11. 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.

3

Methods

All the procedures described below must be performed under

sterile conditions. All instruments touching or in connection to

tissues, cells, scaffolds, and bioreactors have to be sterilized. Cell

manipulation must be carried out under laminar a flow hood and

cell cultures have to be maintained at 37
C during their handling

using thermostatically controlled stages.

All studies were reviewed and approved by the Ethical Com-

mittee of the University of Milan. All animal experiments were

performed in accordance with the Guide for the Care and Use of

Laboratory Animals, published by the US National Institutes of

Health (NIH).

3.1

Porcine Trachea

Collection

1. Collect tracheal segments from gilts weighing approximately

120 kg.

2. Transfer tracheas in cold sterile PBS containing antibiotic/

antimycotic solution (5 mL/500 mL) and transport them to

the laboratory using ice container.

3.2

Generation of the

Decellularized

Tracheal ECM-Based

Porcine Bio-Scaffold

1. Wash extensively tracheal graft in fresh PBS.

2. Completely remove the PBS, place the trachea in an empty

50 mL tube and store organ at 80
C for at least 24 h (see

Note 2).

3. Thaw trachea at 37
C for 30 min using a water bath.

4. Transfer trachea in a bottle containing 500 mL of 1% SDS.

Place the bottle onto an orbital shaker at 200 rpm and incubate

for 3 h at room temperature.

5. Remove SDS solution from the bottle containing the trachea

and wash it with 500 mL of DI-H2O for 40 min using an

orbital shaker at 200 rpm.

6. Remove DI-H2O from the bottle and add 500 mL of 1%

Triton X-100. Incubate trachea for 12 h at room temperature

in 1% Triton X-100, using an orbital shaker at 200 rpm.

7. Remove Triton X-10 solution from the bottle containing the

trachea and add 500 mL of DI-H2O twice.

162

Georgia Pennarossa et al.