Profiling Luminal pH in Three-Dimensional Gastrointestinal Organoids Using Microelectrodes

The overall goal of our research is to understand how gastric epithelial cells, immune cells, stromal cells and their products, such as cytokines and mucus, work together to respond to infection with gastric pathogen Helicobacter pylori. To that end, we are trying to build complex physiologically relevant models of the human gastric mucosa. Previous studies with gastric organoids have shown conflicting results regarding the ability of these models to maintain acid-secreting parietal cells.

Using the pH microelectrodes, we demonstrated that in the absence of specific differentiation or stimulation protocols, human gastric organoids maintain a near-neutral pH in the lumen. Relative in accessibility, the organoid lumen has long restricted our understanding of the microenvironment within. We demonstrate the first successful microelectrode pH measurements for the functional characterization of organoids.

Our microelectrode-based method could provide researchers with a reliable tool that can be adapted to their specific needs. Until now, pH measurement within gastrointestinal organoids has involved the use of pH-sensitive dyes that rely on microscopic imaging for quantification. The main advantage of using microelectrodes is that they provide accurate numerical pH readings.

In addition, intraluminal pH can be recorded in real time and with superior spatial resolution. We are working toward a fully functional model of the human gastric mucus layer with a physiological pH gradient So we can study the role of this barrier in Helicobacter pylori infection. The ultimate goal is to develop new cytoprotective treatments that strengthen the mucus layer and prevent bacterial invasion.

To begin, obtain actively growing human gastric organoids with diameters between 200 and 700 micrometers. Thaw the extracellular matrix or ECM aliquot on ice for at least 45 minutes, and prewarm cell culture plates at 37 degrees Celsius in a 5%carbon dioxide incubator. After removing the media from the wells of the culture plate, pipette ice cold PBS onto each ECM droplet, and scratch the gel with the P1000 pipette tip.

Collect the PBS with the ECM fragments containing organoids into a 15-milliliter conical tube. Centrifuge the tube at 200 g at four degrees Celsius for five minutes. After aspirating the supernatant, add 350 microliters of 0.25%Trypsin-EDTA into each tube, and mix gently.

Incubate the tubes in a water bath tempered at 37 degrees Celsius for two to five minutes. Add 600 microliters of ice cold DMEM supplemented with penicillin and streptomycin to each tube, and vigorously pipette up and down 40 times. Centrifuge the tube as demonstrated, and resuspend the cell palate in ice cold liquid ECM.

For each sample, plate 40 microliters of the liquid ECM containing organoid fragments in a thin horizontal line along the diameter of a 35-millimeter glass-bottom dish. After 15 to 30 minutes of incubation, carefully add two milliliters of organoid expansion media to the plate along the edge of the dish without disturbing the polymerized gel. 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.