Profiling Luminal pH in Three-Dimensional Gastrointestinal Organoids Using Microelectrodes
Summary
The present protocol describes pH measurements in human tissue-derived gastric organoids using microelectrodes for spatiotemporal characterization of intraluminal physiology.
Abstract
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 µm in diameter. For measurements, the pH microelectrode is advanced into the lumen of an organoid (>200 µm) 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 ± 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.
Introduction
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.
Protocol
Representative Results
Discussion
Limited access to the luminal space of organoids has severely restricted our understanding of the physiological dynamics of this microenvironment. A reliable tool for functional analyses of luminal physiology will expand our ability to leverage organoids as in vitro models for physiology, pharmacology, and disease research. Organoids are highly tunable, physiologically relevant models with the added potential to replicate genetic variability within the human population. Existing methods for pH measurement inside…
Divulgations
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge Dr. Ellen Lauchnor, Dr. Phil Stewart, and Bengisu Kilic for their previous work and assistance with the O2 microsensors; Andy Sebrell for training in organoid culture and micromanipulation; Lexi Burcham for assistance in organoid culture, media preparation, data recording, and organization; and Dr. Susy Kohout for general advice in electrophysiology. We would like to thank Dr. Heidi Smith for her assistance with imaging and acknowledge the Center for Biofilm Engineering Bioimaging Facility at Montana State University, which is supported by funding from the National Science Foundation MRI Program (2018562), the M.J. Murdock Charitable Trust (202016116), the US Department of Defense (77369LSRIP & W911NF1910288), and by the Montana Nanotechnology Facility (an NNCI member supported by NSF Grant ECCS-2025391).
Special thanks to the entire Unisense team who made this work possible, especially Dr. Andrew Cerskus, Dr. Laura Woods, Dr. Lars Larsen, Dr. Tage Dalsgaard, Dr. Line Daugaard, Dr. Karen Maegaard, and Mette Gammelgaard. Funding for our study was provided by the National Institutes of Health grants R01 GM13140801 (D.B., R.B.) and UL1 TR002319 (K.N.L), and a Research Expansion Award from the Montana State University Office for Research and Economic Development (D.B.). Figure 1A was created with BioRender.
Materials
| 3 M KCl | Unisense | ||
| 5 mL Wobble-not Serological Pipet, Individually Wrapped, Paper/Plastic, Bag, Sterile | CellTreat | 229091B | |
| 10 mL Wobble-not Serological Pipet, Individually Wrapped, Paper/Plastic, Bag, Sterile | CellTreat | 229092B | |
| 15 mL Centrifuge Tube – Foam Rack, Sterile | CellTreat | 229412 | |
| 24 Well Tissue Culture Plate, Sterile | CellTreat | 229124 | |
| 25 mL Wobble-not Serological Pipet, Individually Wrapped, Paper/Plastic, Bag, Sterile | CellTreat | 229093B | |
| 35 mm Dish | No. 1.5 Coverslip | 20 mm Glass Diameter | Uncoated | MatTek | P35G-1.5-20-C | |
| 50 mL Centrifuge Tube – Foam Rack, Sterile | CellTreat | 229422 | |
| 70% Ethanol | BP82031GAL | BP82031GAL | |
| 70 μm Cell Strainer, Individually Wrapped, Sterile | CellTreat | 229483 | |
| 1,000 µL Extended Length Low Retention Pipette Tips, Racked, Sterile | CellTreat | 229037 | |
| Amphotericin B (Fungizone) Solution | HyClone Laboratories, Inc | SV30078.01 | |
| Biosafety Cabinet | Nuaire | NU-425-600 | Class II Type A/B3 |
| Bovine Serum Albumin | Fisher Bioreagents | BP1605-100 | |
| Cell recovery solution | Corning | 354253 | Cell dissociation solution |
| DMEM/F-12 (Advanced DMEM) | Gibco | 12-491-015 | |
| Dulbecco's Modification of Eagles Medium (DMEM) | Fisher Scientific | 15017CV | |
| Fetal Bovine Serum | HyClone Laboratories, Inc | SH30088 | |
| G418 Sulfate | Corning | 30-234-CR | |
| Gentamycin sulfate | IBI Scientific | IB02030 | |
| HEPES, Free Acid | Cytiva | SH30237.01 | |
| HP Pavillion 2-in-1 14" Laptop Intel Core i3 | HP | M03840-001 | |
| Hydrochloric acid | Fisher Scientific | A144C-212 | |
| Incubator | Fisher Scientific | 11676604 | |
| iPhone 12 camera | Apple | ||
| L-glutamine | Cytiva | SH3003401 | |
| Large Kimberly-Clark Professional Kimtech Science Kimwipes Delicate Task Wipers, 1-Ply | Fisher Scientific | 34133 | |
| M 205 FA Stereomicroscope | Leica | ||
| Matrigel Membrane Matrix 354234 | Corning | CB-40234 | |
| MC-1 UniMotor Controller | Unisense | ||
| Methyl red | |||
| MM33 Micromanipulator | Marzhauser Wetzlar | 61-42-113-0000 | Right handed |
| MS-15 Motorized Stage | Unisense | ||
| Nanoject-II | Drummond | 3-000-204 | nanoliter autoinjector |
| Penicillin/Streptomycin (10,000 U/mL) | Gibco | 15-140-148 | |
| pH Microelectrodes | Unisense | 50-109158, 25-203452, 25-205272, 25-111626, 25-109160 | SensorTrace software is not compatible with Apple computers |
| Reference Electrode | Unisense | REF-RM-001652 | SensorTrace software is not compatible with Apple computers |
| SB 431542 | Tocris Bioscience | 16-141-0 | |
| Smartphone Camera Adapter | Gosky | ||
| Specifications Laboratory Stand LS | Unisense | LS-009238 | |
| Trypsin-EDTA 0.025%, phenol red | Gibco | 25-200-056 | |
| UniAmp | Unisense | 11632 | |
| United Biosystems Inc MINI CELL SCRAPERS 200/PK | Fisher | MCS-200 | |
| Y-27632 dihydrochloride | Tocris Bioscience | 12-541-0 | |
| µSensor Calibration Kit | Unisense/ Mettler Toledo | 51-305-070, 51-302-069 | pH 4.01 and 9.21, 20 mL packets |
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