Analysis of Interleukin 8 Secretion by a Stem-Cell-Derived Human-Intestinal-Epithelial-Monolayer Platform

Abstract

In vitro models of the human intestinal epithelium derived from primary stem cells are much needed for the study of intestinal immunology in health and disease. Here, we describe an intestinal monolayer cultured on a porous membrane with accessible basal and apical surfaces for assay of intestinal cytokine production in response to stimuli. The system was composed of a differentiated, confluent epithelial monolayer derived from human primary stem cells obtained from small or large intestine. Interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) were the most abundant inflammatory cytokines produced by the intestinal epithelium. The epithelium from all five tested regions of the intestine preferentially secreted into the apical reservoir of the monolayer, with a 26-fold greater concentration of IL-8 present in the apical reservoir of the colonic monolayer relative to that in the basal reservoir. Upon application of tumor-necrosis factor α (TNF-α) to the basal surface of the colonic monolayer, the IL-8 concentration significantly increased in the basal, but not the apical, reservoir. A dose-dependent elevation of IL-8 in the basal reservoir was observed for TNF-α-stimulation of the monolayer but not for an organoid-based platform. To demonstrate the utility of the monolayer system, 88 types of dietary metabolites or compounds were screened for their ability to modulate IL-8 production in the basal reservoir of the intestinal monolayer in the absence or presence of TNF-α. No dietary metabolite or compound caused an increase in IL-8 in the basal reservoir in the absence of TNF-α. After addition of TNF-α to the monolayer, two compounds (butyrate and gallic acid) suppressed IL-8 production, suggesting their potential anti-inflammatory effects, whereas the dietary factor forskolin significantly increased IL-8 production. These results demonstrate that the described human-intestinal-monolayer platform has the potential for assays and screening of metabolites and compounds that alter the inflammatory response of the intestine.

An In Vitro Intestinal Model Captures Immunomodulatory Properties of the Microbiota in Inflammation

“…Many of the values reported are below or at the lower end of this range. Other recent reports from primary intestinal cells (as opposed to intestinal cell lines employed here) showed higher levels of some cytokines [44,71], including apical IL8 and MCP1, being more than an order of magnitude greater than what was measured here. These disparities might re ect differences between primary and cell line cultures, and in culture conditions (culture area, media type and volume, time of sampling).…”

“…It has been demonstrated that bacteria differentially modulate production of IL8 by epithelial cells [70]. Polarized (apical) secretion of IL8 by intestinal epithelium has been demonstrated to be stimulated by ligand binding to Toll-like receptor 2 (TLR2) and TLR5 [46,71], which are major receptors for MAMPs including LPS, lipoproteins and agellin [72]. IL12 has been reported to be produced mainly by monocytes in the intestine, but airway epithelium has been demonstrated to produce IL12 in in ammation [73], and elevation of this Th1-inducing factor has been observed in intestinal in ammatory conditions in humans and animal models [74].…”

Photopatterned Membranes and Chemical Gradients Enable Scalable Phenotypic Organization of Primary Human Colon Epithelial Models

“…To enhance adoption by others, incorporation of standardized 96well spacing and geometry is desirable to increase throughput while enabling ease of integration with pre-existing assays and instrumentation. While it is noted that several commercial options based on Boyden chambers exist (e.g., Millicell ® ), 36,[42][43] the basket insert design results in decreased luminal compartment volume preventing the luminal reservoir from being acting as an effective sink during crypt polarization. Furthermore, the limited luminal access area in these commercial assemblies is not compatible with the molding process used to shape crypt scaffolds.…”

“…In brief, Collagen Type 1, rat tail (354236, Corning) was lyophilized for 72 h and resuspended at a concentration of 5 mg mL - All components were cleansed in 75% (v/v) ethanol for 5 min, transferred into a tissue culture hood, and final device assembly took place under aseptic conditions. Diffusion windows for planar monolayer cultures were coated with 1% (v/v) Matrigel (354248, Corning) in 1× phosphate-buffered saline (PBS, Supporting Information) overnight at 37 °C, 36 while micromolded collagen scaffolds for in vitro crypt cultures were coated with 10 µg mL -1 rat tail collagen in 1× PBS under identical conditions. 32 Both surfaces were rinsed once with 1× PBS prior to cell plating.…”

Developingin vitroassays to transform gastrointestinal safety assessment: potential for microphysiological systems

“…23 Replating organoid-derived cultures into transwells or microdevices may be required to allow dual exposure control at both apical and basal surfaces (e.g., ref. 24). Techniques for automated microinjection of organoids with drugs and microbes may be valuable particularly for processes involving highly oxygen sensitive anaerobic microbiota.…”