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

Overview

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 placing it in an ice bucket. The reconstituted basement membrane will become liquid at 4 °C .
6. Fill four Petri dishes with cold phosphate-buffered saline or PBS (4 °C).

2. Isolation of Small Intestinal Crypts

1. Sacrifice by CO₂ inhalation an Lgr5-eGFP-IRES-CreER^t2 mouse on a C57BL6/J background. Dissect the peritoneum longitudinally with a pair of scissors.
2. Hold the stomach with the forceps and cut it transversally in half.
3. Using the intestinal scissors from now on, pull out the intestine and place it in a Petri dish containing PBS.
   NOTE: Intestinal scissors have a sharp and blunt tip. The blunt tip of these scissors is meant to not damage the crypt-villus architecture while opening the intestine.
4. Start by placing the blunt tip into the stomach and gently push it through the pylorus. Proceed by cutting with the scissors and pulling with the forceps.
5. Once the whole small intestine is opened longitudinally, wash it in cold PBS by holding it with the forceps and gently rinse it in the PBS solution with U-shaped movements.
6. Once all the stool remnants are cleared, proceed to flatten the intestine on a cutting board, luminal side up. The luminal side is easily recognizable by the absence of blood vessels and by its pale appearance when compared with the outer part.
7. With a glass slide, gently remove the villi by scraping the flattened intestine. Perform this step twice along the whole length of the tissue.
8. Cut the small intestine with a sterile surgical blade into 2–5 mm pieces.
9. Place the small intestinal fragments in a 50 mL tube containing 10 mL of ice-cold PBS.
10. Clean the tissue fragments, removing any remaining impurities by pipetting them up and down in the PBS. Discard the supernatant and repeat this step until the PBS is completely clear.
11. Add 15 mL cold PBS to bring the total final volume of PBS to 25 mL. Add 2 mM ethylenediaminetetraacetic acid (EDTA) and incubate for 45 min on a roller at 4 °C.
12. Discard the PBS/EDTA.
13. Add 10 mL of PBS and detach the crypts by harshly pipetting the tissue fragments up and down (at least three times). Collect the supernatant.
14. Repeat step 2.13 four times. Add culture medium to reach a final volume of 50 mL.
15. Pellet the cells by centrifugation (300 x g for 5 min).
16. Resuspend the pellet in 10 mL of culture medium and pellet the crypts by centrifugation (80 x g for 3 min).

Materials

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