8. Remove the last washing DI-H2O from the bottle and incubate

trachea in 500 mL of 2% deoxycholate for 12 h at room

temperature, using an orbital shaker at 200 rpm.

9. Remove deoxycholate and wash trachea with DI-H2O for 6 h

at room temperature, using an orbital shaker at 200 rpm.

Changes DI-H2O every 2 h (see Note 3).

10. Sterilize the tracheal ECM-based bio-scaffold in a water solu-

tion containing 70% Ethanol and 2% antibiotic/antimycotic

solution for 30 min at room temperature, using an orbital

shaker at 200 rpm.

11. Extensively wash the ECM-based bio-scaffold with PBS sup-

plemented with 4% antibiotic/antimycotic solution at room

temperature using an orbital shaker at 200 rpm.

12. Before

chondrocyte

repopulation,

immerse

the

tracheal

ECM-based bio-scaffold in chondrocyte culture medium for

at least 1 h at room temperature.

3.3

Human

Chondrocyte

Propagation and

Maintenance

1. Maintain human chondrocyte cell line in 5% CO2 incubator at

37
C.

2. Monitor cells daily.

3. Once cell culture has reached 80% confluency, carefully aspirate

the culture medium from T75 flask (see Note 4).

4. Wash cells three times with 7 mL of PBS supplemented with 1%

antibiotic antimycotic solution.

5. Add 2 mL of trypsin-EDTA solution (see Note 5) and incubate

at 37
C until cell monolayer begins to detach from the bottom

of the tissue culture dish and cells dissociate (see Note 6).

6. Dilute cell suspension in 8 mL of chondrocyte culture medium

to neutralize trypsin action.

7. Dislodge cells by repeatedly and gently pipetting.

8. Collect cell suspension in a 15 mL centrifuge polystyrene tube

and centrifuge at 300  g for 5 min.

9. Remove supernatant and resuspended chondrocytes in 10 mL

of fresh culture medium.

10. Plate cells in a new culture dish and culture at 37
C in 5% CO2

incubator. Keep the passage ratio between 1:2 and 1:4,

depending on growth rate (see Note 7).

11. Change medium every 2–3 days.

12. Maintain cells in culture until they have reached 80% con-

fluency and passage them.

Reconstruction of a Bioprosthetic Trachea

163