Human intestinal organoid models for celiac disease research

The Oral Transglutaminase 2 Inhibitor ZED1227 Accumulates in the Villous Enterocytes in Celiac Disease Patients during Gluten Challenge and Drug Treatment

“…Samples from duodenal biopsies from the Tampere University Hospital (non-celiac patients). Isolation and culture of human epithelial organoids collected from the small intestinal mucosa were carried out as described elsewhere [ 12 ]. Briefly, isolated crypts were mixed with Matrigel (BD Biosciences, San Jose, CA, USA) and cultured in WELR500 medium, containing advanced DMEM/F12 medium (Thermo Fisher Scientific, Waltham, MA, USA) with 100 U/mL penicillin and 100 μg/mL streptomycin (Thermo Fisher Scientific), 10 mM HEPES (Thermo Fisher Scientific), 2 mM GlutaMAX (Thermo Fisher Scientific), 1.25 mM N-acetylcysteine (Sigma-Aldrich, Saint Louis, MO, USA), 1 × B27 supplement (Thermo Fisher Scientific), 1 × N2 supplement (Thermo Fisher Scientific), 10 nM gastrin I (Sigma-Aldrich), 10 % WNT3A-conditioned medium, 50 ng/mL EGF (PeproTech, South Korea), 0.2 µM LDN-193189 (Selleckchem, Planegg, Germany), 500 ng/mL R-spondin-1 (PeproTech), 500 nM A83-01 (Tocris, Bristol, UK), 10 µM SB202190 (Sigma Aldrich) and 10 mM Nicotinamide (Sigma-Aldrich).…”

“…The 2D organoids were differentiated in ELR medium for 48 h and subsequently treated either with 0.02 mg/mL, 0.002 mg/mL ZED1227, or DMSO-vehicle diluted in ELR medium for 30 min at 37 °C in a humidified CO 2 -incubator. 5-BP staining was performed as described [ 12 ].…”

“…The organoids were studied as an in vitro model to support our finding that ZED1227 accumulates on the surface of the epithelial cells and that ZED1227 can inhibit TG2 there. They were maintained as 3D Matrigel-embedded cultures and transformed to 2D monolayers for in-situ staining as described [12]. For cell differentiation, WELR500 medium was changed to ELR1 medium (BCM, EGF 50 ng/mL, LDN-193189 0.1 µM ja R-Spondin-1 250 nng/mL) and incubated for 48 h, allowing enteroid cell lineage differentiation (Dotsenko et al, 2023).…”

“…They were maintained as 3D Matrigel-embedded cultures and transformed to 2D monolayers for in-situ staining as described [12]. For cell differentiation, WELR500 medium was changed to ELR1 medium (BCM, EGF 50 ng/mL, LDN-193189 0.1 µM ja R-Spondin-1 250 nng/mL) and incubated for 48 h, allowing enteroid cell lineage differentiation (Dotsenko et al, 2023). For intestinal stem cell proliferation, R-spondin-1 concentration was increased to 1000 ng/mL, and media was supplemented with 5 µM CHIR 99021 (Tocris) (WELR1000C medium).…”

“…To better understand the mode-of-action of ZED1227, we developed a monoclonal antibody against ZED1227 and studied its accumulation relative to TG2 protein and gliadin peptides using immunofluorescence and confocal microscopy in duodenal biopsies from patients treated with ZED1227. Furthermore, we used human epithelial organoids [ 12 ] to obtain direct functional evidence of transglutaminase inhibition by ZED1227.…”