Materials and Reagents

1. Culture plate, 24 wells (Becton Dickinson Labware, catalog number: 353047 )
   Note: Currently, it is “Corning, Falcon^®, catalog number: 353047”.
   
2. Pipette tips, 1,000 μl, 200 μl and 10 μl volumes (Corning, DeckWorks^TM, catalog numbers: 4124 , 4121 and 4120 )
   
3. Pasteur pipettes, 230 mm length (WHEATON, catalog number: 4500448667 )
   Note: Currently, it is “WHEATON, catalog number: 357335”.
   
4. Conical tube, 50 ml volume (Corning, Falcon^®, catalog number: 352070 )
   
5. Cell strainer, nylon with 100 μm pores (Corning, BD Biosciences, catalog number: 352360 )
   
6. Tissue-culture flask, 150 cm² surface area (T150) (Corning, Falcon^®, catalog number: 355000 )
   
7. Conical microtube, 1.5 ml volume (Bio Basic Canada, catalog number: TC152SN )
   
8. Biopsy punch, circular with 6 mm diameter (Southern Anesthesia & Surgical, Miltex, catalog number: 3336 )
   
9. Bone marrow aspirate, human or ovine, collected through needle aspiration at the iliac crest
   
10. Crystal violet solution (Sigma-Aldrich, catalog number: HT90132 )
    
11. Alpha minimal essential medium (αMEM), containing Earle’s salts, ribonucleosides, deoxyribonucleosides and L-glutamine (Thermo Fisher Scientific, Corning, Mediatech，catalog number: 10022CV )
    
12. Fetal bovine serum (FBS), heat inactivated at 56 °C in the laboratory (Life Technologies, Gibco^®, catalog number: 12483 )
    Note: Currently, it is “Thermo Fisher Scientific, Gibco^TM, catalog number: 12483 ”.
    
13. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (1 M) (Life Technologies, Gibco^®, catalog number: 15630 )
    Note: Currently, it is “Thermo Fisher Scientific, Gibco^TM, catalog number: 15630”.
    
14. Sodium pyruvate, 100 mM (Life Technologies, Gibco^®, catalog number: 11360 )
    Note: Currently, it is “Thermo Fisher Scientific, Gibco^TM, catalog number: 11360”.
    
15. Fibroblast growth factor-two (FGF-2), human recombinant (Neuromics, catalog number: PR80001 )
    
16. Dulbecco’s phosphate buffered saline (PBS), sterile filtered (Sigma-Aldrich, catalog number: D8537 )
    
17. Human serum albumin (Sigma-Aldrich, catalog number: A4327 )
    
18. Transforming growth factor-beta three (TGF-β3), human recombinant, HEK (Prospecbio, ProSpec, catalog number: CYT-113 )
    
19. L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, catalog number: A8960 )
    
20. Dexamethasone-Water Soluble (Sigma-Aldrich, catalog number: D2915 )
    
21. L-proline (Sigma-Aldrich, catalog number: P5607 )
    
22. Trypan blue solution, 0.4% (Sigma-Aldrich, catalog number: T8154 )
    
23. Porous scaffolds, collagen I or esterified hyaluronic acid (described in detail in Bornes et al., 2015)
    
24. Penicillin-streptomycin-glutamine (Life Technologies, Gibco^®, catalog number: 1248310378 ) (see Recipes)
    Note: Currently, it is “Thermo Fisher Scientific, Gibco^TM, catalog number: 1248310378 ”.
    
25. Trypsin-ethylenediaminetetraacetic acid (EDTA) (Thermo Fisher Scientific, Corning, catalog number: 25052 ) (see Recipes)
    
26. Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, catalog number: D6429 ) (see Recipes)
    
27. Insulin-transferrin-selenium (ITS+) premix (Corning, BD Biosciences, catalog number: 354352 ) (see Recipes)
    
28. Expansion medium (see Recipes)
    
29. Serum-free medium (see Recipes)
    
30. TGF-β3 working solution (see Recipes)
    
31. Chondrogenic medium (see Recipes)
    

Equipment

1. Biosafety cabinet (Microzone Corporation, catalog number: BK-2-6 A2 )
   
2. Incubator, containing humidified air at 37 °C with 5% carbon dioxide, and 3% oxygen (Thermo Fisher Scientific, catalog number: Forma Series II Water Jacket CO₂ )
   
3. Centrifuge, 1,500 revolutions per min (rpm) (Beckman Coulter, Allegra^TM, catalog number: X-22R )
   
4. Light microscope (Microscope, Omano, catalog number: OM159T )
   
5. Pipette (Drummond Scientific Company, catalog number: Pipet Aid XP )
   
6. Micropipettes, volumes of 100-1,000 μl, 20-200 μl, 2-20 μl, and 0.5-10 μl (Bio-Rad Laboratories, catalog numbers: 1660508 , 1660507 , 1660506 , and 1660505 )
   
7. Suction source
   
8. Forceps
   
9. Water bath, set to 37 °C (VWR International, catalog number: 89501 )
   
10. Neubauer hemacytometer, 0.1 mm deep (Reichert Bright-Line) (Sigma-Aldrich, catalog number: Z359629 )
    

Procedure