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Materials

• D-PBS without Ca++ and Mg++ (e.g. Catalog #37350)
• DMEM/F-12 with 15 mM HEPES (e.g. Catalog #36254)
• Bovine serum albumin (BSA)
• 50 mL conical tube (e.g. Falcon® Conical Tubes, Catalog #38010)
• 24-well plate (e.g. Costar® 24-Well Plate, Catalog #38017 or Falcon® 24-Well Plate, Catalog #38021)
• 15 mL conical tubes (e.g. Falcon® Conical Tubes, Catalog #38009)
• Microcentrifuge tubes (e.g. Costar® Microcentrifuge Tubes, Catalog #38089)
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• Forceps and scissors
• Gentle Cell Dissociation Reagent (Catalog #07174)
• 70 μm strainer (e.g. Catalog #27260)
• Pipettor (e.g. Corning® Lambda™ Plus Pipettor, Catalog #38060)
• Pipette tips (e.g. Corning® Filtered Pipette Tips, Catalog #38034)
• Serological pipette controller
• Serological pipettes (e.g. Falcon® Serological Pipettes, 10 mL, Catalog #38004)
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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).