Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

The research was approved and performed in compliance with the Institutional Animal Care and Use Committee of Iowa State University (IACUC-19-337; IACUC-18-065; IACUC-19-017). The following section (steps 1.1-1.3) describes the Permeable Support Seeding Protocol, and the procedures are summarized in Figure 3.

Figure 3: Workflow of the permeable support seeding protocol. The permeable support inserts are precoated with a combination of CMGF+ R/G, collagen I, and the extracellular membrane matrix and subsequently incubated. Media from the canine organoid culture is aspirated and replaced with a Cell Recovery Solution, followed by a 30 min incubation at 4 °C. The culture is subsequently transferred to a tube, and organoid dissociation is performed using trypsin-like protease. Undissociated organoids are removed by passage through a strainer to achieve a single-cell suspension, and cell concentration is determined using a hemocytometer or an automated cell counter. The cells are seeded on a permeable support insert, and CMGF+ R/G is added to the basolateral chamber. The culture is then incubated for 24 h, and the remaining liquid is removed from the apical chamber and replaced with CMGF+ R/G. Abbreviations: ROCK = rho-associated kinase; GSKiβ = glycogen synthase kinase beta; CMGF + R/G = Complete medium with growth factors enhanced with ROCK inhibitor and GSKiβ. Please click here to view a larger version of this figure.

1. Permeable support seeding protocol

1. Precoating of the permeable support inserts
   1. Prepare Complete medium with growth factors enhanced with Rho-associated protein kinase (ROCK) inhibitor and glycogen synthase kinase-beta inhibitor (GSKiβ) (CMGF + R/G) according to the information in Table 1.
   2. Prepare an ice bucket and begin thawing the extracellular membrane matrix on ice. Place a 24-well plate containing the needed number of inserts in the incubator to prewarm. Collect rat tail collagen I (3 mg/mL) and place it on ice while protecting from light. Collect CMGF + R/G and place it on ice.
   3. Calculate the total number of inserts and blanks required for the experiment, keeping aside 100 µL of the coating solution for each insert.
      NOTE: Prepare more coating solution than needed; preparing at least 15% more than needed is recommended.
   4. In a 15 mL tube, mix CMGF+ R/G with the extracellular membrane matrix (1%) and collagen I (1%) and gently pipet mix.
   5. Coat each polyester insert with 100 µL of the coating solution and place the inserts in the incubator (37 °C; 5% CO₂ atmosphere) for 1 h.
   6. After the incubation, carefully aspirate the coating solution off each insert using a vacuum aspirator or a P1000 pipette, being careful not to disturb the insert filter. Place a precoated plate in the incubator to keep warm.
2. Canine organoid dissociation
   NOTE: Use canine organoids that have been cultured for at least four days. Before beginning dissociation, refer to Gabriel et al.³⁸ to determine when a sample is healthy, dense, and sufficient for experimentation. It is recommended to dissociate one extra well of organoids for every well plating procedure. Furthermore, increasing the desired number of inserts by ~20% to account for uneven organoid growth or damage caused by improper manipulation is recommended. If planning to use FITC-dextran, prepare extra wells.
   1. Prepare an ice bucket and a vial of cold 1x Advanced DMEM/F12 stock in the biosafety cabinet.
   2. Place the extracellular membrane matrix on ice to begin thawing, submerging it in ice to protect against rapid thawing and avoid solidification. Place a box of pipette tips (P200) in the freezer for the plating of the extracellular membrane matrix.
   3. Prechill a refrigerated centrifuge to 4 °C.
   4. Move CMGF+ R/G from the freezer/refrigerator to a 37 °C water bath. Avoid direct light exposure when possible.
   5. Remove all medium from 24-well plate with organoid culture for an appropriate number of wells (1 well of a 24-well plate per 2-4 inserts) while taking care to not disturb the extracellular membrane matrix.
      NOTE: The volume might vary depending on the cell counting system used.
   6. Add 0.5 mL of prechilled Cell Recovery Solution per well to dissolve the extracellular membrane matrix domes.
   7. Incubate the plate in the refrigerator (4 °C) for 30 min.
   8. Pipette the suspension, collect all organoids and dissolved extracellular membrane matrix, and transfer them to a 15 mL tube.
   9. Centrifuge (700 × g for 5 min at 4 °C) and remove the supernatant until the level reaches the 0.5 mL mark, making sure not to disturb the pellet.
   10. Add 1 mL of the trypsin-like protease and incubate in the 37 °C water bath for 8 min. Flick the tube several times during the incubation period to mix the cells.
   11. Transfer the tube with the sample back to a biosafety cabinet and slowly add 7 mL of prechilled Advanced DMEM/F12 to inactivate the trypsin-like protease and stop cell dissociation.
   12. Prewet a 40 µm cell strainer with 1 mL of Advanced DMEM/F12. Gently pipet the mixture and filter the suspension through; pipette additional Advanced DMEM/F12 to rinse the strainer.
   13. Centrifuge the tube (700 × g for 5 min at 4 °C) and remove the supernatant. Do not disturb the pellet.
   14. Resuspend the cell pellet in ~50-100 µL of culture medium (CMGF+ R/G) for every well of organoids that was disassociated.
   15. Count a subsample of the suspension (~10 µL) using a hemocytometer or appropriate machine and determine the total cell number in the suspension.
3. Canine organoid seeding
   1. Dilute or concentrate the cell suspension to obtain a cell concentration of ~75,000 cells per mL.
   2. Seed 100 µL of the suspension into each insert using BSA (1%) precoated tips to avoid cell adhesion during transfer. Add one coated insert as a cell-free blank without any organoids growing while still receiving regular media changes.
   3. Gently swirl the plate in a circular motion for ~30 s to disperse the seeded cells across the insert. Confirm via light microscopy the even distribution of cells.
   4. Add 700 µL of CMGF + R/G to the basolateral chamber and place the plate in the incubator (37 °C; 5% CO₂ atmosphere) for 24 h.
   5. After 24 h, gently remove the cell suspension from the apical chamber and replace it with 200 µL of CMGF + R/G. Return the plate to the incubator.

2. Organoid cell monolayer maintenance protocol

NOTE: The following section (steps 2.1-2.2) describes the organoid cell monolayer maintenance protocol. The workflow of procedures presented in this protocol is summarized in Figure 4. A table for note-taking that can help with the standardization of TEER value measurements is presented in Supplemental Table 1.

Figure 4: Workflow of the permeable support culture maintenance. TEER values are measured using electrodes (probes) and a volt/ohm meter. Probes must be chemically sterilized with 70% alcohol prior to inserting into the wells. The blank and organoid cell inserts are measured, and TEER values are calculated. Medium is subsequently refreshed in both apical and basolateral chambers, and the canine organoid culture on the insert is visualized using light microscopy. Tears in either organoid culture or microporous membrane are noted and handled according to the protocol. Abbreviation: TEER = transepithelial electrical resistance. Please click here to view a larger version of this figure.

1. TEER value measurement
   NOTE: TEER value measurements are performed using an Epithelial Volt/Ohm Meter with chopstick attachment. See the manufacturer's instructions for use. TEER values provide information on the integrity of the canine organoid monolayer.
   1. Take TEER value measurements on every alternative day during cell culture growth.
   2. Move the Epithelial Volt/Ohm Meter and its electrodes to the biosafety cabinet. Chemically sterilize the electrodes in 70% alcohol before use. Set the function to Ohms. Wait at least one minute until the electrodes dry.
   3. Before taking the first measurement, insert the wire electrode into the port and turn on the power. Ensure that the meter displays 1,000 Ω with the wire electrode insert instead of measuring chopsticks. If this is not the case, adjust the device.
   4. Insert the electrodes in the apical and basolateral chamber of the cell-free insert (blank), so the apical chamber contains the shorter electrode, and the basolateral chamber contains the longer electrode (as seen in Figure 4). Do not touch the membrane, but at the same time, ensure that the electrodes are submerged in the medium.
   5. Wait for a few seconds until the value stabilizes and note the value in a lab book. Measure the remaining canine organoid monolayers, making sure to sterilize the electrodes with 70% alcohol when measuring different samples. Take care not to touch the organoid monolayer with the electrode.
   6. After measurements are taken, sterilize the electrodes with 70% alcohol for the last time. Be sure to protect them from damage caused by inappropriate manipulation and store them according to the manufacturer's instructions.
   7. Calculate the TEER values for every well using Eq (1), where R_sample and R_blank are the ohm (Ω) values from the monolayer and blank wells, respectively, and the area (cm²) is that of the insert.
      (1)
2. Monolayer upkeep
   NOTE: Review the recommended medium change plan in Table 2.
   1. Using sterile disposable 9" Pasteur pipets and a vacuum aspirator, gently aspirate the medium from the apical and then the basolateral chambers. Tilt the plate to see the medium surface clearly. Avoid aspirating too close to the microporous membrane in the apical chamber to prevent damage to the cell monolayer.
      NOTE: Use a new Pasteur pipet when moving between samples. Pipettes are also a possible replacement for this procedure.
   2. Slowly add CMGF+ R/G using P1000 pipettes aiming for a wall of the apical or basolateral chamber. Perform the medium change in the apical chamber very carefully to avoid damaging the monolayer.
   3. Check the wells every other day under a light microscope, evaluating the health of the culture and monitoring for tears in the organoid monolayer or the microporous membrane. Use phase-contrast microscopy to highlight details of the culture.
      NOTE: In the case of canine organoid monolayer tears, the monolayer is given time to recover and regrow. In the case of microporous membrane tears, the well must be excluded from the experiment.

Table 2: Media change recommendation for organoid cultures. CMGF+ R/G is changed in the apical and basolateral chamber of the wells every other day on every alternative day. The longer culture period over the weekend demands an increased volume of medium in both basolateral and apical chambers, which is applied on a Friday afternoon and changed on Monday. Abbreviations: ROCK = rho-associated kinase; GSKiβ = glycogen synthase kinase beta; CMGF + R/G = Complete medium with growth factors enhanced with ROCK inhibitor and GSKiβ. Please click here to download this Table.

3. Permeability experimental protocol

NOTE: The following section (steps 3.1-3.5) describes the Permeability Experimental Protocol. The experimental protocol workflow to measure a drug's in vitro permeability is summarized in Figure 5.

Figure 5: Workflow of the experimental protocol. Organoid medium must be changed from CMGF+ R/G to CMGF+ between eight to twelve days after seeding the inserts, allowing for cellular differentiation. After changing the medium (both apical and basolateral) to CMGF+, the TEER values should still be increasing, with the canine organoid monolayer almost reaching confluency. At least four days after the medium is changed to CMGF+, the readiness of the monolayers is evaluated. When the monolayer is completely formed, and TEER values reach a plateau phase (typically between 1,500 and 2,500 Ω.cm²), the medium is exchanged for the transport buffer for 30 min to allow the monolayer to adjust to the new environment. At time 0 min of the experiment, the FITC-dextran assay is performed, and the 20 min basolateral sample is collected. Results are subsequently analyzed on a plate reader. After the experiment, the apical and basolateral chamber contents are again changed to CMGF+, and TEER value readings are taken. The monolayers are incubated for 24 h, and the integrity of the monolayer is assessed through repeated TEER measurements. Abbreviations: TEER = transepithelial electrical resistance; ROCK = rho-associated kinase; GSKiβ = glycogen synthase kinase beta; CMGF + R/G = Complete medium with growth factors enhanced with ROCK inhibitor and GSKiβ; CMGF+ = Complete medium with growth factors but without ROCK inhibitor or GSKiβ; FITC = fluorescein isothiocyanate; F = fluorescence. Please click here to view a larger version of this figure.

1. Evaluating organoid monolayer readiness
   NOTE: This step occurs 8-14 days after seeding.
   1. Check the monolayer at least every other day under the light microscope (use phase-contrast to visualize the monolayer's integrity). Continue to the next step when the cell monolayer is fully formed without gaps or apparent signs of tears.
   2. Change the organoid medium from CMGF+ R/G to CMGF+ (excluding ROCK inhibitor and GSKiβ from the medium composition). Swapping the medium at least four days prior to the experiment is recommended.
      NOTE: This step will allow for proper differentiation of the organoid monolayer.
   3. Continue measuring TEER values approximately every other day. When TEER values start to plateau at approximately 1,500 to 2,000 Ω × cm² (Figure 6), measure the TEER values every day.
      NOTE: This steady state can be maintained for approximately 2-3 days, which is the optimal window of time to perform permeability testing (usually at days 11 to 13). The plateau TEER values may slightly oscillate based on the intestinal localization of the organoids, measurement temperature, breed, age, and disease state of the dog.
   4. Schedule the drug permeability assay immediately to avoid a rapid decrease in TEER values or overgrowth of the organoid monolayer to multiple cell layers.
2. Preparing for the experiment
   NOTE: Incubator shakers can be used during the experiment to avoid the effects of the unstirred water layer. Measure TEER values at consistent temperatures.
   1. On the day of the experiment, measure TEER values and confirm that the values reached a steady state and are not declining rapidly.
   2. Choose the best monolayers (via light microscopy and TEER values) from the excess of 20% inserts to perform the experiment.
   3. Observe the monolayers under a light microscope and exclude incomplete, torn, or overgrown organoid monolayers.
   4. Prepare the transport buffer and adjust its pH to desired values.
      NOTE: The composition of the experimental buffer differs based on the experimental setup. A frequently used buffer is composed of Hank's Balanced Salt Solution (HBSS), glucose (12.5 mM), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 25 mM). This composition ensures the viability of organoid culture during an experiment.
   5. Carefully aspirate the medium from the apical and basolateral chambers of the selected wells.
   6. Add 200 µL of transport buffer to the apical chamber and 800 µL to the basolateral chamber.
      NOTE: Transport buffer should be first added to the apical chamber and then to the basolateral chamber to avoid detachment of the monolayer from the microporous membrane.
   7. Place the plate in the incubator (37 °C; 5% CO₂ atmosphere) for 30 min to equilibrate.
      NOTE: The canine organoid monolayers are now ready for the drug permeability experiment.
3. Typical experimental layout-IgY concentration solution
   NOTE: The experimental design and layout may change depending on the research question. The permeability of immunoglobulin Y (IgY) through an organoid monolayer is used in the protocol as an example and can be modified. The term donor chamber refers to the chamber where the drug is initially applied, while the receiving chamber refers to the chamber accepting the drug from the donor chamber. A typical experiment collects samples in the receiver chambers over 2 h (e.g., 15, 30, 60, 90, 120 min).
   1. Prepare the IgY solution (drug or solute of choice) by dissolving it in the transport buffer to the desired final concentration. Prepare more drug solution than needed.
      NOTE: Drugs with low aqueous solubility may be first dissolved in an organic solvent (e.g., ethanol, DMSO) before adding to the buffer. The final concentration of the solvent should be less than 1% so as not to damage the cell monolayer.
   2. Remove the buffer from the donor chamber (either apical or basolateral chamber) of each well.
   3. Add the IgY solution (drug solution) to all the donor chambers. Use the remaining solution as the time 0 donor solution for the measurement of the initial drug concentration.
   4. At the required time points, remove 50 µL from the receiving chamber and place it in a labeled tube. At the last time point, remove a sample from the donor chamber. At the end of the experiment, transfer the donor and receiver aliquots to a -20 °C freezer.
      NOTE: If many time points are needed, replacement of buffer in the receiving chamber may be done but must be accounted for in the calculation of the apparent permeability. The concentration in the receiver chamber should not reach more than 10% of the donor chamber at the end of the experiment to maintain sink conditions⁴⁴.
4. Organoid cell monolayer quality control
   NOTE: FITC-dextran solution may be used to confirm monolayer integrity during the experiment. FITC-dextran is used as an example of a monolayer integrity assay. Others include Lucifer yellow, PEG-4000, radiolabeled mannitol, and inulin⁴⁴.
   1. At time 0 min, aspirate the contents of the apical chamber and replace with 250 µL of FITC-dextran solution (5 mg/mL, 4 kDa) in triplicate for each experimental group.
      NOTE: Do not expose FITC-dextran to light.
   2. After 20 min, remove the buffer from the basolateral chamber.
   3. Measure the fluorescence intensity of the basolateral sample using a fluorescence plate reader (use a calibration curve; excitation set at 485 nm and emission value at 528 nm).
      NOTE: P_app of FITC can also be calculated using the method described in the discussion.
   4. After the experiment concludes, carefully aspirate excess buffer from the apical and basolateral chambers.
   5. Add 200 µL of CMGF+ in the apical chamber and 700 µL to the basolateral chamber.
   6. Measure TEER values in the individual wells.
   7. Place the plate in the incubator (37 °C; 5% CO₂ atmosphere) for 24 h.
   8. If applicable, after 24 h, measure TEER values to assess possible damage to the monolayer during the quality control portion of the experiment. Use light microscopy to visualize the integrity of the canine organoid monolayer.
5. Fixing cell monolayers for downstream analysis
   1. Prepare Formalin-Acetic Acid-Alcohol solution (FAA, composition in Table 1).
   2. Remove the transport buffer or CMGF+ from the apical and basolateral chambers using a P1000 pipet or sterile disposable 9" Pasteur pipets and a vacuum aspirator.
   3. Fill the apical and basal chambers with FAA.
   4. After 24 h, aspirate the FAA and replace it with 70% ethanol.
   5. Wrap the plate with flexible laboratory film to prevent evaporation and proceed to block preparation.

Figure 6: TEER values across experimental groups. TEER values were measured in five groups of canine intestinal organoid monolayers. Three groups were composed of canine jejunal enteroids, and two groups consisted of colonoids. Each group included 12 to 22 replicates. The TEER values for jejunal enteroid cultures are shown from Day 4 to Day 14, and colonoid cultures are shown from Day 4 to Day 12 (measurements ended as the organoid monolayer reached steady-state values, indicating the monolayer was ready for experimental use). Error bars express SEM of the measurements. Abbreviation: TEER = transepithelial electrical resistance. Please click here to view a larger version of this figure.