Isolation, Culture and Differentiation of Primary Acinar Epithelial Explants from Adult Murine Pancreas

Materials and Reagents

1. Mice (4-7 weeks old). As a positive control you can always use wild type mice and induce differentiation of acinar explants by treatment with EGF as described. Depending on your application you may use transgenic mice.
   
2. McCoy′s 5A medium (Sigma-Aldrich, catalog number: M8403 )
   
3. Waymouth′medium MB 752/1 medium (Genaxxon, catalog number: C4119 )
   
4. BSA (Sigma-Aldrich, catalog number: A4503 )
   
5. Soybean trypsin inhibitor (SBTI) (Sigma-Aldrich, catalog number: T6522 )
   
6. Collagenase Type VIII (Sigma-Aldrich, catalog number: C2139 )
   
7. 80% EtOH
   
8. Penicillin-streptomycin (P/S) (Life Technologies, Gibco^®, catalog number: 15140-122 )
   
9. Amphotericin B (not critical, for example from Biochrom, catalog number: A2610 )
   
10. FCS (Life Technologies, Gibco^®, catalog number: 10270 )
    
11. Bovine pituary extract (BPE) (Life Technologies, Gibco^®, catalog number: 13028-014 )
    
12. Insulin, Transferrin, Selenium, Ethanolamine solution (ITS-X) (Life Technologies, Gibco^®, catalog number: 51500 )
    
13. Rat Tail Collagen Type I (BD Biosciences, catalog number: 354236 )
    
14. Sterile 10x PBS (BIOCHROM, catalog number: 182-50 )
    
15. Sterile dH₂O
    
16. Sterile 1 N NaOH
    
17. rhTGFa (R&D systems, catalog number: 239-A )
    
18. mEGF (BD Biosciences, catalog number: 354010 )
    
19. SBTI stock solution (see Recipes)
    
20. Collagenase VIII stock solution (see Recipes)
    
21. Wash solution (see Recipes)
    
22. 0.1% Culture medium (Waymouth′s medium) (Genaxxon, catalog number: C4119) (see Recipes)
    
23. 30% Culture medium (Waymouth′s medium) (Genaxxon, catalog number: C4119) (see Recipes)
    

Equipment

1. Stericups filter units (EMD Millipore)
   
2. 50 ml Falcon tubes
   
3. Tissue culture dishes (10 mm)
   
4. 6-well plates
   
5. 24- or 48-well plates
   
6. Dissecting forceps and scissors
   
7. Disposable scalpels
   
8. 100 μM cell strainer (BD Biosciences, Falcon^®, catalog number: 352360 )
   
9. Syringe plungers
   

Procedure