out from the bypass tubing for the lower microchannel.
8. Reconnect the outlet tubing for the lower microchannel.
9. Secure the lower microchannel's inlet, bypass, and outlet tubing by using binder clips to clamp them. Also clamp the inlet tubing for the upper microchannel.
10. Disconnect the outlet tubing for the upper microchannel. Ensure that the bypass tubing of the upper microchannel remains open.
11. Using a P100 micropipette, introduce 100 µL of a 1% PEI solution through the outlet hole of the upper microchannel.
    NOTE: Observe the PEI solution flowing through the microchannel and the PEI solution drops to come out from the bypass tubing for the upper microchannel.
12. Reattach the outlet tubing of the upper microchannel.
13. Once both upper and lower microchannels are filled with 1% PEI solution, allow the device to incubate at room temperature (RT) for 10 min.
14. Perform steps 1.5-1.12 with 0.1% GA solution.
15. Once both upper and lower microchannels are filled with 0.1% GA solution, allow the device to incubate at RT for 20 min.
16. Perform steps 1.5-1.12 with DI water to remove any excess surface activation solution.
17. Place the chip in a dry oven at 60 °C and allow it to dry overnight.
    ​NOTE: Make sure to remove the binder clips from all the tubing to avoid any impingement of the tubing. This drying process is critical to the even surface activation of the microchannel within the Gut-on-a-Chip¹².

2. Extracellular matrix (ECM) coating and culture medium preparation for Gut-on-a-Chip culture

1. Prepare an ECM mixture with organoid basal medium so that the final concentration of Collagen I and Matrigel are 60 µg/mL and 2 % (vol/vol), respectively.
   NOTE: Prepare 50 µL of ECM mixture per gut chip (i.e., 20 µL of ECM mixture for each upper and lower microchannel and 10 µL extra). Prepare this on the day of use and store this solution at 4 °C or place it on ice until it is ready for use.
2. Take out the Gut-on-a-Chip that has been treated with UV/Ozone, PEI, and GA from the dry oven.
   NOTE: Inspect under a phase-contrast microscope to see if there is any residual moisture.
3. Allow the Gut-on-a-Chip to cool down in a biosafety cabinet for 10 min.
4. Secure the upper microchannel's inlet, bypass, and outlet tubing by using binder clips to clamp them. Also clamp the inlet tubing for the lower microchannel.
5. Disconnect the outlet tubing for the lower microchannel.
6. Using a P100 micropipette, introduce 20 µL of ECM mixture through the outlet hole of the lower microchannel.
   NOTE: Observe the ECM mixture flowing through the microchannel without any air bubble entrapment. If there is any air bubble present, introduce additional ECM mixture to the lower microchannel until the bubble is gone.
7. Reattach the outlet tubing for the lower microchannel.
8. Secure the lower microchannel's inlet, bypass, and outlet tubing by using binder clips to clamp them. Also clamp the inlet tubing for the upper microchannel.
9. Disconnect the outlet tubing for the upper microchannel.
10. Using a P100 micropipette, introduce 20 µL of ECM mixture through the outlet hole of the upper microchannel.
11. Reattach the outlet tubing for the upper microchannel.
12. Place the chip in a humidified CO₂ incubator with 5% CO₂ at 37 °C for 1 h to form the ECM layer onto the PDMS membrane treated with PEI and GA (Figure 3A).
    NOTE: Make sure all the tubing is clamped during the incubation.
13. During the incubation of the ECM coating, prepare two 1 mL syringes containing cold seeding medium, which is the organoid culture medium lacking A8301 but containing 10 µM Y-27632 and 2.5 µM CHIR99021, as previously reported in Gut-on-a-Chip derived from human organoids².
    NOTE: A8301 was removed from the medium to enhance the attachment of cells to the coated ECM as previously reported². Day 0 of Gut-on-a-Chip culture only requires upper microchannel flow. Therefore, prepare a full syringe for the upper channel while a minimum of 0.2 mL for the lower channel.
14. Once the ECM coating is completed, take the Chip out of the incubator and release the clamp to the bypass tubing connected to the lower microchannel.
15. Connect a 1 mL syringe filled with seeding medium to the blunt-end needle that is linked to the lower microchannel of the Gut-on-a-Chip. Carefully introduce the seeding medium (~50 µL) to the bypass tubing. Once the tubing is filled with the introduced medium, secure the bypass tubing connected to the lower microchannel with a binder clip.
16. Release the clamp of the outlet tubing connected to the lower microchannel. Carefully introduce the seeding medium into the lower microchannel allowing it to flow smoothly through the system. Secure the outlet tubing connected to the lower microchannel with a binder clip after perfusion.
17. Next, open the bypass tubing connected to the upper microchannel.
18. Connect a 1 mL syringe filled with seeding medium to the blunt-end needle that is linked to the upper microchannel of the Gut-on-a-Chip. Carefully introduce the seeding medium (~50 µL) to the bypass tubing. Once the tubing is filled with the introduced medium, secure the bypass tubing connected to the upper microchannel with a binder clip.
19. Release the outlet tubing connected to the lower microchannel. Carefully introduce the seeding medium into the upper microchannel allowing it to flow smoothly through the system. Secure the outlet tubing connected to the upper microchannel with a binder clip after perfusion.

3. Canine intestinal organoid cell preparation for seeding

NOTE: To generate the Gut-on-a-Chip model, canine colon organoids (referred to as colonoids) derived from IBD patient dogs were utilized in this protocol. These colonoids were derived from three to five small fragments of biopsied colonic tissue, following a previously reported method^15,18. For optimal results, it is crucial to use canine colonoids that have undergone a minimum of three culture passages to establish stable organoids suitable for in vitro applications. It is recommended to culture the canine colonoids for a duration of at least 3-4 days to facilitate adequate differentiation of multi-lineage cells within the organoids, ensuring their functional maturity and suitability for subsequent experiments in the Gut-on-a-Chip model. The maximum limit of the passage for this work is fewer than 20, as indicated by a previous study that demonstrated the unaltered phenotype and karyotype throughout 20 consecutive passages²⁵. The signalment of these donors is presented in Supplementary Table S1.

1. Culture the canine colonoids in 24-well plates embedded in 30 µL of Matrigel^15,18 with organoid culture medium listed in the Table of Materials, which is modified from previously reported medium^15,26,27. The conditioned medium was obtained by cultivating Rspo1 cells and HEK293 cells engineered to secrete Noggin²⁸.
2. Discard the organoid culture medium through vacuum suction and introduce 500 µL of Cell Recovery Solution at an ice-cold temperature into each well. Incubate for 30 minutes at 4 °C.
   NOTE: Typically, three wells out of a 24 well-plate of mature canine intestinal organoids provide a sufficient quantity of dissociated cells to seed a single Gut-on-a-Chip device with 40-50 organoids/one field of view at x10 magnification.
3. Mechanically disrupt the Matrigel domes for 5 s using a P1000 micropipette. Subsequently, gather the organoid suspension in a 15 mL conical tube.
4. Centrifuge the conical tube at 200 × g and 4 °C for 5 min, followed by the removal of the supernatant.
5. Introduce 1 mL of trypsin-like protease at room temperature, supplemented with 10 µM Y-27632, and resuspend the cell pellet by pipetting using a P1000 micropipette.
6. Place the cell suspension in a water bath set at 37 °C and incubate it for 10 min while periodically shaking the mixture.
7. Introduce 5 mL of warm organoid basal medium and vigorously pipette the cell suspension using a P1000 micropipette until it becomes cloudy, without any visible cell clumps.
8. Pass the cell suspension through a cell strainer with a 70 µm cutoff to eliminate any Matrigel debris and large cell clusters.
9. Centrifuge the conical tube at 200 × g and 4 °C for 5 min, followed by resuspension of the pellet in seeding medium. For the seeding of one canine Gut-on-a-Chip device, use 20 µL of seeding medium to resuspend the cell pellet (i.e., use 20 µL of seeding medium when seeding one Gut-on-a-Chip device).
10. Modify the concentration of viable cells to 1 × 10⁷ cells/mL with seeding medium as previously reported². Conduct viability assessment using a hemocytometer by combining 10 µL of the cell suspension with 10 µL of Trypan blue, and then observe the cells under a microscope.
    ​NOTE: It is crucial to adjust the cell concentration appropriately to ensure optimal cell attachment and the formation of a uniform monolayer on the chip membrane. If the initial number of cells is insufficient, it may result in delayed or unsuccessful establishment of a confluent monolayer. Conversely, if the number of cells is excessive, non-adherent cells can aggregate into clumps within the channel, leading to undesirable concentration effects.

4. Seeding and formation of a 2D cell monolayer

1. Disconnect the outlet tubing for the upper microchannel. Ensure that the bypass tubing of the upper microchannel remains open. Secure both the inlet and outlet of the lower microchannel by using binder clips to clamp them.
2. Using a P100 or P20 micropipette, introduce 20 µL of the cell suspension from Protocol 3 to the outlet hole of the upper microchannel (Figure 3B Seeding).
3. Secure the bypass and inlet tubing of the upper microchannel by using binder clips to clamp it. Then, reattach the outlet tubing to the outlet hole of the upper microchannel, ensuring that the tubing remains open throughout the process to avoid any pressure being applied to the upper microchannel. After this step, secure the outlet tubing of the upper channel slowly using a binder clip to clamp it.
4. Verify using a microscope that the cells are evenly distributed throughout the upper microchannel.
   NOTE: It is important to clamp the tubing to stop the movement of the medium within the channel to allow stable static conditions until the desirable cell attachment is accomplished.
5. Place the chip in a humidified CO₂ incubator at 37 °C.
   NOTE: It takes approximately 3 h for canine intestinal organoid cells to attach to the ECM coating (Figure 3B Attachment).
6. Connect the syringe attached to the upper microchannel of the Gut-on-a-Chip to a syringe pump positioned within a CO₂ incubator.
   NOTE: Carefully flush the medium into the microchannel using the knob of the syringe pump and remove unbound cells. Examine the chip under a microscope to ensure that any unattached cells have been effectively washed away.
7. Initiate the flow of culture medium at 30 µL/h with seeding medium. This continuous flow is only for the upper microchannel initially until the 2D monolayer establishment on a Gut-on-a-Chip. For the lower microchannel, leave the microchannels clamped and the medium unflushed.
8. From the day after the cells are seeded, change the culture medium to organoid culture medium, which contains A8301 and lacks 10 µM Y-27632 and 2.5 µM CHIR99021, from the seeding medium.
9. Once the monolayer is established, initiate the continuous medium flow to the lower microchannel as well. It usually takes 2 to 3 days for canine intestinal organoid epithelial cells to establish epithelial monolayers (Figure 3C).

5. Establishment of 3D morphogenesis in canine Gut-on-a-Chip

NOTE: After the confluent monolayers are formed in Gut-on-a-Chip, medium flow of both the upper channel and lower channel and cell strain were introduced to initiate 3D morphogenesis to 2D monolayer as presented in Figure 2.

1. Introduce the organoid culture medium into both the upper and lower microchannels to initiate the development of 3D morphogenesis in a Gut-on-a-Chip system. To achieve shear stress of 0.02 dyne/cm² in the existing Gut-on-a-Chip design (i.e., 500 µm height microchannel), increase the flow rate to 50 µL/h²⁹.
2. Initiate 10% of cell strain and 0.15 Hz of frequency, as recommended before², using a computer-regulated bioreactor applying cyclic strain to cells cultured in vitro. This process would apply vacuum suction to the Gut-on-a-Chip device.
3. Sustain these culture conditions for a minimum of 2 to 3 days. The 3D morphogenesis of the canine intestinal monolayer usually occurs 2 to 3 days after Dural channel flow and vacuum suction have been started (Figure 3C).

6. Characterization of canine Gut-on-a-Chip

1. Live cell imaging
   1. Remove any air bubbles in the Gut-on-a-Chip by gently flowing the medium using the syringe pump.
   2. Disconnect the Gut-on-a-Chip device from the syringe pump.
      NOTE: Avoid any maneuver that can apply pressure within the Gut-on-a-Chip.
   3. Position the device onto a microscope to capture images of the established 3D epithelium. For visualizing the structure of the 3D epithelial layers using phase-contrast, use 10x and 20x objectives (Figure 3C).
2. Immunofluorescence staining
   1. Prepare the blocking solution by dissolving 1 g of BSA in 50 mL of Phosphate buffer saline (PBS) to make 2% BSA. Pass the solution through a syringe filter of 0.2 µm for filtration. Keep this solution stored at 4 °C.
   2. Prepare the permeabilizing solution by combining 150 µL of Triton X-100 with 50 mL of blocking solution, resulting in a final concentration of 0.3% Triton X-100. Pass the solution through a syringe filter of 0.2 µm for filtration. Keep this solution stored at 4 °C.
   3. Perform fixation of the cells by injecting 100 µL of a 4% PFA into both the upper and lower microchannels as described in steps 2.4-2.11.
   4. Rinse the cells by injecting 100 µL of PBS into both upper and lower microchannels as described in steps 2.4-2.11.
   5. Perform permeabilization of the cells by injecting 100 µL of 0.3% Triton into both upper and lower microchannels as described in steps 2.4-2.11. Place the device at RT for 30 min.
   6. Rinse the cells by injecting 100 µL of PBS into both upper and lower microchannels as described in steps 2.4-2.11.
   7. Perform blocking of the cells to prevent nonspecific binding by injecting 100 µL of 2% BSA to both upper and lower microchannels as described in steps 2.4-2.11. Place the device at RT for 1 h.
   8. Inject 20 µL of the primary antibody solution diluted in 2% BSA and place it at RT for 3 h, followed by overnight incubation at 4 °C.
      NOTE: Make sure all the tubing is clamped during the incubation at 4 °C for overnight. The concentration of a primary antibody should be 2-5 times higher than the recommended concentration for monolayer or 3D organoid staining (Supplementary Figure S1).
   9. Rinse the cells by injecting 100 µL of PBS to both upper and lower microchannels as described in steps 2.4-2.11.
   10. Inject 20 µL of the secondary antibody solution diluted in 2% BSA and place it at RT for 1 h.
       NOTE: Make sure all the tubing is clamped during the incubation. Starting from this step, it is necessary to shield the device setup with aluminum foil to prevent photobleaching.
   11. Rinse the cells by injecting 100 µL of PBS to both upper and lower microchannels as described in steps 2.4-2.11.
   12. Prepare a combined solution for F-actin and DAPI (diamidino-2-phenylindole) counterstaining. Inject 20 µL of the combined solution into both upper and lower microchannels as described in steps 2.4-2.11.
   13. Rinse the cells by injecting 100 µL of PBS into both upper and lower microchannels as described in steps 2.4-2.11.
       Perform fluorescence imaging of the architecture of the 3D epithelial cells using a fluorescence microscope or a confocal microscope.

7. Epithelial barrier function

1. Remove any air bubbles in the Gut-on-a-Chip by gently flowing the medium using the syringe pump. Make sure all the tubing is open during the measurement.
2. Remove the Gut-on-a-Chip from the syringe pump and place it at RT for at least 10 min.
3. Remove Ag/AgCl electrodes from 70% EtOH solution.
4. Place two Ag/AgCl electrodes into the upper inlet and lower outlet, respectively, to measure the resistance of the epithelial layer using a multimeter.
5. Place two Ag/AgCl electrodes into the lower inlet and upper outlet, respectively. Report the mean of these two values as a resistance value for the Gut-on-a-Chip.
   NOTE: The blank TEER should be measured on a Gut-on-a-Chip with only ECM coating without the epithelium.
6. Calculate transepithelial electrical resistance (TEER) value (kΩ × cm²) can be calculated using equation (1).
   TEER = (Ω_t - Ω_blank) × A     (1)
   ​Where Ω_t is a resistance value measured at the specific time point since the start of the experiment, Ω_blank is a resistance value measured at the time without the epithelium, and A is the area of the surface covered by a cell layer (approximately 0.11 cm² for this Gut-on-a-Chip design²⁹).
   1. Calculate normalized TEER using equation (2).
      TEER =       (2)
      Where Ω₀ is a resistance value at the initial reading time point of the experiment as reported previously³⁰ (Figure 4C).