An organotypic slice model for ex vivo study of neural, immune, and microbial interactions of mouse intestine

Abstract

Organotypic tissue slices provide seminatural, three-dimensional microenvironments for use in ex vivo study of specific organs and have advanced investigative capabilities compared with isolated cell cultures. Several characteristics of the gastrointestinal tract have made in vitro models for studying the intestine challenging, such as maintaining the intricate structure of microvilli, the intrinsic enteric nervous system, Peyer's patches, the microbiome, and the active contraction of gut muscles. In the present study, an organotypic intestinal slice model was developed that allows for functional investigation across regions of the intestine. Intestinal tissue slices were maintained ex vivo for several days in a physiologically relevant environment that preserved normal enterocyte structure, intact and proliferating crypt cells, submucosal organization, and muscle wall composure. Cell death was measured by a membrane-impermeable DNA binding indicator, ethidium homodimer, and less than 5% of cells were labeled in all regions of the villi and crypt epithelia at 24 h ex vivo. This tissue slice model demonstrated intact myenteric and submucosal neuronal plexuses and functional interstitial cells of Cajal to the extent that nonstimulated, segmental contractions occurred for up to 48 h ex vivo. To detect changes in physiological responses, slices were also assessed for segmental contractions in the presence and absence of antibiotic treatment, which resulted in slices with lesser or greater amounts of commensal bacteria, respectively. Segmental contractions were significantly greater in slices without antibiotics and increased native microbiota. This model renders mechanisms of neuroimmune-microbiome interactions in a complex gut environment available to direct observation and controlled perturbation.

Vasoactive intestinal peptide regulates ileal goblet cell production in mice

“…The mechanisms of signaling between neural fibers and crypt epithelia remains unclear. Incorporation of the thymidine analog EdU into DNA has previously been used to quantify the rate of epithelial cell production in mouse small intestine both in vivo (Krndija et al, ()) and ex vivo ( Schwerdtfeger et al, ). In this study, the number of glycan synthesizing goblet cells that were also undergoing DNA synthesis as marked by EdU was substantially decreased in slices treated with the VIP receptor antagonist VPACa.…”

“…This study uses organotypic intestinal slices (Schwerdtfeger, Nealon, Ryan, & Tobet, 2019;Schwerdtfeger, Ryan, & Tobet, 2016) as a platform for investigating neural -goblet cell interactions in mouse ileum ex vivo. This intestinal slice method allowed for use of three pharmacological tools for altering goblet cell function ex vivo in a cellularly heterogenous tissue.…”

Organoids, organs-on-chips and other systems, and microbiota

“…PCTS are believed to have advantages over organoids and organ-on-a-chip (discussed later), as they represent all regions of the tissue [91]. It has been reported that murine intestine is maintained for 6 days in the presence of normal neuronal, muscular, and mucosal structures with contractibility functions and some immune responses, therefore, representing a more holistic intestinal organ [93]. Additionally, in the absence of antibiotic media, some aerobic commensal microbes can be maintained [93].…”

“…It has been reported that murine intestine is maintained for 6 days in the presence of normal neuronal, muscular, and mucosal structures with contractibility functions and some immune responses, therefore, representing a more holistic intestinal organ [93]. Additionally, in the absence of antibiotic media, some aerobic commensal microbes can be maintained [93]. Although this system holds great promise to study holistically the complex mechanisms that underpin healthy gut functions and disease in a 3D physiologically relevant environment, further work is required to maintain anaerobic microbes that are predominately found within the intestinal environment [94].…”

From organotypic culture to body‐on‐a‐chip: A neuroendocrine perspective

“…A number of intestinal explant models were focused on characterising and maintaining healthy tissue in vitro. A modified organotypic slice model, using a similar protocol to that previously used in brain,71 was capable of maintaining 250-μm thick mouse intestinal slices in serum-free media, without antibiotics, for up to 6 days ex vivo 97. Unfortunately, details of tissue health were minimal in the early colon studies, which also used serum (5%-15% foetal calf serum).…”