This article details a method for isolating small intestinal crypts from mice, focusing on the anatomical structures involved and the procedural steps necessary for successful isolation. The process is critical for studying intestinal biology and related research.
The intestinal lining is covered with projections or villi and invaginations or crypts. Crypts are the intestinal glands that primarily secrete digestive enzymes.
To isolate small intestinal crypts, begin by positioning an anesthetized mouse in the supine position on a dissection board. Using scissors, dissect the peritoneum longitudinally to expose the digestive system.
Locate the small intestine and gently pull it up from the mouse's body. Snip the intestine at its base. Transfer the harvested intestine to a dish containing a suitable buffer. Cut through the intestine longitudinally, exposing the lumen.
Place the intestine, luminal side up, on a cutting board to expose the villi. Move an angled glass slide over the intestinal surface to scrape off the villi, exposing the whole crypts.
Cut the intestine into tiny pieces, and transfer the pieces into a tube containing an ice-cold buffer to ensure minimal damage to the crypts.
Next, treat with EDTA and incubate. EDTA causes the whole crypts to loosen from the intestinal lining. Remove the spent EDTA-containing solution. Add fresh buffer and triturate using a pipette to detach the crypts from the intestinal lining.
Collect the supernatant containing the crypts into a fresh tube. Supplement the tube with a suitable culture medium and centrifuge to pellet the crypts.
Dissect the peritoneum longitudinally with a pair of scissors. Hold the stomach with the forceps and cut it transversely in half using intestinal scissors from this point forward. Pull out the intestine and place it in a Petri dish containing PBS. 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 open longitudinally, wash it in cold PBS by holding it with the forceps and gently rinsing 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 removed the villa by scraping the flattened intestine. Perform this step twice along the whole length of the tissue. Then, cut the small intestine with a sterile surgical blade into 2- to 5-millimeter pieces.
Place the small intestinal fragments in a 50-milliliter tube containing 10 milliliters 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. Next, add 15 milliliters of cold PBS to bring the total final volume of PBS to 25 milliliters. Add 2 millimolar EDTA and incubate for 45 minutes on a roller at 4 degrees Celsius.
After discarding the PBS/EDTA, add 10 milliliters of PBS and detach the crypts by harshly pipetting the tissue fragments up and down. Then, collect the supernatant. Repeat the step four times. Add culture medium to reach a final volume of 50 milliliters. Pellet the cells by centrifugation. Finally, resuspend the pellet in 10 milliliters of culture medium before pelleting the crypts by centrifugation.
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