(c)

Use a saw to open the sternum and expose the mediastinal

contents.

(d)

Dissect the midline neck and strap muscles, connective

tissue and mobilize the thyroid to expose the trachea.

(e)

Dissect circumferentially around the trachea as well as

proximally and distally into the chest to expose it above

the carina (note: there is variability in anatomy with pigs

having a separate bronchus above the carina).

(f)

Use forceps to grasp the proximal portion of the trachea

and using scissors make an incision in the trachea below

the larynx.

(g)

Lift the trachea caudally and snip any attachments of the

mediastinal tissue.

(h)

Make an incision in the trachea at the carina or above the

bronchus to separate it from the lungs.

6. Wipe with 10% bleach, spray 70% ethanol, then dry with one

paper towel at a time before putting any non-autoclaved

objects inside the BSC.

7. Make the tubing circuitries on the bench and send them for

autoclaving, including the rubber stoppers with the tubes in

their holes. Replace the tubing with new ones when they start

to wear off, perhaps every two or three experiments.

8. Make one package with the bioreactor main components and

another one with the extra components. Package the tray sepa-

rately and autoclave the silicone-based lubricant in a 50 mL

glass bottle. Have extra packages with sterile surgical instru-

ments, tubing, and tube fittings just in case they are needed.

9. Prevent leakage by using Teflon tape which may be needed for

the barbed swivel adapter tube fitting and the barbed tube

fitting adapter that goes into the anchors.

10. Position cable ties and silk sutures to prevent the communica-

tion between the inner and outer chambers; this will prevent

detergents from being in contact with the outer wall of the

trachea.

11. Place the clamp on the tubing protruding from the top of the

stopper.

12. The estimated time for each of the steps is as follows:

(a)

Preparation of media, antibiotics, and detergents: 4 h.

(b)

Graft procurement and preparation: 4 h.

(c)

Bioreactor setup for de-epithelialization.

l

Detergent circuitries: 30 min.

l

Bioreactor setup: 10 min.

De-Epithelialization of Tracheal Grafts

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