Validation and Optimization of an Ex Vivo Assay of Intestinal Mucosal Biopsies in Crohn’s Disease: Reflects Inflammation and Drug Effects

Abstract

Crohn’s disease (CD) is a chronic illness demanding better therapeutics. The marketed biologics only benefit some patients or elicit diminishing effect over time. To complement the known methods in drug development and to obtain patient specific drug responses, we optimized and validated a known human explant method to test drug candidates and pathophysiological conditions in CD intestinal biopsies. Mucosal biopsies from 27 CD patients and 6 healthy individuals were collected to validate an explant assay test where the polarized tissue was cultured on a novel metal mesh disk, slightly immersed in medium imitating an air-liquid interphase. After culture in high oxygen for 24 hours with or without biological treatment in the medium, biopsy integrity and penetration of antibodies was measured by immunohistochemistry (IHC). Nine cytokines were quantified in the conditioned medium as a read-out for degree of inflammation in individual biopsies and used to evaluate treatment efficacy. The biopsies were well-preserved, showing few structural changes. IHC revealed tissue penetration of antibodies demonstrating ability to test therapeutic antibodies. The cytokine release to the medium showed that the assay can distinguish between inflammation states and then validate the known effect of two treatment biologics confirmed by a detection panel of five specific cytokines. Our data also suggest that the assay would be able to indicate which patients are responders to anti-TNF-α therapeutics, and which are non-responders. This study demonstrates this version of an ex vivo culture as a valid and robust assay to assess inflammation in mucosal biopsies and test of the efficacy of novel drug candidates and current treatments on individual patients–potentially for a personalized medicine approach.

Health-Promoting Properties of Proanthocyanidins for Intestinal Dysfunction

“…Nevertheless, advantages related to the usefulness of in vitro models for screening of bioactives and exploring action mechanisms are offset by limitations regarding the mimicking of the in vivo situation and translation to the human [133]. Thus, some human ex vivo models have been proposed to test immunomodulatory properties of drug candidates in intestinal explants from IBD patients [134,135]. Intestinal function can also be studied with UCh-based protocols.…”

“…An advantage of UCh models over explant-based models is that UCh models make it possible to measure the electrophysiological parameters, including TEER [136]. All these set-ups permit analyzing the cytokine profiling of intestinal explants or biopsies retaining their in-situ conditioning in a polarized fashion [135,138]. We have employed the UCh to determine TEER and cytokine release (TNF-α) in intestinal tissues from cafeteria diet-induced obese rats treated with GSPE [8,15].…”

Beneficial Effects of Proanthocyanidins on Intestinal Permeability and Its Relationship with Inflammation

“…Nevertheless, advantages related to the usefulness of in vitro models for screening of bioactives and exploring action mechanisms, are offset by limitations regarding the mimicking of the in vivo situation and translation to the human [103]. Thus, some human ex vivo models have been proposed to test immunomodulatory properties of drug candidates in intestinal explants from IBD patients [104,105]. Intestinal function can also be studied with UCh-based protocols.…”

“…An advantage of UCh models over explant-based models is that UCh models make it possible to measure the electrophysiological parameters, including TEER [106]. All these setups permit analyzing the cytokine profiling of intestinal explants or biopsies retaining their in situ conditioning in a polarized fashion [105,108]. We have employed the UCh to determine TEER and cytokine release (TNF-α) in intestinal tissues from cafeteria diet-induced obese rats treated with GSPE [8,15].…”

Evaluation of Compound Activity in Primary Human Intestinal Organoids Using Gene Expression and Histology

“…Human epithelial cells can also be studied as isolated intact tissue in explant models, where all intestinal cell populations are present in the correct spatial orientation. One major limitation is the narrow window during which the tissue can be used (24 to 48 hr) before it degrades and tissue architecture is lost (Fiocchi, ; Oshima et al., ; Randall, Turton, & Foster, ; Vadstrup et al., ). Furthermore, although the presence of various immune cells, fibroblasts, and other non‐epithelial cell types provides a more complete repertoire of cells, it can also add a level of complexity that may obscure results when one is specifically investigating the biology of the epithelium.…”