preparation of human gastric organoids for ph profiling to study luminal environment

Exakt Luminal pH-mätning i 3D magorganoider med mikroelektroder

Organoids-miniature multicellular structures derived from stem cells-have revolutionized our ability to study human physiology and are starting to replace animal models, even in regulatory settings1. Since the initial description of intestinal organoids by Sato et al. in 2009, organoid technology has become immensely popular2. A large number of studies have characterized the cellular composition and function of organoid models in great detail3,4,5,6. However, the luminal space of these 3D multicellular structures remains largely undefined7,8. The lumen is the central cavity of organoids derived from mucosal tissues that is surrounded by the apical portions of polarized epithelial cells. Since cellular secretion and absorption predominantly occur at the apical epithelial surface, the luminal microenvironment of organoids is controlled by these important physiological processes. Currently used organoid models demonstrate variations in cell signaling patterns, overall stemness, metabolite concentration gradients, and environmental conditions9. Understanding organoid luminal physiology is therefore necessary for the accurate modeling of organ function and pathology. Unfortunately, the relative inaccessibility of the lumen significantly hinders functional analyses of luminal physiology in 3D organoids10.
The ability to examine pH profiles is especially important in the stomach, which is notorious for having the steepest proton gradient in the body, ranging from approximately 1-3 in the lumen, to near neutral at the epithelium11,12,13.There remains a significant gap in our understanding of the microscale maintenance of the gastric pH gradient, and the relevance of organoid models in recapitulating this dynamic milieu across the gastric mucus layer. Traditional approaches for the analysis of organoid pH have involved the use of pH-sensitive dyes, which can be fluorescent or colorimetric indicators. McCracken et al. used a luminal injection of SNARF-5F-a ratiometric pH indicator-into organoids to analyze a drop in luminal pH in response to histamine treatment. Such dyes can be incorporated into the culture media, allowing for real-time, non-invasive monitoring of pH. Not only do pH-sensitive dyes require complex calibration steps that contribute to poor reliability and accuracy with measurements, but such dyes also tend to operate within specific detection ranges that may not be representative of the full pH range within the microenvironment of interest14,15. It could be considered reasonable, however, to use indicator dyes for confirmatory experiments. Optical nanosensors that use fluorescent optode-based, pH-sensing approaches have also been developed; however, such sensing techniques require microscopic imaging and are also susceptible to photobleaching, phototoxicity, as well as imaging bias16,17. Additionally, Brooks et al. have 3D-printed multiwell plates containing microelectrodes on top of which organoids may be plated18. This approach, however, does not allow for measurements directly inside the organoid lumen.
Electrode-based pH measurements can achieve improved accuracy compared to other methods, as well as provide real-time pH monitoring. In addition, pH electrodes mounted on micromanipulators allow for superior spatial resolution of pH measurements as the precise location of the electrode tip can be finely controlled. This enables the highest possible flexibility and reproducibility in the analyses of organoid models. The electrodes used here are miniaturized pH microelectrodes that operate based on the diffusion of protons through selective pH glass that surrounds a thin platinum wire. The microelectrode is connected to an external Ag-AgCl reference electrode and then connected to a high-impedance millivolt meter. The electrical potential between the two electrode tips when submerged in the same solution will reflect the pH of the solution19. Such microprofiling systems have been used in the metabolic analysis of biofilms20,21, planktonic algae22, human sputum samples23, and even in mesenchymal stem cell spheroids24. Both our lab and Murphy et al. have previously used micromanipulator-controlled O2 microelectrodes to evaluate the oxygen concentrations in the luminal spaces of organoids. Murphy et al. paired this method with mathematical modeling to reveal an oxygen gradient within their spheroids. Our group was able to find reduced luminal oxygen levels in tissue-derived gastric organoids compared to the surrounding extracellular matrix25.
Here, we provide a detailed method for the manual microelectrode profiling of the luminal pH in spherical gastrointestinal tract organoids that will enable enhanced physiological understanding of their complex luminal microenvironment. We anticipate that this technique will add a new dimension to the exploration of organoid physiology through real-time, high-resolution measurements of pH levels at a microscale. Furthermore, the following protocol could be easily adapted for the analysis of O2, N2O, H2, NO, H2S, redox, and temperature in various types of organoid models. Physiological profiling serves as a valuable tool for optimizing organoid culture conditions to better mimic in vivo environments, thereby enhancing the relevance and utility of organoid models in biomedical research.

Författare i fokus: Gastric Epithelial Cell Responses in Helicobacter pylori-infektion

Profilering av luminalt pH i tredimensionella gastrointestinala organoider med hjälp av mikroelektroder

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 the 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.

Förberedelse av humana magorganoider för pH-profilering för att studera luminal miljö

preparation-human-gastric-organoids-for-ph-profiling-to-study-luminal

Research

JoVE Journal

Medicine

Preparation of Human Gastric Organoids for pH Profiling to Study Luminal Environment

Profiling Luminal pH in Three-Dimensional Gastrointestinal Organoids Using Microelectrodes

The optimization and detailed characterization of gastrointestinal organoid models require advanced methods for analyzing their luminal environments. This paper presents a highly reproducible method for the precise measurement of pH within the lumina of 3D human gastric organoids via micromanipulator-controlled microelectrodes. The pH microelectrodes are commercially available and consist of beveled glass tips of 25 &#181;m in diameter. For measurements, the pH microelectrode is advanced into the lumen of an organoid (&gt;200 &#181;m) that is suspended in Matrigel, while a reference electrode rests submerged in the surrounding medium in the culture plate. 
Using such microelectrodes to profile organoids derived from the human gastric body, we demonstrate that luminal pH is relatively consistent within each culture well at ~7.7 &plusmn; 0.037 and that continuous measurements can be obtained for a minimum of 15 min. In some larger organoids, the measurements revealed a pH gradient between the epithelial surface and the lumen, suggesting that pH measurements in organoids can be achieved with high spatial resolution. In a previous study, microelectrodes were successfully used to measure luminal oxygen concentrations in organoids, demonstrating the versatility of this method for organoid analyses. In summary, this protocol describes an important tool for the functional characterization of the complex luminal space within 3D organoids.

The present protocol describes pH measurements in human tissue-derived gastric organoids using microelectrodes for spatiotemporal characterization of intraluminal physiology.

The optimization and detailed characterization of gastrointestinal organoid models require advanced methods for analyzing their luminal environments. This ...

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 P-1000 pipette tip. Collect the PBS with the ECM fragments containing organoids into a 15 milliliter conical tube.Centrifuge the tube at 200G 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 re-suspend the cell palette 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.

High-Resolution pH Profiling in Gastric Organoids Using Microelectrodes

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.