We aim to develop and characterize an organoid-based model to mimic the gastrointestinal tract and compare it with living pigs, and to focus on the transport characteristics. The change from a classical animal experiment to. In our field of the gastrointestinal physiology to an.
Organoid-based in-vitro model, which mimics the in-vivo situation. We combined our model of the small and large intestine of organoids with. Which can be used to investigate the transport characteristics in real time to allow a compare-ment of our model and the living pig situation.
Especially in the field of livestock-related research, alternative models to the classical animal experiments are rare. We overcome this limitation by using our organoid-based model of the porcine intestinal tract. We plan to use our organoid-based intestinal model to study the effects of swine pathogenic bacteria.
This aims to understand the pathogenicity mechanisms of these bacteria. To begin, mix the freshly-thawed basement membrane at a ratio of 1:40 with ice-cold sterile PBS in a conical tube. Add 200 microliters of this mix to the apical compartment of each insert within the sterile plates.
Replace the lid of the well plate and incubate for at least 1.5 hours at 37 degrees Celsius with 5%carbon dioxide. After incubation, carefully aspirate the solution without touching the membrane. Remove the organoid medium from the wells containing three-dimensional crypt organoids.
To dissolve the basement membrane, add one milliliter of ice-cold PBS to the well and pipette up and down with a P1000 tip. Collect all dissolved organoids in a 15-milliliter tube prefilled with 10 milliliters of ice-cold PBS. Centrifuge the tube at 250 G for 10 minutes at four degrees Celsius.
After aspirating the supernatant, re-suspend the pellet in one milliliter of warm 0.05%trypsin EDTA. Incubate the tube for five minutes at 37 degrees Celsius in a water bath, and then, place it on ice to stop the reaction. With a P1000 tip, pipette up and down 20 times to thoroughly re-suspend the organoids, and then, repeat for another 15 times using a P1000 tip with a P200 tip attached on top.
Now, add 10 milliliters of ice-cold DMEM supplemented with 10%fetal calf serum, or FCS. Centrifuge the tube at 1000 G for 10 minutes at four degrees Celsius. After discarding the supernatant, re-suspend the pellet in one milliliter of monolayer medium.
Using a Neubauer chamber, follow the manufacturer's instructions to determine the number of living cells per milliliter. Now, replace the coating solution from the apical compartment with 500 microliters of the monolayer medium, tempered at 37 degrees Celsius, and add three milliliters of the monolayer medium to the basal lateral compartment. Remove the apical monolayer medium.
For 2D culture, add 500 microliters of monolayer medium containing an appropriate amount of cells to the apical chamber of each transwell and incubate the cells. For trans-epithelial electrical resistance, or TEAR measurement, clean the chopstick electrode with 70%ethanol and let it dry completely. Introduce the short arm of the chopstick electrode to the apical compartment, and the long arm into the basal lateral compartment of the inserts.
Next, turn on and let the. Meter equilibrate for a stable measurement, then click the store button to record TEAR values. After measuring the last well, click save to store data on a USB device.
Clean the electrode after each plate and at the end of the measurement. Allow the electrode to dry completely before storage. Subtract blank TEAR values determined before seeding the cells from the measured cellular values.
Change the medium and measure TEAR every two to three days, ensuring TEAR is measured before the medium is changed. Carefully add 500 microliters, or three milliliters, of fresh, warm differentiation medium to the apical and basal compartments. On day 18 for jejunal organoids, or day nine for colonic organoids, after seeding, proceed with functional experiments.
Warm up mucosal, and serosal buffer solutions to 37 degrees Celsius and aerate with carbogen. Assemble the single chambers using an empty insert for each individual chamber, ensuring the apical sides of the transwells are all facing in the same direction. Fill all chambers with five milliliters of pre-warmed mucosal buffer solution.
Connect all electrodes from the voltage clamp to the individual chambers following the manufacturer's instructions. To calibrate the Ussing chamber software, click the RFDPI button in the voltage clamp software. Confirm that the resistance of all empty inserts is approximately 70 ohms and the current is around zero millivolts.
Remove all electrodes and discard the used buffer solution. Open the individual chambers, remove the empty inserts, and maintain the order of the chambers. Now, under the safety cabinet, carefully aspirate the basolateral and apical medium from the plate.
Wash the cells with 500 microliters of warm mucosal buffer in the apical chamber, and with three milliliters of serosal buffer in the basolateral chamber. Remove the inserts from the plate and gently remove their supports. Place the inserts into the Ussing chambers, ensuring the orientation matches the calibration phase.
After assembling the individual chambers, fill the chambers facing the basolateral side of the cells with five milliliters of serosal buffer, and those facing the apical side with five milliliters of mucosal buffer. Connect the electrodes and aeration tubes to each individual chamber, and start the measurement in the Ussing software. Change the conditions from open-circuit to short-circuit mode in the software.
After five to 10 minutes of equilibration, add 10 micromolar forskolin to the serosal chamber. After 15 minutes, stop the measurement, remove the aeration tubes and electrodes from the chambers, and disassemble the chambers. Trans-epithelial electrical resistance values progressively increased during cultivation, peaking at 150 ohms times centimeter squared for jejunum after 18 days, and 200 ohm centimeter square for colon organoids after nine days.
The basal short-circuit current values were significantly lower in jejunum organoids than in colon organoids, while basal resistance values showed no significant differences. Forskolin treatments significantly increased basal short-circuit current values in jejunal organoids from 0.86 to 27.78 microamperes per square centimeter, and in colonic organoids from 3.83 to 28.78 microamperes per square centimeter.
