Materials and Reagents

1. Hank’s balanced salt solution (HBSS) without calcium and magnesium (Sigma-Aldrich, catalog number: H2387 )
   
2. Collagenase type II (Sigma-Aldrich, catalog number: C6885-500MG )
   
3. Fetal bovine serum (FBS) (EuroClone, catalog number: ECS0180L )
   
4. Trypan blue (Sigma-Aldrich, catalog number T6146-5G )
   
5. Dulbecco’s phosphate buffered saline (D-PBS) (Life Technologies, catalog number: 14190-250 )
   
6. Fibronectin from human plasma 0.1% solution (cell culture tested) (Sigma-Aldrich, catalog number: F0895 )
   
7. Modified Eagle’s medium (MEM) Joklik without NaHCO₃ (Sigma-Aldrich, catalog number: M0518 )
   
8. Hepes (powder) (Sigma-Aldrich, catalog number: H3375 )
   
9. L-glutamine (Sigma-Aldrich, catalog number: G7513 )
   
10. Taurine (Sigma-Aldrich, catalog number: T0625 )
    
11. Penicillin-streptomycin (100x solution stabilized, sterile-filtered, suitable for cell culture) (Sigma-Aldrich, catalog number: P4333-100ML )
    
12. Insulin (Sigma-Aldrich, catalog number: I5523-50MG )
    
13. Dulbecco’s modified Eagle’s medium (DMEM) low glucose (Life Technologies, Gibco^®, catalog number: 31600-091 )
    
14. MCDB-201 (Sigma-Aldrich, catalog number: M6770 )
    
15. Linoleic acid-bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: L9530 )
    
16. Dexamethasone (powder) (Sigma-Aldrich, catalog number: D2915 )
    
17. Ascorbic acid-2 phosphate (powder) (Sigma-Aldrich, catalog number: A8960 )
    
18. 100x insulin-transferrin-selenium (ITS) (Life Technologies, Gibco^®, catalog number: 41400-045 )
    
19. Stem cell tested fetal bovine serum (Stem Cells Technologies, catalog number: 10M37180 )
    
20. Human platelet derived growth factor-BB (hPDGF-BB) (Pepro Tech, catalog number: 100-14B )
    
21. Human epidermal growth factor (hEGF) (Pepro Tech, catalog number: AF-100-15 )
    
22. TrypLE-express dissociation reagent (Life Technologies, Gibco^®, catalog number: 12605 )
    
23. Basic buffer (BB) (see Recipes)
    
24. Incubation buffer (IB) (see Recipes)
    
25. 0.025% Collagenase type II (see Recipes)
    
26. 0.4% Trypan blue solution (see Recipes)
    
27. Fibronectin-coated 100 mm petri dish (see Recipes)
    
28. Multipotent adult stem cell (MASC) medium (see Recipes)
    

Equipment

1. Sterile tissue culture dishes 100 x 20 mm style (BD Biosciences, Falcon^®, catalog number: 353003 )
   
2. Scalpel blades (Sigma-Aldrich, catalog number 27046 )
   
3. Sterile serological transfer pipettes
   
4. Serological pipettes
   
5. Sterile Falcon tubes
   
6. Mesh filter (70 µm) (BD Biosciences, Falcon^®, catalog number 352350 )
   
7. Biological hood (Faster, model: Safe FAST Elite )
   
8. Agitation rotor (Celbio, model: MINI OVEN )
   
9. Burker counting chamber (Sigma-Aldrich, catalog number: Z359629-1EA )
   
10. 37 °C, 5% CO₂, 5% O₂ force air incubator (New Brunswick, model: Galaxy 170 R )
    
11. Centrifuge (Heraeus Instruments, model: Megafuge 1.0 R )
    
12. Light microscope (Leica Microsystems, model: DMIL )
    

Procedure