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

<div><p>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 <i>ex vivo</i> 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.</p></div

Antibodies in the culture medium diffuse into the biopsy and bind to target cells.

<p>Inflamed colonic biopsies were cultured in the presence of anti-CD31 and anti-CD3 antibodies (10 μg/ml each) for 20 hours followed by IHC staining of consecutive sections with isotype-specific anti-IgG2a (A) and anti-IgG1 (B) antibodies for detection of anti-CD31 and anti-CD3 antibodies, respectively. Arrows indicate blood vessels, which were stained in (A) by the anti-IgG2a secondary antibody specifically recognizing the anti-CD31 antibody. Blood vessels were not stained by the anti-IgG1 secondary antibody (B) that instead recognized the binding of the anti-CD3 antibody to T cells in the lamina propria (indicated by arrowheads). Bar = 100 μm.</p

Explant assay setup.

<p>(A) Reusable T-disk with a central metal mesh designed to fit into the Falcon center-well organ culture dish. (B) Cartoon of a biopsy placed on the T-disk in air/liquid interphase oriented with the epithelial cell layer upwards and the cut surface downwards, slightly submerged in the medium. (C) Picture of the open incubator. (D) Picture of the sealed incubator.</p