3.4

Repopulation of

the Decellularized

Tracheal ECM-Based

Porcine Bio-Scaffold

with Human

Chondrocytes and

Bioreactor Setup

1. Carefully remove culture medium from culture dish, wash cells

three times with PBS and incubate in 2 mL of trypsin-EDTA

solution at 37
C until cell monolayer begins to detach (for

detailed procedure see Subheading 3.3, steps 3 and 4).

2. Add 8 mL of chondrocyte culture medium to cell suspension

and

collect

chondrocytes

in

a

15

mL

centrifuge

polystyrene tube.

3. Count cells using a counting chamber under an inverted opti-

cal microscope at room temperature.

4. Calculate the volume of medium needed to resuspend chon-

drocytes to obtain a concentration of 1  10⁶ cells/cm² of

tracheal lumen bio-scaffold (see Note 8).

5. Centrifuge cell suspension at 300  g for 5 min. Carefully

aspirate supernatant and resuspend cell pellet in the previously

calculated volume (see Subheading 3.2, step 4) of chondrocyte

culture medium.

6. Repopulate the tracheal ECM-based bio-scaffold by injecting

chondrocytes into the tracheal lumen and carefully transfer it

into CO2 incubator.

7. Allow chondrocyte adhesion for 2 h under static conditions.

8. Connect the repopulating tracheal ECM-based bio-scaffold to

an autoclaved bioreactor, set a rotation of 5 rpm (see Note 9)

and culture at 37
C in 5% CO2 incubator.

9. Change culture medium every 48 h.

4

Notes

1. It is possible to apply the protocol here described to other cell

types, such as tracheal epithelial cells (HTEpCs).

2. Tracheal segments can be stored at 80
C for longer periods

without causing ECM alteration.

3. At the end of decellularization protocol, it is a good practice to

verify the efficiency of the process, by confirming cellular com-

partment removal (e.g., DNA quantification, DAPI and/or

hematoxylin and eosin staining) and the retention of intact

ECM components (e.g., collagen, elastin, glycosaminoglycans,

etc.).

4. Confluency normally takes between 7–10 days. If cells are not

confluent after 10 days, they are not successfully growing.

5. The trypsin volume here reported is necessary for detaching

cell cultured in a T75 flask. When working with smaller flask or

dish, scale down the volumes accordingly.

6. It usually takes 3–5 min.

164

Georgia Pennarossa et al.