How to Isolate Human Colonic Crypts from Intestinal Biopsies

This protocol describes how to isolate human colon crypts from biopsies. The isolated crypts can subsequently be used for intestinal organoid culture using IntestiCult™ Organoid Growth Medium (Human).

Materials


Protocol

  1. Place the following reagents on ice: D-PBS (without Ca++ and Mg++) and DMEM + 1% BSA.

    Preparation of DMEM + 1% BSA

    Use sterile technique to prepare DMEM + 1% BSA. The following example is for preparing 50 mL of DMEM + 1% BSA. If preparing other volumes, adjust accordingly.

    1. Add 2 mL of 25% BSA to 48 mL of DMEM/F-12 with 15 mM HEPES in a 50 mL conical tube.
    2. Mix well by inversion. Place on ice.

    NOTE: If not used immediately, store at 2 - 8°C for up to 6 months.

  2. Warm a tissue culture-treated 24-well plate in a 37°C incubator for at least 2 hours.
  3. In a 15 mL conical tube, wash the tissue sample obtained from colon biopsies with 10 mL of ice-cold PBS. Allow the tissue to settle by gravity (~5 seconds), then aspirate the supernatant.
  4. Repeat step 3, leaving 1 mL of supernatant in the tube.
  5. Using a 1 mL pipettor, transfer the tissue and remaining supernatant to a 1.5 mL microcentrifuge tube.
  6. Using sterile scissors, thoroughly mince the tissue into the smallest pieces possible. Transfer the tissue fragments to a new 15 mL conical tube using a 1 mL pipettor. Rinse the microcentrifuge tube with D-PBS and add the rinse to the tissue fragments.
  7. Allow the tissue fragments to settle by gravity (~5 seconds), then aspirate supernatant.
  8. Add 10 mL of Gentle Cell Dissociation Reagent, then incubate on ice on a rocking platform set at medium speed (~40 rpm) for 30 minutes.
  9. Centrifuge at 290 x g for 5 minutes. Aspirate supernatant.

    Note: For the remainder of the protocol, pre-wet pipette tips with DMEM + 1% BSA before manipulating the tissue sample. This prevents crypts from sticking to the wall of the pipette tip.

  10. Add 1 mL of ice-cold DMEM + 1% BSA. Vigorously pipette up and down 20 times with a 1 mL pipettor to remove crypts from tissue.

    Note: Avoid touching the side/bottom of the tube with the pipette tip.

  11. Using a 1 mL pipettor, pass the contents of the tube through a 70 µm strainer (tilted on its side) into a new 15 mL conical tube. Rinse the original tube with 1 mL of DMEM + 1% BSA and pass through the strainer into the tube. This final tube contains isolated crypts that can be used for establishing intestinal organoid culture using IntestiCult™ Organoid Growth Medium (Human).
  • Document #PR00045
  • Version 1.0.0
  • May 2021


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