13. Chondrocyte culture medium: Ham’s F-12 Nutrient Mixture,

10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 50 μg/mL

ascorbic acid-2-phosphate (ASC), 5.0 ng/mL basic fibroblast

growth factor (bFGF), 10,000 U/mL penicillin G; 2.5 μg/mL

Amphotericin B.

2.3

Human

Chondrocyte

Propagation and

Maintenance

1. Human chondrocyte cell line (see Note 1).

2. T75 cell culture flasks.

3. 15 mL centrifuge polystyrene tubes.

4. Centrifuge.

5. CO2 incubator.

6. Inverted microscope.

7. Antibiotic/Antimycotic Solution.

8. Dulbecco’s phosphate-buffered saline (PBS): dissolve 8 g of

NaCl (137 mM), 200 mg of KCl (2.7 mM), 1.44 g of

Na2HPO4 (8 mM) and 240 mg of KH2PO4 (2 mM) in

800 mL of distilled water. Adjust pH to 7.4. Add distilled

water until volume is 1 L. Sterilize solution with autoclave

and store at +4
C.

9. Trypsin-EDTA solution: dissolve 0.5 g of porcine trypsin and

0.2 g of EDTA 4Na in 1 L of HBSS with phenol red.

10. Chondrocyte culture medium: Ham’s F-12 Nutrient Mixture,

10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 50 μg/mL

ascorbic acid-2-phosphate (ASC), 5.0 ng/mL basic fibroblast

growth factor (bFGF), 10,000 U/mL penicillin G; 2.5 μg/mL

Amphotericin B.

2.4

Repopulation of

the Decellularized

Tracheal ECM-Based

Porcine Bio-Scaffold

with Human

Chondrocytes and

Bioreactor Setup

1. Autoclave.

2. 15 mL centrifuge polystyrene tubes.

3. Centrifuge.

4. CO2 incubator.

5. Cell counting chambers.

6. Inverted microscope.

7. Sterile decellularized tracheal ECM-based bio-scaffold.

8. Sterile rotating bioreactor.

9. Dulbecco’s phosphate-buffered saline (PBS): dissolve 8 g of

NaCl (137 mM), 200 mg of KCl (2.7 mM), 1.44 g of

Na2HPO4 (8 mM) and 240 mg of KH2PO4 (2 mM) in

800 mL of distilled water. Adjust pH to 7.4. Add distilled

water until volume is 1 L. Sterilize solution with autoclave

and store at +4
C.

10. Trypsin-EDTA solution: dissolve 0.5 g of porcine trypsin and

0.2 g of EDTA 4Na in 1 L of HBSS with phenol red.

Reconstruction of a Bioprosthetic Trachea

161