4. Change the trachea from one bottle to the new one using

sterile forceps.

5. Place the tracheal bottle on the rocker at low-medium speed for

24 h at RT.

Please see Note 12 for the approximate time period expected

for each of the described steps.

4

Notes

1. Make solutions, media, and detergents at least the day before

setting up the bioreactor. Filter the deionized water as well. It

may take 3 or 4 h.

2. Sterilization of biological scaffolds for tissue engineering appli-

cations carries the risk of unwanted changes in physical and

chemical

structure

[11].

We

designed

an

antimicrobial

solution-based disinfection method for partially decellularized

tracheal grafts. The concentrations of antibiotics and antimy-

cotics were optimized for further in vitro and ex vivo airway

epithelial cell differentiation protocols (human tracheal epithe-

lial cells and human induced pluripotent derived airway

progenitors).

3. Verify and adjust the pH to 7.4: If the pH is higher than 7.4,

add a few drops of hydrochloric acid solution. If the pH is

lower than 7.4, adjust it using sodium hydroxide solution.

Use a plastic pipette with the correct solution and add a few

drops to the solution in the beaker and wait at least 20 s before

reading the pH on the meter.

4. Make different packages to autoclave for ease of handling.

Packages of surgical instruments include the following:

(a)

For OR use, include one of each of the following: forceps,

spatula, scissors, and scalpel.

(b)

For lab use and graft preparation, make two packages with

each of the following instruments: forceps, spatulas.

(c)

For the tracheal positioning step, make one package with

the following: three clamps, one forceps, one spatula, and

a pair of scissors.

(d)

For the graft decontamination step, make a package with

only one forceps.

5. Procure the trachea aseptically in a sterile fashion using stan-

dard surgical procedures [12]:

(a)

Place the pig in supine position.

(b)

Make a skin incision along the anterior neck and overlying

the sternum.

180

Alba E. Marin-Araujo et al.