Obtain previously prepared human gastric organoids for pH profiling. To begin, connect the reference electrode to the pH electrode cable via the connector. Then, connect the microelectrode to the amplifier and the amplifier to a grounded PC with the software via USB cable.
Place the microelectrode and the reference electrode in deionized water to submerge both tips at least one centimeter in the liquid. Fill 50-milliliter conical tubes with desired calibration buffers. Using a delicate laboratory wipe, gently blot the protective tubing along with the reference electrode.
After opening the software, under the calibration tab, select the Y-Zoom box and then set the sensor signal reading to millivolts. Starting with the electrodes in the pH 4.01 buffer, enter 4.01 as the known pH value. Select Add point once the millivolt reading stabilizes, then place both electrodes back in deionized water to rinse them.
After repeating the process for the pH 9.21 buffer, click Add point when the signal is stable at around 83 millivolts. Check that the microelectrodes respond linearly between pH 4.01 and 9.21 for a two-point calibration curve. Carefully lay the protection case flat on the bench and pull the case off in a swift, quick motion to remove the microelectrode.
Mount the microelectrode on a micromanipulator and arrange the stereoscope and micromanipulator so the microelectrode can advance toward the culture dish freely. Position the culture dish containing the organoids for profiling. Lightly secure the reference electrode to the clamp on the ring stand to the left of the stereoscope.
Visually advance the tip of the microelectrode until it is sufficiently submerged in the media. Once the signal has stabilized, record three pH readings of the media. Then, slowly advance the microelectrode into the ECM without touching any organoids.
Record at least three pH readings to calculate the average. Position the microelectrode along the perpendicular axis to the organoid surface, gently making a minor indentation on the basolateral organoid surface without penetrating it. Carefully advance the microelectrode into the organoid and measure the luminal pH.
After completion, proceed to measure the pH for the next organoid. Place the electrode to be cleaned back in its protection tube. Flush the electrode serially with deionized water, ethanol, and buffer.
Place the reference electrode in a beaker filled with a three-molar potassium chloride solution. Backfill a two microliter glass capillary with sterile mineral oil and load it onto a micromanipulator-controlled nanoliter autoinjector. Finally, fill it with a solution containing 0.02%methyl red and 150 millimolar hydrochloric acid and perform the injection with 9.2 nanoliters of the solution.
Variation in luminal pH across 10 organoids on the same culture plate was minimal, with measurements consistently around 8.16. The intraluminal pH across five different organoid lines was consistent within each culture, but significant variability was observed between organoid lines. The organoid luminal pH was consistently lower than the ECMs which in turn was lower than the media's pH, suggesting that the luminal pH is physiologically relevant and not solely determined by the surrounding culture environment.
Methyl red injection into the organoid lumen confirmed that it had a pH greater than 6.2, consistent with the microelectrode measurements that showed a near neutral pH.
