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

微小電極を用いた3D胃オルガノイドの精密管腔pH測定

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.

著者スポットライト: ヘリコバクター・ピロリ 感染における胃上皮細胞応答

微小電極を用いた三次元消化管オルガノイドの管腔pHプロファイリング

管腔環境を研究するためのpHプロファイリングのためのヒト胃オルガノイドの調製

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

45 ビュー. まず、直径が200〜700マイクロメートルの活発に成長しているヒト胃オルガノイドを入手します。細胞外マトリックス(ECM)を氷上で少なくとも45分間解凍します。また、5%二酸化炭素インキュベーターで摂氏37度のプレウォーム細胞培養プレートを調製します。培養プレートのウェルから培地を取り除いた後、氷冷したPBSを各ECM液滴にピペット?...

ビデオ: 管腔環境を研究するためのpHプロファイリングのためのヒト胃オルガノイドの調製

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 ...