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Protocol

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Biology

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

Published: June 2nd, 2023

DOI:

10.3791/64804

1Division of Pediatric Gastroenterology, Hepatology and Nutrition, UPMC Children’s Hospital of Pittsburgh, 2Division of Pediatric Surgery, UPMC Children’s Hospital of Pittsburgh, 3Department of Surgery, University of Pittsburgh School of Medicine
* These authors contributed equally

We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic cell layer provides the first line of defense between microbial and immune populations and helps to modulate the intestinal immune response. Disruption of the epithelial barrier is a hallmark of inflammatory bowel disease (IBD) and is of interest for therapeutic targeting. The 3-dimensional colonoid culture system is an extremely useful in vitro model for studying intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. Ideally, establishing colonoids from the inflamed epithelial tissue of animals would be most beneficial in assessing the genetic and molecular influences on disease. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice with acute inflammation. To address this limitation, we have developed a protocol to treat colonoids with a cocktail of inflammatory mediators that are typically elevated during IBD. While this system can be applied ubiquitously to various culture conditions, this protocol emphasizes treatment on both differentiated colonoids and 2-dimensional monolayers derived from established colonoids. In a traditional culture setting, colonoids are enriched with intestinal stem cells, providing an ideal environment to study the stem cell niche. However, this system does not allow for an analysis of the features of intestinal physiology, such as barrier function. Further, traditional colonoids do not offer the opportunity to study the cellular response of terminally differentiated epithelial cells to proinflammatory stimuli. The methods presented here provide an alternative experimental framework to address these limitations. The 2-dimensional monolayer culture system also offers an opportunity for therapeutic drug screening ex vivo. This polarized layer of cells can be treated with inflammatory mediators on the basal side of the cell and concomitantly with putative therapeutics apically to determine their utility in IBD treatment.

Inflammatory bowel disease (IBD) is a chronic, remitting, and relapsing disease characterized by episodes of inflammation and clinical quiescence. The etiology of IBD is multifactorial, but key characteristic features of the disease include defective barrier function and increased permeability of the intestinal epithelium, in addition to proinflammatory signaling cascades activated within the epithelial compartment1,2. Several in vitro and in vivo models have been used to recapitulate the epithelial response during IBD, including cell culture and murine models of inflammation

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All the experimentation using murine tissues described herein was approved by the Institutional Review Board at the University of Pittsburgh and conducted in accordance with the guidelines set forth by the Animal Research and Care Committee at the University of Pittsburgh and UPMC.

1. Preparation for culture

NOTE: All the reagents are listed in the Table of Materials section and all solution compositions can be found in the solution c.......

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The 3D intestinal colonoid culture system is an invaluable tool to study the intrinsic contribution of the epithelium to intestinal mucosal homeostasis. The described protocol provides detailed instructions on how to isolate crypts from C57BL/6J (WT) mice at 8 weeks of age and establish a long-term colonoid culture system that can be manipulated for multiple downstream applications. Upon the isolation and plating of crypts in the basement membrane matrix, the crypts appear dense and multicellular in structure when visual.......

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Organoid development has revolutionized the way the scientific community studies organ systems in vitro with the ability to partially recapitulate cellular structure and function from an animal or human in a dish. Further, organoid systems derived from humans with diseases offer a promising tool for personalized medicine that could guide therapeutic decision-making. Here, we describes a crypt isolation protocol that works well and introduces key steps that allow for cleaning up excess debris in the isolatio.......

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This work was supported by National Institutes of Health Grants R01DK120986 (to K.P.M.).

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Name Company Catalog Number Comments
0.4-μM transparent transwell, 24-well Greiner Bio-one 662-641
15-mL conical tubes Thermo Fisher  12-565-269
50-mL conical tubes Thermo Fisher  12-565-271
70-μM cell strainer VWR 76327-100
Advanced DMEM/F12 Invitrogen 12634-010 Stock Concentration (1x); Final Concentration (1x)
B-27 supplement  Invitrogen 12587-010 Stock Concentration (50x); Final Concentration (1x)
Chopsticks Electrode Set for EVO World Precision Instruments STX2
Corning Matrigel GFR Membrane Mix Corning 354-230 Stock Concentration (100%); Final Concentration (100%)
Dithiothreitol (DTT) Sigma-Aldrich D0632-5G Stock Concentration (1 M); Final Concentration (1.5 mM); Solvent (ultrapure water)
DMEM high glucose Thermo Fisher 11960-069 Stock Concentration (1x); Final Concentration (1x)
Dulbecco's phosphate-buffered saline without Calcium and Magnesium Gibco  14190-144 Stock Concentration (1x); Final Concentration (1x)
Ethylenediaminetetraacetic acid (ETDA) Sigma-Aldrich E7889 Stock Concentration (0.5 M); Final Concentration (30 mM)
Fetal Bovine Serum Bio-Techne S11150H Stock Concentration (100%); Final Concentration (1%)
Fisherbrand Superfrost Plus Microscope Slides, White, 25 x 75 mm Thermo Fisher  12-550-15
G418 InvivoGen ant-ga-1 Final Concentration (400 µg/µL)
Gentamicin Reagent Gibco/Fisher 15750-060 Stock Concentration (50 mg/mL); Final Concentration (250 μg/mL)
GlutaMAX-1 Fisher Scientific 35050-061 Stock Concentration (100x); Final Concentration (1x)
HEPES 1 M Gibco 15630-080 Stock Concentration (1 M); Final Concentration (10 mM)
hIFNγ R&D Systems 285-IF Stock Concentration (1000 ng/µL); Final Concentration (10 ng/mL); Solvent (ultrapure water)
hIL-1β R&D Systems 201-LB Stock Concentration (10 ng/µL); Final Concentration (20 ng/mL); Solvent (ultrapure water)
hTNFα R&D Systems 210-TA Stock Concentration (10 ng/µL); Final Concentration (40 ng/mL); Solvent (ultrapure water)
Hydrogen Peroxide  Sigma H1009 Stock Concentration (30%); Final Concentration (0.003%); Solvent (Mouse wash media)
Hygromycin B Gold InvivoGen ant-hg-1 Final Concentration (400 µg/µL)
L-WRN Cell Line ATCC CRL-3276
mEGF Novus NBP2-35176 Stock Concentration (0.5 µg/µL); Final Concentration (50 ng/mL); Solvent (D-PBS + 1% BSA)
N-2 supplement Invitrogen 17502-048 Stock Concentration (100x); Final Concentration (1x)
N-Acetyl-L-cysteine Sigma  A9165-5G Stock Concentration (500 mM); Final Concentration (1 mM); Solvent (ultrapure water)
Noggin Peprotech 250-38 Stock Concentration (0.1 ng/µL); Final Concentration (100 ng/mL); Solvent (UltraPure water + 0.1% BSA)
Penicillin-Streptomycin (10,000 U/mL) Thermo Fisher 15140-122 Stock Concentration (100x); Final Concentration (1x)
Petri dishes (sterilized; 100 mm x 15 mm) Polystrene disposable VWR 25384-342
Polystyrene Microplates, 24 well tissue culture treated, sterile Greiner Bio-one 5666-2160
R-Spondin R&D Systems 3474-RS-050 Stock Concentration (0.25 µg/µL); Final Concentration (500 ng/mL); Solvent (D-PBS + 1% BSA)
Tryp LE Express Thermo Fisher 12604-013 Stock Concentration (10x); Final Concentration (1x); Solvent (1 mM EDTA)
UltraPure Water  Invitrogen 10977-023 Stock Concentration (1x); Final Concentration (1x)
Y-27632 dihyddrochloride  Abcam ab120129 Stock Concentration (10 mM); Final Concentration (10 µM); Solvent (UltraPure Water)

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