Enterochromaffin Cell-Enriched Monolayer Platform for Assaying Serotonin Release from Human Primary Intestinal Cells

Abstract

Enteroendocrine (EE) cells within the intestinal epithelium produce a range of hormones that have key roles in modulating satiety and feeding behavior in humans. The regulation of hormone release from EE cells as a potential therapeutic strategy to treat metabolic disorders is highly sought after by the pharmaceutical industry. However, functional studies are limited by the scarcity of EE cells (or surrogates) in both in vivo and in vitro systems. Enterochromaffin (EC) cells are a subtype of EE cell that produce serotonin (5HT). Here, we explored simple strategies to enrich EC cells in in vitro monolayer systems derived from human primary intestinal stem cells. During differentiation of the monolayers, the EC cell lineage was significantly altered by both the culture method (air-liquid interface [ALI] vs. submerged) and the presence of vasoactive intestinal peptide (VIP). Compared with traditional submerged cultures without VIP, VIP-assisted ALI culture significantly boosted the number of EC cells and their 5HT secretion by up to 430% and 390%, respectively. The method also increased the numbers of other subtypes of EE cells such as L cells. Additionally, this method generated monolayers with enhanced barrier integrity so that directional (basal or apical) 5HT secretion was measurable. For all donor tissue, the enriched EC cells improved the signal-to-background ratio and reliability of 5HT release assays. The enhancement in 5HT secretion behavior was consistent over time from a single donor, but significant variation in the amount of secreted 5HT was present among tissues derived from 5 different donors. To demonstrate the utility of the EC-enriched monolayer system, 13 types of pungent food ingredients were screened for their ability to stimulate 5HT secretion. Curcumin found in the spice turmeric derived from the Curcuma longa plant was found to be the most potent secretagogue. This EC-enriched cell-monolayer platform can provide a valuable analytical tool for highthroughput screening of nutrients and gut microbial components that alter the secretion of 5HT.

Potential Roles of Enterochromaffin Cells in Early Life Stress-Induced Irritable Bowel Syndrome

“…Moreover, it was found that a large proportion of ECs renewed relatively rapidly, with a turnover rate of approximately 16 days in the gut ( de Bruïne et al, 1992 ). Although these disadvantages severely impede the in-depth study of ECs physiology ( Wang et al, 2020 ), in addition to the selection of human pancreatic neuroendocrine tumor cell line (BON) cells ( Christofi et al, 2004 ; Wu et al, 2011 ) and the malignant ECs carcinoid cell line ( Siddique et al, 2009 ), the ECs model has performed well, despite the fact that tumor cell lines have not proven to be suitable mimics of intestinal ECs physiological responses ( Wang et al, 2020 ). In addition, a study revealed that a human pancreatic endocrine cell line, one islet carcinoma cell line (QGP-1), also expressed various ECs marker genes, including TRPA1 and TPH1, suggesting that QGP-1 is a new model for the investigation of ECs ( Doihara et al, 2009 ).…”

Magnetically-Propelled Fecal Surrogates for Modeling the Impact of Solid-Induced Shear Forces on Primary Colonic Epithelial Cells

“…To promote long-term stability of the platform under repeated shear stress over time, a porous membrane with a surface coating of extracellular matrix (ECM) was adopted for cellular support. We have previously demonstrated hanging basket insert systems with basal porous membranes supply critical nutrients and growth factors to the basal tissue side and support a healthy and functional cell monolayer that is representative of primary intestinal tissue ( e.g ., proliferative regions, differentiated lineages, transport, and barrier function) [35-37, 42]. The epithelial monolayer also establishes proper intercellular focal adhesions creating a high resistance barrier that segregates the luminal and basal culture compartments, enabling measurements of directional transport or secretion.…”

“…These results agree with our observations of secreted protein modulation after shear force induction. Further investigations that utilize newly developed primary culture techniques in which goblet cell lineages [36] or enterochromaffin cell lineages [37, 80] are enriched will be important platforms when used in combination with the magnetic agarose fecal mimic to understand the role of Piezo2 mediated mechanotransduction in response to shear forces.…”

“…Once extracted from primary tissue, long-lived proliferative cells can be subjected to serial expansion over collagen hydrogel scaffolds or within Matrigel patties, maintaining a stable karyotype while in culture [32, 34]. When cultured over porous membranes, these primary epithelial cells form a high-resistance barrier as occurs in vivo and can be used for high-content analytical screens in which the basal and luminal compartments are segregated [35-37]. In this report, this primary cell-derived culture format is extended to include the application of solid-induced shear forces, rather than those induced by liquid flow, at user-defined velocities and frequencies that are representative of human colonic luminal contents.…”

Role of Ion Channels in the Chemotransduction and Mechanotransduction in Digestive Function and Feeding Behavior

“…Mechanosensitive ion channels expressed in neurons act as primary mechanosensors, regulating food intake and nutritional responses. A specific knockout of Piezo and its homologs were found to increase feeding in Drosophila [ 49 , 50 ] and C. elegans [ 51 ], suggesting that Piezo is able to inhibit feeding through stress sensation. In addition, Piezo was also found to be expressed in the digestive tract, nerves, and brain, revealing a direct gut-brain neural loop that uses Piezo as a vital mediator of signal sensation to transmit signals for the digestive tract distension [ 49 , 50 ].…”