Materials and Reagents

1. Cover glass (Corning, catalog number: 2998075X25 )
   
2. 48-Well Tissue Culture Plate (Corning, Falcon^®, catalog number: 351178 )
   
3. 50 ml and 15 ml Conical centrifuge tubes (Corning, Falcon^®, catalog number: 352098 and 352097 )
   
4. 10 ml Syringe (BD, catalog number: 309604 )
   
5. 21G Needle (BD, catalog number: 305165 )
   
6. Pipette Tips
   
7. 70 μM Cell Strainer (Corning, Falcon^®, catalog number: 352350 )
   
8. 100 μM Cell Strainer (Corning, Falcon^®, catalog number: 352360 )
   
9. Mice to be harvested for this protocol must be approved for use by the Institutional Animal Care and Use Committee (IACUC) at the institution, which sponsors the laboratory research
   
10. Phosphate Buffered Saline (PBS) (Invitrogen, catalog number: 10010023 )
    Note: Currently, it is “ Thermo Fisher Scientific, Gibco^TM, catalog number: 10010023”.
    
11. Growth Factor Reduced Matrigel (BD, catalog number: 356230 )
    Note: Currently, it is “Corning, Matrigel^®, catalog number: 356230”.
    
12. Fetal Bovine Serum (Thermo Fisher Scientific, Gibco^TM, catalog number: 16000-044 )
    
13. Collagenase Type IV (Worthington, catalog number: LS004188 )
    
14. Bovine Serum Albumin (Sigma-Aldrich, catalog number: A2058 )
    
15. 1% BSA-PBS (sterile)
    
16. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
    
17. 5 mM EDTA-PBS
    
18. Recombinant DNase I, RNase Free (which is provided at 10 U/μl) (Sigma-Aldrich, catalog number: 4716728001 )
    
19. Optional: 10 μM Rho Kinase Inhibitor Y-27632, provided as a 5 mM Solution (Merck Millipore Corporation, catalog number: 68801 )
    
20. Advanced DMEM F/12 (Thermo Fisher Scientific, Gibco^TM, catalog number: 12634-010 )
    
21. Streptomycin (Gibco, catalog number: 15140-22 )
    
22. N-Acetylcysteine (Sigma-Aldrich, catalog number: A9165 )
    
23. HEPES (Sigma-Aldrich, catalog number: H3375 )
    
24. EGF 50 ng/ml (Invitrogen, catalog number: PMG8043 )
    Note: Currently, it is “ Thermo Fisher Scientific, Gibco^TM, catalog number: PMG8043”.
    
25. Recombinant Murine EGF 50 ng/ml (Invitrogen, catalog number: PMG8043)
    
26. Recombinant Murine Noggin 50 ng/ml (Peprotech, catalog number: 250-38 )
    
27. Recombinant Human R-Spondin 1,500 ng/ml (R&D Systems, catalog number: 3474-RS-050 )
    
28. Recombinant Murine Wnt-3A 100 ng/ml (Merck Millipore Corporation, catalog number: GF-160 )
    
29. 10 mM Nicotinamide (Sigma-Aldrich, catalog number: N3376 )
    
30. Intestinal basal medium (see Recipes)
    
31. Small intestinal organoid growth media (see Recipes)
    
32. Large intestinal organoid growth media (see Recipes)
    

Equipment

1. Pipettes, Pipetaid and Micro-pipettes
   
2. Dissection Forceps and Scissors (Fine Science Tools, catalog number: 11150-10 and 14058-09 )
   
3. Centrifuge 5810R (Eppendorf, model: 5810R )
   
4. Brightfield inverted Microscope
   
5. Tabletop Roller in 4 °C Room (Bibby Scientific Limited, Stuart Equipment, model: SRT9D )
   
6. 37 °C, 5% CO₂ cell culture incubator
   
7. Biosafety cabinet (Tissue culture hood)
   
8. For mouse sacrifice by carbon dioxide asphyxiation, a carbon dioxide source, regulated dispenser, and euthanasia chamber, must be used in accordance with approved animal use protocols at the laboratory’s sponsoring institution.
   

Procedure