1. Physiological 3D Tissue Model of the Airway Wall and Mucosa Melanie M. Choe Alice A. Tomei Melody A. Swartz Press return to continue

2. Normal human bronchial epithelial cells (NHBEs) 2 x T175 Fetal human lung fibroblasts (HLFs) 4 x T175 70% confluent PBS trypsin for HLFs trypsin for NHBE co-culture media hemacytometer 6 well transwell plate at least 12 ml collagen solution (7.25 < pH < 7.3) 6 porous PE cylindrical inserts (acid treated to make hydrophilic) Cells & reagents needed for 6 models Press return to continue

3. 1.Harvest fibroblasts (> 6*10 6 cells) 2. Dissociate pellet in 1 ml co-culture medium 1 ml 3. Add collagen and co- culture medium to make a final solution of 500,000 cells /ml and 2.5 mg/ml collagen >12 ml 4. Mix well by gently pipetting up and down Avoid bubbles Press return to continue

4. Place the PE constructs into each transwell insert Pipette 100 µl HLF suspension in collagen into each insert to create a plug Incubate (37°C, 5% CO 2 ) 5 min Press return to continue

5. Pipette the HLF-collagen suspension to completely fill insert (~2 ml/insert) Avoid bubbles Incubate (37°C, 5% CO 2 ) 15 min (collagen turns opaque upon gelation) Press return to continue

6. Coat the top of collagen gel with a thin layer of acellular collagen (2.5 mg/ml) Avoid bubbles Press return to continue

7. Gently level the surface of the gel with a cell scraper Incubate (37°C, 5% CO 2 ) 5 min Press return to continue

8. Aspirate excess fluid from the bottom of the well Press return to continue

9. Gently pipette 2 ml medium to the bottom chamber Incubate (37°C, 5% CO 2 ) Press return to continue

10. PAUSE POINT The model can be stored in the incubator for several hours Press return to continue

11. 2. Dissociate pellet in 1 ml co-culture medium 1 ml 1. Harvest NHBE Press return to continue

12. Pipet 500,000 NHBE cells (200 µl) onto each PE well Incubate (37°C, 5% CO 2 ) 2 hours Press return to continue

13. Every ~20 minutes, examine the wells to ensure the top surface is covered by medium (otherwise pipet extra medium, ~100µl) Incubate (37°C, 5% CO 2 ) Press return to continue

14. After 2 hours, add medium to cover the surface and submerge the model Incubate (37°C, 5% CO 2 ) Culture in submersion for at least 7 days Refresh medium every 2 days Press return to continue

15. Check every few days under phase contrast to determine when epithelium is confluent Press return to continue

16. When the epithelium is confluent, create air-liquid interface (ALI) Aspirate the medium Press return to continue

17. Wash the epithelial surface with warm PBS (~500µl) gently (not directly on the cells) Aspirate the PBS Press return to continue

18. Change medium in the bottom well daily (maintain level for ALI) Wash epithelial surface with PBS daily Culture in air liquid interface for at least 7 days (21 days of optimal epithelial differentiation) END