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

Published: April 30, 2023

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 Autoclave one set of intestinal scissors, normal scissors, and forceps in a sterile container. Place a 96-well (flat bottom) dish in an incubator at 37 °C. Prepare 10 mL of complete culture medium with the reagents listed in the Table…

開示

The authors have nothing to disclose.

Materials

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

タグ

Play Video

記事を引用
Intestinal Crypts Isolation: A Method to Isolate Whole Crypts from Small Intestine of Murine Model. J. Vis. Exp. (Pending Publication), e20427, doi: (2023).

View Video