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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 of Materials.
- Incubate the complete medium at 37 °C in a water bath.
- Thaw reconstituted basement membrane by placing it in an ice bucket. The reconstituted basement membrane will become liquid at 4 °C .
- Fill four Petri dishes with cold phosphate-buffered saline or PBS (4 °C).
2. Isolation of Small Intestinal Crypts
- Sacrifice by CO2 inhalation an Lgr5-eGFP-IRES-CreERt2 mouse on a C57BL6/J background. Dissect the peritoneum longitudinally with a pair of scissors.
- Hold the stomach with the forceps and cut it transversally in half.
- 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.
- 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.
- 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.
- 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.
- With a glass slide, gently remove the villi by scraping the flattened intestine. Perform this step twice along the whole length of the tissue.
- Cut the small intestine with a sterile surgical blade into 2–5 mm pieces.
- Place the small intestinal fragments in a 50 mL tube containing 10 mL of ice-cold PBS.
- 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.
- 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.
- Discard the PBS/EDTA.
- 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.
- Repeat step 2.13 four times. Add culture medium to reach a final volume of 50 mL.
- Pellet the cells by centrifugation (300 x g for 5 min).
- Resuspend the pellet in 10 mL of culture medium and pellet the crypts by centrifugation (80 x g for 3 min).