Intestinal Crypts Isolation: A Method to Isolate Whole Crypts from Small Intestine of Murine Model

Abstract

Source: Schewe, M. et al. The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells. J. Vis. Exp. (2017)

In this video, we demonstrate a technique to isolate whole crypts from a mouse’s small intestine. The isolated whole crypts can be used to propagate long-lived and self-renewing 3D organoids in vitro.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of Instruments, Culture Media, and Dishes

1. Autoclave one set of intestinal scissors, normal scissors, and forceps in a sterile container.
2. Place a 96-well (flat bottom) dish in an incubator at 37 °C.
3. Prepare 10 mL of complete culture medium with the reagents listed in the Table of Materials.
4. Incubate the complete medium at 37 °C in a water bath.
5. Thaw reconstituted basement membrane by placin....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Advanced DMEM/F-12	Thermo Fisher Scientific	12634-010
Penicillin/Streptomycin	Thermo Fisher Scientific	15140122	Final concentration: 1%
Glutamax	Thermo Fisher Scientific	35050061	Final concentration: 10 mM
Hepes	Thermo Fisher Scientific	15630080	Final concentration: 10 mM
Recombinant murine EGF	Thermo Fisher Scientific	PMG8041	Final concentration: 50 ng/ml
Recombinant murine Noggin	Peprotech	250-38	Final concentration: 100 ng/ml
y27632	Sigma	Y0503	Final concentration: 10 µM
Jagged-1	Anaspec Bio	AS-61298	Final concentration: 1 µM
Recombinant murine R-spondin	R&D systems	3474-RS-050	Final concentration: 1 mg/ml
B6.129P2-Lgr5tm1(cre/ERT2)Cle/J	Jackson Laboratories	8875
Low binding tubes	Eppendorf	Z666548-250EA