15. Remove the detergents with 140 mL of PBS for 30 min using

the syringe pump at a flow rate of 4.5 mL/min (Fig. 6). Now,

the waste bottle should be filled with H2O, Triton X-100,

and PBS.

16. Remove the motor and small pulley from the system using an

Allen key.

17. Take the bioreactor system to the BSC using the tray, disinfect

the surfaces with 70% ethanol.

18. Disinfect a sterile hybridization bottle with fresh DMEM +

Antibiotics and place it inside the BSC.

19. Loosen and remove the two worm-drive hose clamps placed

around the bioreactor body and lid using the screwdriver.

20. Remove the lid on top of the body and expose the trachea.

21. Cut the 6⁰⁰ long cable ties using the scissors and remove

both rods.

22. Cut the trachea close to the anchors transversely. Use sterile

forceps to determine the edge of each anchor.

23. Put the trachea into the bottle with DMEM + Antibiotics.

3.5

Decontamination

of the De-

epithelialized Graft

1. Place the bottle with the trachea in a rocker at low-medium

speed for 24 h at RT.

2. Disinfect the bottle with 70% ethanol and place it inside

the BSC.

3. Disinfect a new bottle of fresh DMEM + Antibiotics with 70%

ethanol and place it in the BSC.

Fig. 6 PBS step schematic representation. H2O and Triton X-100 tubes are clamped on top of their respective

reservoirs. PBS flows through the tubing main circuitry and ends in the waste bottle. This is the final step of the

tracheal de-epithelialization

De-Epithelialization of Tracheal Grafts

179