12. Introduce the 15 cm tubing into the short anchor fitting

protruding at the left of the bioreactor (Subheading 3.3.1,

step 12b, i.e., bioreactor LEFT side tubing).

3.3.3

Positioning the

Trachea

1. Disinfect the hybridization bottle (with the trachea) before

placing it inside the BSC (see Note 6).

2. Open a sterile package with clamps, forceps, and scissors (see

Note 4).

3. Clamp the tubing attached to both anchors.

4. Fill the bioreactor with 225 mL of DMEM + Antibiotics using

a plastic pipette.

5. Put on sterile gloves.

6. Place the trachea inside the bioreactor’s body and keep it

submerged in DMEM + Antibiotics, using a forceps.

7. Fit the two ends of the trachea over each anchor with grooved

tips to prevent slippage. Secure the trachea with one tight

4⁰⁰-long cable tie, using the pliers and tweezers to make it

tight. Then make a triple knot using a double-looped 20 cm

silk suture on each end of the trachea. Place the suture adjacent

to the cable tie on each end (see Note 10).

8. Connect the two anchors using two rods, specifically by placing

the rods’ washers into the small anchor’s grooves (Fig. 1b).

9. Secure the rods with one tight 6⁰⁰ long cable tie at each end.

10. Position and secure the lid with the silicone gasket on top of

the body by tightening two worm-drive hose clamps around

the bioreactor body and lid. Use a screwdriver for this step.

11. Remove the clamps.

12. Place all flasks, tubing, and clamps on the tray; then carefully

transfer the bioreactor system to the incubator.

3.3.4

Motor and Pumps

Installation

1. Place the bioreactor system inside the incubator at 37 ^C.

2. Place the small pulley onto the motor and secure it by tighten-

ing its screw.

3. Attach the programmable gear motor to the motor bracket

using four screws.

4. Make sure that the timing belt is connecting the large and small

pulleys.

5. Place the Tygon^® 3-stop tubing (SDS circuitry) onto the peri-

staltic pump. Set its flow to 7.5 mL/min.

6. Secure the 60 mL syringe onto the syringe pump.

7. Connect the motor to the Arduino motor shield. Then, con-

nect the Arduino to the electric power using an electric coaxial

cable and a 12 V 5 A switching power supply adapter.

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