Mechanical Stimuli Affect E. Coli Heat Stable Enterotoxin (ST)-Cyclic GMP Signaling in a Human Enteroid Intestine-Chip Diarrhea Model

Abstract

Modeling host-pathogen interactions with human intestinal epithelia using enteroid 20 monolayers on permeable supports (such as Transwells) represents an alternative to animal studies or 21 use of colon cancer-derived cell lines. However, the static monolayer model does not expose epithelial 22 Introduction: 43Human adult intestinal stem cell-derived enteroids/organoids retain intestinal segment-specific 44 transcriptional as well as phenotypic characteristics, that enable study of untransformed, non-cancer 45 epithelia (1)(2)(3)(4) . Enteroids are generally propagated as 3-dimensional (3D) basement membrane matrix-46 embedded spheroids that are polarized such that the inward facing apical cell surface is not directly 47 accessible. This places limitations on host-pathogen interaction studies. Enteroids grown as monolayers 48 on collagen IV-coated permeable supports overcome the challenges of 3D culture, by permitting direct 49 application of commensal organisms, pathogens, toxins, nutrients, electrolytes and drugs to the apical 50 cell surface and enabling subsequent sampling of exported metabolites from both apical and basolateral 51 surfaces (2, 5-8). 52Static enteroid monolayers do not, however, recreate mechanical forces acting on intestinal epithelia in 53 vivo (4), lacking luminal flow and blood flow (shear stress forces) and the rhythmic contractions that are 54 part of peristalsis. Although multiple efforts have yielded microengineered cell culture systems with flow 55 to recapitulate physical forces of flow and stretch in Organ-on-Chip models (9-12), evidence of discrete 56 advantages associated with inclusion of mechanical forces for modeling host-pathogen interactions 57 within human intestinal epithelia in these systems is not yet clear. In this study, we evaluated the effects 58 of mechanical forces on both baseline characteristics and the responses to heat stable enterotoxin (ST), 59 a virulence factor of enterotoxigenic E. coli (ETEC) infection that is associated with a large burden of 60 diarrheal illness in low-middle income countries (13). The model was a micro-engineered 61 polydimethylsiloxane (PDMS)-based 14, 15). The chip contains two parallel hollow 62 channels separated by a flexible permeable membrane that enables human intestinal epithelial cells to 63 be cultured in the presence of physiologically-scaled fluid shear stress forces and repetitive lateral 64 mechanical deformation (14, 15). 65 5 CA). Enteroids were cultured in a 5% CO2 atmosphere at 37°C and passaged every 7-12 days. Expansion 90 medium was supplemented with 10 μmol/L Y-27632 (Tocris) and 10 μmol/L CHIR99021 (Tocris) during 91 the first 2 days after passaging. Studies were performed with a jeunal enteroid line (passage numbers 92 30-45). 93Intestine-Chip: Organ-Chips were fabricated from PDMS and assembled as described (14, 23, 24) 94 (Emulate Inc.,Boston, MA). The top (1 mm high × 1 mm wide) and bottom (0.2 mm high x 1 mm wide) 95 channels were separated by a thin (50 μm) flexible, PDMS membrane containing 7 μm diam...

Research in a time of enteroids and organoids: how the human gut model has transformed the study of enteric bacterial pathogens

“…ST was also used to probe the contribution of fluid flow and mechanical stretch to functional responses using microfluidic Intestine Chips, an innovative tool in the advancement of the enteroid model. 31 The chip establishes an enteroid monolayer within a flexible polymer chamber containing channels that enable luminal surface fluid flow and application of culture membrane deformation to mimic peristalsis-like forces. Under continuous flow conditions, exposure of jejunal enteroid monolayers to ST increased the secreted but not intracellular cGMP concentrations and increased expression of cyclic nucleotide transporter MRP4, compared to monolayers under static conditions.…”

“…Repetitive stretch did not have an additive effect on cGMP levels. When considering the aforementioned studies, 30,31 the choice of experimental model, such as enteroids vs. immortalize cell lines or application of static vs. shear fluid forces, can give different results, and it is not yet clear which model is to be considered most reflective of native intestinal physiology or pathophysiology of ETEC infection.…”