The human gut microbiome has emerged as a powerful determinant of host health and has been associated with several important comorbidity factors and disease states including obesity, metabolic syndrome, bowel cancer, and neurodegenerative disorders such as Alzheimer's and Parkinson's disease. As key players in the human large intestinal environment, the interplay between the gut microbiota, intestinal mucosa and colonocytes has been shown to play an important role in modulating host immunity and conferring resistance to infection. Previous works have demonstrated enterocytes' utilisation of short-chain fatty acid (SCFAs) produced by gut microbes. However, large gaps remain in our understanding of the multidirectional interplay between colonocytes, the mucosa and commensal bacteria, largely due to the lack of highthroughput models and methods able to study these processes at a molecular level while also reproducing key physiological traits of the human large intestine. Currently existing techniques for probing the exchange between colonocytes and gut microbial species largely rely on specialist equipment and coculture methods that can be difficult to set up. Examples include, gut-microbiome physiomimetic platforms , organ-onchip technologies , diffusion chambers. All of these techniques lack spatial resolution, real time pH measurement and SCFA profilingkey parameters for the detection of metabolic pathogenesis. Chemical shift imaging nuclear magnetic resonance (CSI NMR) allows for the quantitative spatial mapping of complex mixtures and heterogeneous samples on a molecular level. Through continuous CSI NMR measurements, changes in the molecular and spatial composition of dynamic heterogeneous samples can be captured. In this work we create a model of the human large intestine directly in a standard NMR tube, featuring vertically oriented luminal, mucosal and colonocyte regions. Our model features colonocytes of four different linages together in 3-D culture (Caco-2, T84, HT-29 and SW620) a mixture of mucus-secreting and absorptive enterocytes/colonocyteswhich represent the major cell types in the large intestine. The model features both loosely and tightly bound mucosal layers , and is inoculated using faecal samples from a healthy human. We apply CSI techniques to probe changes in pH in real time using standard, widely available NMR equipment, and in-house written automation scripts for rapid data processing. Furthermore, our method allows for the spatially resolved quantification of key metabolites such as SCFAs at 0.5 mm spatial resolution, and the assessment of how they mediate crosstalk between commensal bacteria and colonocytes. Finally, we show how our methodology can be applied to probe drug transit through a model of the intestinal luminal space and mucosakey for optimal drug development and delivery to physiologically relevant regions, such as the distal parts of the human gastrointestinal tract (GIT). RESULTS AND DISCUSSION Continuous pH measurement. Using CSI techniques, we record changes in the 1 H NMR chemical shifts of acetate (pKa = 4.58) and maleate (pKa = 5.93) as NMR pH indicators in 8-minute experiments, allowing continuous observation of pH changes in the NMR tube over 24 hours. Since on an NMR time scale, the observed chemical shift δobs of an indicator is a weighted average of its protonated and deprotonated chemical shifts (δH and δL, respectively), the pH of the solution is related to the pKa of the indicator molecules through equation 1 pH = pK a + log 10 (