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 |
