Journal: Journal of visualized experiments : JoVE
Article Title: Three-Dimensional Morphogenesis in Canine Gut-on-a-Chip Using Intestinal Organoids Derived from Inflammatory Bowel Disease Patients
doi: 10.3791/65720
Figure Lengend Snippet: (A) Immunofluorescence imaging of a canine IBD Gut-on-a-Chip, showcasing a top-down view of a fully developed 3D epithelium after 12 days of culture, which is evaluated by a fluorescence microscope. The tight junction protein (ZO-1) is visualized in yellow; the brush-border membrane (F-actin) appears in red; and the nuclei are stained with DAPI and appear blue. (B) Immunofluorescence imaging of a canine IBD Gut-on-a-Chip using a confocal microscope with a long-distance lens. As shown in the schematic, a fluorescent image of a cross section of a villus-like structure of a fully developed 3D epithelium after 12 days of culture is shown. In addition, Z-stacking shows a side view of the 3D epithelium, which reveals the formation of villus-like structures. The brush-border membrane (F-actin) appears in red, and the nuclei are stained with DAPI and appear blue. (C) Intestinal barrier function was evaluated and measured by TEER in patient-derived canine Gut-on-a-Chips. Stable TEER values were reached on Day 5 of culture on Gut-on-a-Chip. Error bars express the SEM of the measurements. TEER value was measured among two biological replicates with one technical replicate. Scale bars = 50 μm (A), 25 μm (B). Abbreviations: IBD = inflammatory bowel disease; DAPI = 4’,6-diamidino-2-phenylindole; TEER = transepithelial electrical resistance.
Article Snippet: Name of Material/Equipment Company Catalog Number Final Concentration/Dilution Organoid basal medium Advanced DMEM/F12 Gibco 12634–010 GlutaMAX Gibco 35050–061 2 mM 1M HEPES VWR Life Science J848–500ML 10 mM 100×penicillin–streptomycin Corning MT30009CI 1x Organoid medium A-83–01 PeproTech 9094360 500 nM B27 supplement Gibco 17504–044 1x CHIR99021 Reprocell 04–0004-base 2.5 μM Murine EGF PeproTech 315-09-1MG 50 ng/ml Murine Wnt-3a PeproTech 315-20-10UG 100 ng/ml N-Acetyl-L-cysteine Sigma A9165–25G 1 mM N2 MAX Media supplement Gibco 17502–048 1x Nicotinamide Sigma N0636–100G 10 mM Noggin Conditioned Medium NA NA 10 vol/vol % Primocin InvivoGen ant-pm-1 100 μg/ml R-Spondin-1 Conditioned Medium NA NA 20 vol/vol % SB202190 Sigma-Aldrich S7067–25MG 10 μM Y-27632 StemCellTechnologies 72308 10 μM [Leu15]-Gastrin I human Sigma-Aldrich G9145-.5MG 10 nM Reagents 4% Paraformaldehyde solution Fisher Scientific AAJ19943K2 Alexa Fluor ™ 647 Phalloidin Thermo Fisher Scientific A22287 x250 dilution Anti-Rabbit IgG H&L labeled with Alexa Fluor 555 Abcam ab150078 x1000 dilution Anti-ZO-1 polyclonal antibody Thermo Fisher Scientific 61–7300 x50 dilution Cell Recovery Solution Corning 354253 Collagen I, Rat Tail 3 mg/mL Gibco A10483–01 Diamidino-2-phenylindole (DAPI) Thermo Fisher Scientific 62248 x1000 dilution EMS Glutaraldehyde Aqueous 50% Electron Microscopy Sciences 16320 Matrigel Matrix Corning 356255 Poly(ethyleneimine) solution Sigma 408700–250ML TrypLE Express Gibco 12604–021 Materials and Equipment 24-well culture plates Corning 3524 87V Industrial Multimeter Fluke Corporation Centrifuge Eppendorf 5910R CO2 incubator Eppendorf C170i DMi8 fluorescence microscope Leica microsystems DMi8 Dry oven Fisher Scientific 15-103-0519 FlexCell FX-5000 Tension system Flexcell International Corporation Inverted phase-contrast microscope Leica microsystems DMi1 SP8-X inverted confocal microscope Leica microsystems SP8-X Syringe pump Braintree Scientific model no. BS-8000 120V Syringe, 3 mL sterile BD Biosciences 14-823-435 Syringes, 1 mL sterile BD Biosciences 14-823-434 UV/ozone generator Jelight Company model no. 30 Software LAS X imaging software Leica microsystems Open in a separate window Table of Materials.
Techniques: Immunofluorescence, Imaging, Fluorescence, Microscopy, Membrane, Staining, Derivative Assay
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