Engineering Decellularized Extracellular Matrix-Incorporated Apical-Out Airway Organoids
构建整合脱细胞细胞外基质的顶端向外气道类器官
发布: 2026年08月20日第16卷第16期 DOI: 10.21769/BioProtoc.5799 浏览次数: 143
评审: Neha SaxenaShanmugaPriyaa MadhukaranPankaj Mogha
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参见作者原研究论文
The authors used this protocol in:
Aug 2026
Abstract
The airway epithelium interfaces with the external environment through its apical surface and with the extracellular matrix (ECM) through its basolateral surface. To model this organization in vitro, we developed a decellularized ECM-incorporated apical-out airway organoid (dECM-AoAO) platform in which human bronchial epithelial cells (HBECs) self-assemble around human lung-derived decellularized ECM microparticles (dECM-MPs). This configuration preserves apical-out polarity while enabling direct epithelial–ECM interactions. Here, we describe a protocol for the vacuum filtration and quantification of dECM-MPs, the generation of dECM-AoAOs, and ultimately, whole-mount immunofluorescence staining for organoid characterization.
Key features
• This protocol incorporates dECM as size-refined microparticles, enabling a consistent and reproducible ECM input.
• The workflow uses suspension culture to generate apicalout airway organoids, eliminating the need for matrix embedding while preserving ECM–cell interactions.
• dECM-MPs are directly wrapped by epithelial cells, simplifying handling and supporting native-like epithelial–ECM integration.
• The system is compatible with wholemount immunofluorescence staining for comprehensive epithelial lineage analysis.
Keywords: Decellularized extracellular matrix microparticles (dECM-MPs) (脱细胞细胞外基质微粒（dECM-MPs）)
Apical-out airway organoids (AoAOs) (顶端向外气道类器官（AoAOs）)
Human bronchial epithelial cells (HBECs) (人支气管上皮细胞（HBECs）)
Airway epithelial differentiation (气道上皮分化)
Epithelial-ECM interactions (上皮细胞-细胞外基质相互作用)
Whole-mount immunofluorescence (整体免疫荧光染色)
Background
The airway epithelium is a polarized barrier that protects the respiratory tract from inhaled pathogens and environmental particulates while sustaining mucociliary clearance. Its function relies on the coordinated activity among multiciliated, secretory, and basal stem/progenitor cells, which collectively maintain epithelial homeostasis and support injury repair [1,2]. In native tissue, the apical surface faces the airway lumen, whereas the basal surface interfaces with the extracellular matrix (ECM), a source of structural and biochemical cues that regulate epithelial survival, differentiation, polarity, signaling, and morphogenesis [3–7].
Airway organoids provide a tractable platform for modeling epithelial biology, yet current systems impose a trade-off between ECM engagement and apical accessibility. Hydrogel-embedded, apical-in organoids preserve epithelial–ECM interactions but restrict direct access to the apical surface [2,8–10]. Conversely, apical-out organoids exhibit proper surface polarization and enable direct interrogation of apical stimulation and ciliary function, but they are typically generated under ECM-free or ECM-withdrawal conditions and therefore lack the capacity to model epithelial–ECM crosstalk [11–13].
Decellularized extracellular matrix (dECM) retains tissue-specific matrix composition and provides native-like biochemical cues that support epithelial morphogenesis [14–17]. To integrate ECM exposure with apical-out polarity, we developed decellularized ECM-incorporated apical-out airway organoids (dECM-AoAOs), in which primary human bronchial epithelial cells self-assemble around size-controlled airway-derived dECM microparticles. This protocol details the preparation, vacuum filtration, and quantification of dECM microparticles, the generation of dECM-AoAOs, and downstream whole-mount staining workflows for organoid characterization.
Materials and reagents
Biological materials
1. Human lung tissue, acquired from the University of Vermont (UVM)
2. Healthy donor-derived human bronchial epithelial cells (HBECs) (Lonza, catalog number: CC-2541)
3. 804G cells (a gift from Dr. Hongmei Mou, Massachusetts General Hospital and Harvard Medical School)
Reagents
1. Dulbecco’s phosphate-buffered saline (DPBS), 1×, without calcium and magnesium (Corning, catalog number: 21-031-CV)
2. Cell-culture-grade water (Corning, catalog number: 25-055-CV)
3. Bronchial epithelial cell growth medium (Lonza, catalog number: CC-3171)
4. A83-01 (Sigma-Aldrich, catalog number: SML0788)
5. Y-27632 (Cayman Chemical, catalog number: 129830-38-2)
6. DMH-1 (Tocris, catalog number: 4126)
7. CHIR99021 (REPROCELL, catalog number: 04000402)
8. RPMI 1640 (Corning, catalog number: 10-040-CV)
9. Fetal bovine serum (FBS) (Fisher Scientific, catalog number: FB12999102)
10. Penicillin-streptomycin (Thermo Fisher Scientific, catalog number: 15140122)
11. TrypLE Express (Gibco, catalog number: 12604013)
12. CryoStor® CS10 cell freezing medium (STEMCELL Technologies, catalog number: 07930)
13. PneumaCult-ALI medium (STEMCELL Technologies, catalog number: 05001)
14. Heparin solution (STEMCELL Technologies, catalog number: 07980)
15. Hydrocortisone stock solution (STEMCELL Technologies, catalog number: 07925)
16. Paraformaldehyde (PFA), 4% (Santa Cruz Biotechnology, catalog number: sc-281692)
17. Triton X-100 (Sigma-Aldrich, catalog number: X100-500mL)
18. Tween-20 (Sigma-Aldrich, catalog number: P2287)
19. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: 9048-46-8)
20. Mouse anti-E-cadherin antibody (1:200) (Cell Signaling Technology, catalog number: 14472S)
21. Alexa Fluor 647-conjugated donkey anti-mouse IgG (1:1,000) (Thermo Fisher Scientific, catalog number: A-31571)
22. DAPI (4′,6-diamidino-2-phenylindole) in H2O, 10 mg/mL (Biotium, catalog number: 40043)
23. Mouse anti-acetylated α-tubulin antibody (1:500) (Sigma-Aldrich, catalog number: T6793)
24. Mouse anti-MUC5AC antibody (1:100) (Thermo Fisher Scientific, catalog number: MA5-12178)
25. Rabbit anti-SCGB1A1 antibody (1:100) (Thermo Fisher Scientific, catalog number: PA5-78215)
26. Mouse anti-TP63 antibody (1:100) (Biocare Medical, catalog number: cm163a)
27. Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:1,000) (Thermo Fisher Scientific, catalog number: R37118)
28. N-hydroxysuccinimide-Cyanine5 (NHS-Cy5) (Tocris Bioscience, catalog number: 5436-10)
29. Biotin-NHS (Millipore Sigma, catalog number: H1759-5MG)
30. Streptavidin-488 (Thermo Fisher Scientific, catalog number: S11223)
31. Human IL-13 recombinant protein (PeproTech, catalog number: 200-13-1MG)
32. Sodium deoxycholate (Sigma-Aldrich, catalog number: S1827)
33. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888)
34. Calcium chloride (Sigma-Aldrich, catalog number: C4901)
35. Deoxyribonuclease I (DNase I) from bovine pancreas (Sigma-Aldrich, catalog number: DN25)
36. Peracetic acid (Sigma-Aldrich, catalog number: 269336)
37. Gentamicin (50 mg/mL) (Thermo Fisher Scientific, catalog number: 15750060)
38. Amphotericin B (Sigma-Aldrich, catalog number: A9528)
39. Pepsin from porcine gastric mucosa (Sigma-Aldrich, catalog number: P6887)
40. 10× Phosphate-buffered saline solution (PBS, Thermo Fisher Scientific, catalog number: 70011044)
41. Hydrochloric acid (HCl) (Fisher Scientific, catalog number: SA49)
42. Sodium hydroxide (NaOH) solution (Fisher Scientific, catalog number: SS255)
43. DNeasy Blood and Tissue kit (Qiagen, catalog number: 69504)
44. Dimethyl sulfoxide (DMSO), anhydrous (Sigma-Aldrich, catalog number: 276855-100ML)
Solutions
1. HBEC expansion medium (see Recipes)
a. 10 mM A83-01 solution
b. 10 mM Y-27632 solution
c. 10 mM DMH-1 solution
2. 804G conditioned medium (see Recipes)
3. dECM-AoAO differentiation media (see Recipes)
a. PneumaCult-ALI complete medium
b. dECM-AoAO seeding medium
c. dECM-AoAO differentiation medium
4. Lung decellularization solutions (see Recipes)
a. Triton-X solution
b. SDC solution
c. NaCl solution
d. DNase solution
e. Peracetic acid (PAA) solution
f. Storage solution
5. Digestion and neutralization solutions (see Recipes)
a. Pepsin digestion solution
b. Neutralization solution
Recipes
1. HBEC expansion medium
Note: For all small molecules (such as A83-01, Y-27632, and DMH-1), aliquot before freezer storage to avoid multiple freeze-thaw cycles. Do not freeze-thaw more than three times.
a. 10 mM A83-01 solution
Dissolve 5 mg of A83-01 (MW: 421.52 Da) in 1.186 mL of sterile DMSO to constitute a 10 mM A83-01 stock solution. Store at -20 °C for up to 6 months.
b. 10 mM Y-27632 solution
Dissolve 10 mg of Y-27632 dihydrochloride (MW: 320.3 Da) in 3.122 mL of sterile DMSO to constitute a 10 mM Y-27632 dihydrochloride stock solution. Store at -20 °C for up to 6 months.
c. 10 mM DMH-1 solution
Dissolve 1 mg of DMH-1 (MW: 380.44) in 262.85 μL of sterile DMSO to constitute a 10 mM DMH-1 stock solution. Store at -20 °C for up to 6 months.
d. HBEC expansion medium
| Reagent | Final concentration | Volume |
|---|---|---|
| Bronchial epithelial cell growth basal medium | N/A | 500 mL |
| BEGM bronchial epithelial cell growth medium SingleQuots | N/A | 1 kit |
| 10 mM A83-01 | 1 μM | 50 μL |
| 10 mM Y-27632 | 5 μM | 250 μL |
| 10 mM DMH-1 | 0.2 μM | 10 μL |
| 10 mM CHIR99021 | 0.5 μM | 25 μL |
| Total | ~506 mL |
Note: Store at 4 °C for up to 4 weeks.
2. 804G conditioned medium
804G rat bladder epithelial cells are thawed and expanded in 175-cm2 flasks using RPMI 1640 complete medium, which serves as the base medium of 804G conditioned medium. Once confluent, the cultures are used to generate 804G-conditioned medium as described in Procedure D1.
| Reagent | Final concentration | Volume |
|---|---|---|
| RPMI 1640 with L-glutamine | N/A | 267 mL |
| FBS | 10% | 30 mL |
| Penicillin-Streptomycin | 1% | 3 mL |
| Total | 300 mL |
Note: RPMI 1640 complete medium can be stored at 4 °C for up to 3 weeks.
3. dECM-AoAO differentiation media
a. PneumaCult-ALI complete medium
| Reagent | Final concentration | Volume |
|---|---|---|
| PneumaCult-ALI basal medium | N/A | 450 mL |
| PneumaCult-ALI 10× supplement | N/A | 50 mL |
| PneumaCult-ALI maintenance supplement (100×) | N/A | 5 × 1 mL |
| Heparin solution (0.2%) | 0.0004% | 1 mL |
| Hydrocortisone stock solution (200×) | 1% | 2.5 mL |
| Penicillin-Streptomycin | 1% | 5 mL |
| Total | ~513.5 mL |
Note: Aliquot 45 mL per tube and store at 4 °C for up to 3 weeks.
b. dECM-AoAO seeding medium (day 0 only)
| Reagent | Final concentration | Volume |
|---|---|---|
| PneumaCult-ALI complete medium | N/A | 44.973 mL |
| 10 mM A83-01 | 1 μM | 4.5 μL |
| 10 mM Y-27632 | 5 μM | 22.5 μL |
| Total | 45 mL |
c. dECM-AoAO differentiation medium (day 2 onward)
| Reagent | Final concentration | Volume |
|---|---|---|
| PneumaCult-ALI complete medium | N/A | 44.9955 mL |
| 10 mM A83-01 | 1 μM | 4.5 μL |
| Total | 45 mL |
Note: dECM-AoAO assembly begins by seeding HBECs into ultra-low attachment plates using PneumaCult-ALI complete medium supplemented with 1 μM A83-01 and 5 μM Y-27632 (referred to as the dECM-AoAO seeding medium). Y-27632 is included only on day 0 to enhance single-cell viability during the initial aggregation phase. At the first medium change on day 2, cultures are switched to dECM-AoAO differentiation medium containing PneumaCult-ALI complete medium with 1 μM A83-01 only, as Y-27632 does not show improved subsequent organoid differentiation. Medium is then replaced every other day using differentiation medium without Y-27632.
4. Lung decellularization solutions
a. Triton-X solution
| Reagent | Final concentration | Volume |
|---|---|---|
| Triton-X | 0.1% | 12 mL |
| Penicillin-Streptomycin (10,000 U/mL) (100×) | 5× (i.e., 500 U/mL) | 600 mL |
| DI water | N/A | 11,388 mL |
| Total | ~12 L |
b. SDC solution
| Reagent | Final concentration | Quantity |
|---|---|---|
| Sodium deoxycholate powder | 2% | 240 g |
| Penicillin-Streptomycin (10,000 U/mL) (100×) | 1× (i.e., 100 U/mL) | 120 mL |
| DI water | N/A | 11,880 mL |
| Total | ~12 L |
c. NaCl solution
| Reagent | Final concentration | Quantity |
|---|---|---|
| Sodium chloride powder | 1 M | 701.28 g |
| Penicillin-Streptomycin (10,000 U/mL) (100×) | 5× (i.e., 500 U/mL) | 600 mL |
| DI water | N/A | 11,400 mL |
| Total | ~12 L |
d. DNase solution
| Reagent | Final concentration | Quantity |
|---|---|---|
| Bovine pancreatic DNase | 0.03 mg/mL | 0.36 g |
| Calcium chloride powder | 2 mM | 2.664 g |
| Magnesium sulfate powder | 1.3 mM | 1.878 g |
| Penicillin-Streptomycin (10,000 U/mL) (100×) | 5× (i.e., 500 U/mL) | 600 mL |
| DI water | N/A | 11,400 mL |
| Total | ~12 L |
e. PAA solution
| Reagent | Final concentration | Volume |
|---|---|---|
| Peracetic acid | 0.1% | 12 mL |
| Ethanol (200 Proof) | 4% | 480 mL |
| DI water | N/A | 11,508 mL |
| Total | ~12 L |
f. Storage solution
| Reagent | Final concentration | Quantity |
|---|---|---|
| Gentamicin | 50 μg/mL | 12 mL |
| Amphotericin B | 2.5 μg/mL | 30 mg |
| Penicillin-Streptomycin (10,000 U/mL) (100×) | 5× (i.e., 500 U/mL) | 600 mL |
| 1× PBS | 1× | 11.388 L |
| Total | ~12 L |
5. Digestion and neutralization solutions
a. Pepsin digestion solution
| Reagent | Final concentration | Quantity |
|---|---|---|
| Pepsin powder | 1 mg/mL | 50 mg |
| HCl | 0.01 M | 50 mL |
| Total | 50 mL |
b. Neutralization solution
| Reagent | Final concentration | Volume |
|---|---|---|
| NaOH | 0.1 M | 0.5 mL |
| DI water | N/A | 49.5 mL |
| Total | 50 mL |
Laboratory supplies
1. Millipore Steriflip vacuum tube top filter, 40 μm (Millipore Sigma, catalog number: SCNY00040)
2. Nalgene rapid-flow sterile disposable filter units, 0.45-μm pore size (Thermo Fisher Scientific, catalog number: 124-004)
3. Stainless steel laboratory spatula
4. Wide-orifice pipette tips, 200 μL (Axygen, catalog number: T-205-WB-C-R-S)
5. 25 cm2 cell culture flask (Greiner Bio-One, catalog number: 690175)
6. 75 cm2 cell culture flask (Greiner Bio-One, catalog number: 658175)
7. 175 cm2 cell culture flask (Greiner Bio-One, catalog number: 660175)
8. 96-well cell-repellent, U-bottom microplate (Greiner Bio-One, catalog number: 650970)
9. Water bath (Fisher Scientific, catalog number: FSGPD10)
10. Incubator for Cell Culture (Fisher Scientific, catalog number: 11676604)
11. Mr. FrostyTM freezing container (Thermo Fisher Scientific, catalog number: 5100-0001)
12. M-20 microplate swinging bucket rotor (Thermo Fisher Scientific, catalog number: 75003624)
13. μ-slide 18-well chambered coverslip (ibidi, catalog number: 81816)
14. Sorvall Legend X1 Centrifuge (Thermo Fisher Scientific, catalog number: 75004220)
15. EVOS M7000 imaging system (Thermo Fisher Scientific, catalog number: AMF7000)
16. ECLIPSE Ts2 Inverted Cell Culture Microscope (Nikon, N/A)
17. AXR confocal microscope (Nikon, N/A)
18. Roller Pump (Stockert-Shiley, catalog number: 10-00-00)
19. Spex Sample Prep 6775 Freezer Mill
20. Scalpel
21. Forceps
22. Scissors
23. V-shaped magnetic stir bar
24. Magnetic stir plate
Procedure
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文章信息
稿件历史记录
提交日期: May 28, 2026
接收日期: Jul 20, 2026
在线发布日期: Aug 6, 2026
出版日期: Aug 20, 2026
版权信息
© 2026 The Author(s); This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
如何引用
Gong, Z., Ikpechukwu, C. S., Bhattaram, D., Ryan, A. L., Weiss, D. J. and Ren, X. (2026). Engineering Decellularized Extracellular Matrix-Incorporated Apical-Out Airway Organoids. Bio-protocol 16(16): e5799. DOI: 10.21769/BioProtoc.5799.
Download Citation in RIS Format
分类
干细胞 > 类器官培养
细胞生物学 > 细胞工程 > 组织工程
细胞生物学 > 细胞分离和培养 > 3D细胞培养
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