Culture and Characterization of Differentiated Airway Organoids from Fetal Mouse Lung Proximal Progenitors

Materials and reagents

Biological materials

1. 10–12-week-old adult male mice and sexually attracted female mice; obtained from the Animal Experimental Center of Anhui Medical University

1. PBS (Thermo Fisher, catalog number: 20012027), store at RT

2. Ethanol absolute (Hushi, catalog number: 10009218), store at RT

3. Sucrose (Sigma-Aldrich, catalog number: V900116), store at RT

4. Triton X-100 (Sigma-Aldrich, catalog number: T8787), store at RT

5. Normal goat serum (Gibco, catalog number: 16210-064), shelf-life is 3 months, store at -20 °C

6. NaN₃ (Sigma-Aldrich, catalog number: S2002), dissolve 2 g in 1 mL of sterile Milli-Q-treated water, store at -20 °C.

   Caution: NaN₃ is hazardous. Avoid inhalation, ingestion, and skin contact.

7. Tween-20 (Sigma-Aldrich, catalog number: P1379), add 10 µL of Tween-20 in PBS to 10 mL, store at RT

8. Optimal cutting temperature compound (OCT) (Sakura, catalog number: 4853), store at RT

9. Chloral hydrate (MACKLIN, catalog number: 302-17-0), dissolve 10 g in 90 mL of sterile Milli-Q-treated water, store at 2–8 °C

10. Matrigel basement membrane matrix, growth factor reduced (GFR) (Corning, catalog number: 354230), store at -80 °C

    Note: Thaw Matrigel on an icebox in a 4 °C refrigerator, aliquot in 200 μL vials upon arrival, and minimize freeze and thaw cycles; it will polymerize at temperatures above 10 °C.

11. Paraformaldehyde (PFA) (MACKLIN, catalog number: 30525-89-4), dissolve 20 g of PFA in 500 mL of sterile PBS to prepare 4% PFA. Shelf-life is 2 months; store at 2–8 °C

    Caution: PFA is hazardous. Avoid inhalation, ingestion, and skin contact. Use protective equipment and work in a fume hood.

12. Type I rat tail collagen (Corning, catalog number: 354236), dissolve 4 mg of Type I rat tail collagen in 1 mL of 0.02 N acetic acid to prepare 4 mg/mL Type I rat tail collagen, then add 50 μL of 4 mg/mL Type I rat tail collagen in 1.95 mL of 0.02 N acetic acid to prepare 0.1 mg/mL Type I rat tail collagen. Store at -20 °C

13. Acetic acid (Sigma-Aldrich, catalog number: 695092), dissolve 1 mL in 87 mL of sterile Milli-Q-treated water. Use a syringe filter to filter the acetic acid solution; store at -20 °C

14. Penicillin/streptomycin (PS) (Life Technologies, catalog number: 15140-163), with 10,000 units penicillin and 10 mg streptomycin per milliliter in 0.9% NaCl; store at -20 °C

15. DMEM (high glucose, L-glutamine, sodium pyruvate, sodium bicarbonate, phenol red, w/o HEPES) (VivaCell, catalog number: 113-0500), store at 2–8 °C

16. DMSO (Solarbio, catalog number: D8370), store at RT

17. Advanced DMEM/F-12 (Gibco, catalog number: C11330500BT), store at 2–8 °C

18. GlutaMAX, 0.4 M (Gibco, catalog number: 35050061), store at 2–8 °C

19. HEPES, 1 M (Gibco, catalog number: 15630-080), store at 2–8 °C

20. Primocin^TM, 50 mg/mL (InvivoGen, catalog number: ant-pm), store at -20 °C aliquoted. Avoid multiple freeze-thaw cycles

21. NaHCO₃ (Sigma-Aldrich, catalog number: S6014), dissolve 0.75 g in 10 mL of sterile Milli-Q-treated water. Use a syringe filter to filter the solution; store at -20 °C

22. FBS (Biowest, catalog number: S1580-500), store at -20 °C

23. HBSS (Gibco, catalog number: 14175), store at RT

24. BSA (Sigma-Aldrich, catalog number: V900933), dissolve 0.1 g in 10 mL of PBS to prepare 1% BSA, store at 2–8 °C

25. EGF (Corning, catalog number: 354001), dissolve 100 μg in 4 mL of HBSS + 0.1% BSA. Shelf-life is 3 months; store at -20 °C aliquoted

26. CHIR99021 (Tocris, catalog number: 4423), dissolve 1 mg in 716.3 μL of DMSO. Shelf-life is 3 months; store at -20 °C aliquoted

27. Y-27632 (Tocris, catalog number: 1254), dissolve 1 mg in 624.5 μL of PBS. Shelf-life is 1 year; store at -20 °C aliquoted

28. Retinoic acid (Sigma-Aldrich, catalog number: R2625), dissolve 1 mg in 13.31 mL of DMSO. Make fresh each time and store at -20 °C

    Note: Vortex retinoic acid stock before making the working solution. This is light sensitive; store in the dark.

29. Transferrin (Sigma-Aldrich, catalog number: T1147-100 mg), dissolve 100 mg in 20 mL of HBSS + 0.1% BSA. Shelf-life is 2 months; store at -20 °C aliquoted

30. A-8301 (AbMole, catalog number: M5037), dissolve 5 mg in 23.72 mL of DMSO. Shelf-life is 2 months; store at -20 °C aliquoted

31. Insulin (Sigma-Aldrich, catalog number: I6634), dissolve 100 mg in 50 mL of 4 mM HCl. Shelf-life is 28 days; store at -20 °C aliquoted

32. Noggin (Sino Biological, catalog number: 10267), dissolve 1 mg in 50 μL of PBS. Shelf-life is 1 year; store at -20 °C aliquoted

33. Cholera toxin (Sigma-Aldrich, catalog number: 9012-63-9), dissolve 1 mg in 4 mL of HBSS + 0.1% BSA. Shelf-life is 1 year; store at -20 °C aliquoted

34. Pronase (Sigma-Aldrich, catalog number: 9036-06-0), dissolve 15 mg of Pronase in 10 mL of Ham’s F12

    Note: Use a 0.22 µm sterile filter to filter the Pronase solution and make fresh each time.

35. Ham’s F12 (Corning, catalog number: 10-080-cv), store at 2–8 °C

36. Anti-fluorescence quenching agent (Beijing Biotopped Technology, catalog number: Top0702). Shelf-life is 1 year; store at 2–8 °C. This is light sensitive; store in the dark

1. Anti-mouse Tp63 (Santa Cruz Biotechnology, catalog number: sc-25268), we recommend dilution at 1:400 with dilution buffer and storing at -20 °C aliquoted. Fresh dilution is prepared before staining

2. Anti-mouse acetylated α Tubulin (Abcam, catalog number: ab-24610), we recommend dilution at 1:400 with dilution buffer and storing at -20 °C aliquoted. Fresh dilution is prepared before staining

3. Anti-rabbit Krt5 (Abcam, catalog number: ab-64081), we recommend dilution at 1:250 with dilution buffer and storing at -20 °C aliquoted. Fresh dilution is prepared before staining

4. Goat anti-rabbit 555 (Life Technologies, catalog number: A21430), we recommend dilution at 1:1,000 with dilution buffer and storing at -20 °C aliquoted. This is light sensitive; store in the dark. Fresh dilution is prepared before staining

5. Goat anti-mouse 488 (Life Technologies, catalog number: A11029), we recommend dilution at 1:1,000 with dilution buffer and storing at -20 °C aliquoted. This is light sensitive; store in the dark. Fresh dilution is prepared before staining

6. DAPI (Sigma-Aldrich, catalog number: D9542), we recommend dilution at 1:1,000 with dilution buffer and storing at -20 °C aliquoted. This is light sensitive; store in the dark. Fresh dilution is prepared before staining

1. Buffer solution (see Recipes)

2. Suspension medium (see Recipes)

3. mTEC/basic medium (see Recipes)

4. mTEC/Plus1 medium (see Recipes)

5. mTEC/Plus2 medium (see Recipes)

6. Blocking buffer (see Recipes)

7. Dilution buffer (see Recipes)

1. Buffer solution (50 mL)

   Shelf-life is 1 month; store at 2–8 °C.

   Reagent	Final concentration	Volume
   PBS	n/a	49.5 mL
   Penicillin/streptomycin	500 U/mL	500 μL




2. Suspension medium (50 mL)

   Make fresh each time and store at 2–8 °C.

   Reagent	Final concentration	Volume
   DMEM	n/a	47 mL
   FBS	5%	2.5 mL
   Penicillin/streptomycin	500 U/mL	0.5 mL




3. mTEC/basic medium (50 mL)

   Shelf-life is 1 month; store at 2–8 °C.

   Reagent	Stock concentration	Final concentration	Volume
   Advanced DMEM/F-12	n/a	n/a	48.05 mL
   HEPES	1 M	15 mM	0.75 mL
   Penicillin/streptomycin	500 U/mL	500 U/mL	0.5 mL
   GlutaMAX	0.4 M	4 mM	0.5 mL
   NaHCO3	0.9 M	3.6 mM	0.2 mL




4. mTEC/Plus1 medium (3 mL)

   Shelf-life is 1 month; store at 2–8 °C.

   Reagent	Stock concentration	Final concentration	Volume
   MTEC/basic medium	n/a	n/a	2.82 mL
   FBS	100%	5%	150 μL
   EGF	25 μg/mL	25 ng/mL	3 μL
   Insulin	2 mg/mL	10 μg/mL	15 μL
   Transferrin	5 mg/mL	5 μg/mL	3 μL
   Cholera toxin	250 μg/mL	0.1 μg/mL	1.2 μL
   PrimocinTM	50 mg/mL	100 μg/mL	6 μL




5. mTEC/Plus2 medium (1 mL)

   Make fresh each time and store at 2–8 °C.

   Reagent	Stock concentration	Final concentration	Volume
   MTEC/Plus1 medium	n/a	n/a	996.4 μL
   Retinoic acid	250 μM	50 nM	0.2 μL
   Y-27632	5 mM	5 μM	1 μL
   CHIR99021	3 mM	3 μM	1 μL
   A8301	500 μM	500 nM	1 μL
   Noggin	20 mg/mL	20 μg/mL	1 μL




6. Blocking buffer (10 mL)

   Make fresh each time and store at 2–8 °C.

   Reagent	Stock concentration	Final concentration	Volume
   PBS	n/a	n/a	7.98 mL
   Normal goat serum	100%	20%	2 mL
   Triton X-100	100%	0.1%	10 μL
   NaN3	2 g/mL	0.2 mg/mL	1 μL
   Tween-20	100%	0.05%	5 μL




7. Dilution buffer (10 mL)

   Make fresh each time and store at 2–8 °C.

   Reagent	Stock concentration	Final concentration	Volume
   PBS	n/a	n/a	9.78 mL
   Normal goat serum	100%	2%	0.2 mL
   Triton X-100	100%	0.1%	10 μL
   NaN3	2 g/mL	0.2 mg/mL	1 μL
   Tween-20	100%	0.05%	5 μL

Laboratory supplies

1. 1 mL syringe (Jiufekang, catalog number: 045X15RWLB), containing 4.5-gauge syringe needle

2. 24-well plates (Corning, catalog number: 353047)

3. 35 mm culture dish (Sparkjade, catalog number: GF0006)

4. 60 mm culture dish (Sparkjade, catalog number: GF0007)

5. 1.5 mL centrifuge tube (Sparkjade, catalog number: NZ-86009)

6. Falcon, 50 mL centrifuge tube (Wocas, catalog number: NZ-86011)

7. Falcon, 15 mL centrifuge tube (Wocas, catalog number: NZ-86010)

8. Pipette tip, 10, 200, 1,000 µL (Wocas, catalog numbers: NZ-86001, NZ-86002, NZ-86003)

9. Parafilm M wrapping film (Thermo Fisher, catalog number: 1337416)

10. 300-mesh cell strainers (Sangon biotech, catalog number: F513453)

11. Adhesion microscope slides (Citotest, catalog number: 80312-3161)

12. Syringe filter (SHIGOUYI, catalog number: SFMCE013022NA)

13. Dyeing tank (Biosharp, catalog number: BS-WB-20B)

Equipment

1. Cell culture hood (Nuaire, model: NU-425-400S)

2. Cell culture incubator (Nuaire, model: NU-5510E)

3. Cell counter (Biosharp, model: BS-QT-1103)

4. Cryostat (Yidi, model: YD-1900), set from 0 to -40 °C

5. Surgical scissors (Merck, catalog numbers: S3146)

6. Surgical forceps (Biolab, catalog numbers: Z168777 and Z225614)

7. Vortex mixer (Kylin-bell, model: VORTEX-5)

8. Ice machine (PHCbi, model: SIM-F140AY65-PC)

9. Autoclave (TOMY, model: SX-500)

10. Brightfield microscope (Leica, model: DMI3000B)

11. Cell culture benchtop centrifuge (Merck, model: MGL-16MA)

12. Micropipettes (Dragon, catalog numbers: 7010101004, 7010101005, 7010101006, 7010101009, 7010101016)

13. Pure water technology (Zhiang, model: Best-R)

14. 37 °C water bath (Changzhou Yuexin, model: HH-1)

15. Stereomicroscope (Olympus, model: SZX16)

16. Multiphoton laser scanning microscope (Leica, model: TCS SP8 DVE)

Procedure