patient specific in vitro platforms that provide increased similarity to

the in vivo physiology and pathology. The 3D semi-xenografts may

be used for the development of new therapies against upper airway

diseases and inflammation and can contribute to advances in the

prevention and treatments of life-threatening infections such as

COVID-19.

2

Materials

Prepare all solutions immediately before use (unless indicated

otherwise).

2.1

Porcine Trachea

Collection

1. Tracheas collected from a slaughterhouse.

2. Sterile plastic containers.

3. Ice container.

4. Dulbecco’s phosphate-buffered saline (PBS): dissolve 8 g of

NaCl (137 mM), 200 mg of KCl (2.7 mM), 1.44 g of

Na2HPO4 (8 mM), and 240 mg of KH2PO4 (2 mM) in

800 mL of distilled water. Adjust pH to 7.4. Add distilled

water until volume is 1 L. Sterilize solution with autoclave

and store at +4
C.

5. Antibiotic/Antimycotic Solution.

2.2

Generation of the

Decellularized

Tracheal ECM-Based

Porcine Bio-Scaffold

1. 50 mL centrifuge polypropylene tubes.

2. Water bath.

3. Orbital shaker.

4. 500 mL plastic or glass bottles.

5. Deionized water (DI-H2O).

6. Sterile water.

7. Ethanol.

8. Antibiotic/Antimycotic Solution.

9. 1% sodium dodecyl sulfate (SDS): dissolve 5 g of SDS in

500 mL of DI-H2O.

10. 1% Triton X-100: add 5 mL in 495 mL of DI-H2O.

11. 2% deoxycholate: dissolve 10 g of deoxycholate in 500 mL of

DI-H2O.

12. Dulbecco’s phosphate-buffered saline (PBS): dissolve 8 g of

NaCl (137 mM), 200 mg of KCl (2.7 mM), 1.44 g of

Na2HPO4 (8 mM) and 240 mg of KH2PO4 (2 mM) in

800 mL of distilled water. Adjust pH to 7.4. Add distilled

water until volume is 1 L. Sterilize solution with autoclave

and store at +4
C.

160

Georgia Pennarossa et al.