Fig 1.
(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.
Table 1.
Clinical features of patients and healthy controls.
Fig 2.
Preservation of tissue morphology and robust cytokine release in the explant assay.
Non-inflamed (A, C) and inflamed (B,D) biopsies from the same patient were cultured on T-disks (A, B) or transwells (C,D) for 24 hours and processed for histology. Representative pictures of H&E-stained paraffin-sections (n = 5 patients). Bars = 600 μm. (E) Spontaneous release of the cytokines IL-1β, IL-10, GM-CSF, IFN-γ and TNF-α from inflamed biopsies into the culture medium measured by Bio-Plex. Biopsies from the same patients were cultured on either T-disk or transwell as indicated. No difference in cytokine release was observed when comparing T-disk and transwell for all 5 cytokines in combination (P > 0.05, two-way ANOVA), but when analyzed alone, IL-10 and IFN-γ were significantly increased with T-disk (*P < 0.05, t-test). 2 biopsies per patient (n = 6). (F) Spontaneous release of GM-CSF from 6 inflamed patients, 4 biopsies per patients taken as closely as possible from each site, all shown individually. Data show mean ± SEM.
Fig 3.
Cytokine release in conditioned medium relative to degree of inflammation as determined by endoscopy and histomorphology.
Spontaneous release of the cytokines IL-1β, IL-10, GM-CSF, IFN-γ and TNF-α from (A) inflamed biopsies (n = 12 patients (2 biopsies/patient)), non-inflamed (n = 10 (2)) and normal controls (n = 6 (4)) into the culture medium measured by Bio-Plex. The inflammation states of CD biopsies were determined by the endoscopist. (B) Spontaneous release from severely inflamed (n = 3 biopsies), mildly/moderately inflamed (n = 6) and non-inflamed (n = 9) CD biopsies from 4 patients as determined by histology. Cytokine levels (pg/ml/100 mg tissue) are expressed as mean ± SEM. Statistical analyses (two-way ANOVA) were performed on the levels of the 5 cytokines in combination.
Fig 4.
Antibodies in the culture medium diffuse into the biopsy and bind to target cells.
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.
Fig 5.
The assay responds to stimulation of the T cells and to a known biologic.
(A) Cytokine release from inflamed colonic biopsies incubated with either anti-CD3 (10 μg/ml) or isotype control (10 μg/ml) in the explant assay for 24 hours (n = 12 patients, 2–4 biopsies each). Significant differences are observed for GM-CSF, IFN-γ and TNF-α (paired t-test) as well as across the panel, P = .0026 (two-way ANOVA). (B) Cytokine release from inflamed colonic biopsies cultured in the presence of 10 μg/ml isotype control or 10 μg/ml IFX activated by 10 μg/ml anti-CD3 for 24 hours (n = 6 patients, 4 biopsies each). IL-17a, IL-12p40 and IL-2 now reaches detection threshold under anti-CD3 stimulation. IL-10, GM-CSF and IL-17a release were significantly decreased by IFX treatment in the activated test (paired t-test). (C) Cytokine release from inflamed colonic biopsies cultured in the presence of 10 μg/ml Infliximab (IFX) or isotype control for 24 hours (n = 10 patients, 2–4 biopsies each). IL-10 release was significantly decreased by IFX treatment (paired t-test). (D) Cytokine release from healthy colonic control biopsies cultured in the presence of 10 µg/ml Infliximab (IFX) or isotype control for 24 hours (n = 6 patients, 4 biopsies each). Of the patients in (C), three were non-responders (had no effect) to IFX. All 10 are showed here divided into responders (or untested) and non-responders for TNF-αrelease (E) and GM-CSF release (F) (paired t-test within a group and un-paired t-test between responders and non-responders). *P < .05, **P < 0.01.