8. Program the Arduino for a speed rotation of 5 rpm. The

bioreactor system is ready for de-epithelialization as shown in

Fig. 2.

3.4

Tracheal Graft

De-epithelialization

1. Start the tracheal rotation at 5 rpm inside an incubator at

37 ^C.

2. Clamp Tube-A and Tube-B from Subheading 3.3.1, steps 12

and 15.

3. Fill up the SDS closed-loop circuitry and tracheal luminal

chamber with 1% SDS at a flow rate of 5 mL/min using the

peristaltic pump (Fig. 3).

4. Tilt the bioreactor at 45^ for 5 min to remove the air inside the

trachea. Continue the SDS step for 3 h.

5. Stop the peristaltic pump, then remove the clamps from Tube-

A and Tube-B. The main one-way flow detergent-waste system

is now open.

6. Close the SDS circuitry by placing a clamp onto both SDS

tubing (see Note 11). Used SDS will remain in its reservoir

until the end of the process.

7. Clamp both Triton X-100 tubing (see Note 11).

8. Clamp both PBS tubing (see Note 11).

9. Wash the tracheal lumen with 240 mL of filtered deionized

H2O for 40 min using the syringe pump at flow rate of 6 mL/

min (Fig. 4). Used H2O should be seen in the waste bottle.

10. Remove the Triton X-100 tubing clamps.

Fig. 3 SDS step schematic representation. Two clamps are positioned on each side of the tubing main circuitry

(Tube-A and Tube-B) to make an SDS closed system

De-Epithelialization of Tracheal Grafts

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