Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

Summary

The present protocol describes establishing a murine colonic organoid system to study the activity and functioning of colonic stem cells in a claudin-7 knockout model.

Abstract

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.

Introduction

Intestinal organoid culture is a three-dimensional (3D) ex vivo system in which stem cells are isolated from the intestinal crypts of primary tissue and plated into a gel matrix^1,2. These stem cells are capable of self-renewal, self-organization, and organ functionality². Organoids mimic the tissue microenvironment and are more similar to in vivo models than two-dimensional (2D) in vitro cell culture models, although less manipulatable than cells^3,4. This model eliminates obstacles encountered in 2D mo....

Protocol

All animal experiments and procedures were approved by the East Carolina University (ECU) Animal Care and Use Committee (IACUC) and conducted in compliance with guidelines from the National Institutes of Health and ECU on laboratory animal care and use. Inducible, intestinal-specific claudin-7 knockout mice were generated by crossing C57BL6 claudin-7-flox transgenic mice with Villin-CreERT2 mice¹⁹. Male and female mice aged 3 months were used in this study.

1. Rea.......

Discussion

Organoid culture is an excellent model for studying stem cell function, intestinal physiology, drug discovery, human intestinal diseases, and tissue regeneration and repair^{7,8,9,10,11,26}. While it has many advantages, it can be challenging to establish. Care must be taken in all steps throughout the protocol, but most importa.......

Materials

Name	Company	Catalog Number	Comments
0.09 cubic feet space-saver vacuum desiccator	United States Plastic Corp	78564	anesthesia chamber
0.5 M EDTA pH 8.0	Invitrogen	AM9261
1.5 mL microcentrifuge tubes	ThermoFisher	69715
15 mL conical centrifuge tubes	Fisher Scientific	14-959-53A
1x Dulbecco’s Phosphate buffered saline	Gibco	14190-144
2-methylbutane	Sigma	277258
4% paraformaldehyde	ThermoFisher	J61899.AK
4-hydroxytamoxifen (4OH-TAM)	Sigma	579002
50 mL conical centrifuge tubes	Fisher Scientific	14-432-22
70 µm nylon cell strainer	Corning	352350
96 well culture plate	Greiner Bio-One	655180
B-27 Supplement (50x)	Gibco	12587-010
Bovine serum albumin	Fisher Scientific	BP1605-100
Claudin-7 anti-murine rabbit antibody	Immuno-Biological Laboratories	18875
Cover glass (24 x 50-1.5)	Fisher Scientific	12544E
Cryomolds	vwr	25608-916
Cultrex RCF BME, Type 2	R&D Systems	3533-005-02	gel matrix
Cy3 anti-rabbit antibody	Jackson Immunoresearch	111-165-003
Dewar Flask	Thomas Scientific	1173F61
DMEM High Glucose with L-Glutamine	ATCC	30-2002
EVOS FLoid Imaging System	ThermoFisher	4477136
Fluoro-Gel II with DAPI	Electron Microscopy Sciences	17985-50
GlutaMAX (100x)	Gibco	35050-061
Glycine	JT Baker	4059-02
HEPES (1 M) Buffer Solution	Gibco	15630-080
Hoechst	ThermoFisher	62249
In situ cell death detection kit, TMR Red	Roche	12156792910
Isoflurane	Pivetal	07-893-8440
L-WRN Media	Harvard Medical School Gastrointestinal Organoid Derivation and Culture Core	N/A
Mouse surgical kit	Kent Scientific Corporation	INSMOUSEKIT
Murine EGF	PeproTech	315-09-500UG
N2 Supplement (100x)	Gibco	17502-048
Optimum cutting temperature (OCT) compound	Agar Scientific	AGR1180
Penicillin-Streptomycin	Gibco	15140-122
Sequenza Rack	vwr	10129-584
Sodium Citrate	Fisher Scientific	S-279
Sucrose	Sigma	S9378
Triton X-100	Sigma	X100
Vacuum filter (0.22 µm; cellulose acetate)	Corning	430769
Y-27632 dihydrochloride	Tocris Bioscience	1254