肺类器官培养：气道与肺泡模型

以下为英文全文（English full text）

1. Introduction to Lung Organoid Technology

The lung is a structurally intricate organ characterized by a highly branched airway tree that terminates in approximately 300 million alveolar units responsible for gas exchange. The airway tree is organized into distinct regions: the proximal conducting airways (trachea, bronchi, and bronchioles) lined by a pseudostratified epithelium containing basal cells, ciliated cells, secretory club cells, and goblet cells; and the distal respiratory zone (alveoli) composed of alveolar type 1 (AT1) cells (thin, squamous cells responsible for gas exchange) and alveolar type 2 (AT2) cells (cuboidal cells that produce surfactant and function as stem cells for alveolar regeneration). This regional specialization, combined with the lung's exposure to environmental pollutants, pathogens, and allergens, makes it a challenging but critically important target for in vitro modeling [1,2].

Lung organoid technology has enabled the in vitro modeling of both proximal airway (trachea, bronchi) and distal alveolar epithelium, providing physiologically relevant platforms for studying lung development, chronic obstructive pulmonary disease (COPD), cystic fibrosis, asthma, pulmonary fibrosis, acute respiratory distress syndrome (ARDS), and respiratory infections including influenza, respiratory syncytial virus (RSV), and SARS-CoV-2 [1,2,3]. The first adult lung organoids were established by Barkauskas and colleagues in 2013, who demonstrated that alveolar type 2 (AT2) cells co-cultured with PDGFRα+ mesenchymal cells could form alveolospheres containing both AT2 and AT1 cells, recapitulating the cellular composition of the alveolar epithelium [4]. Subsequently, airway organoids derived from basal cells were established and shown to contain basal, ciliated, secretory/club, and goblet cells that could be maintained for over a year [5].

Lung organoids can be generated from adult stem cells (basal cells for airway, AT2 cells for alveoli) or from pluripotent stem cells (PSCs) through directed differentiation that recapitulates embryonic lung development. This article provides comprehensive protocols for both airway and alveolar lung organoid culture, including media formulations, differentiation protocols, and functional validation assays.

2. Principles of Lung Organoid Development

Lung development proceeds through a series of precisely orchestrated stages: foregut endoderm → anterior foregut endoderm → ventral anterior foregut endoderm (VAFEC) → NKX2-1+ lung progenitors → airway and alveolar lineages. The key signaling pathways that govern these developmental transitions are:

• TGF-β/Nodal signaling: Activin A and TGF-β activate the Smad2/3 pathway to induce definitive endoderm formation from pluripotent stem cells. This is the first step in the directed differentiation of PSCs toward lung fate [6].
• Wnt signaling: Wnt/β-catenin signaling promotes the specification of posterior foregut endoderm and the expansion of lung progenitors. CHIR99021 (a GSK-3β inhibitor that activates Wnt signaling downstream of the receptor) is used at 3–10 µM during the early stages of lung organoid differentiation [6,7].
• BMP signaling: Bone morphogenetic proteins promote anterior foregut fate at early stages but must be inhibited (by Noggin) during lung progenitor expansion to prevent differentiation toward other lineages. BMP4 is also used later to promote proximal airway differentiation [6,7].
• FGF signaling: Fibroblast growth factors are essential for lung bud outgrowth and branching morphogenesis. FGF7 promotes airway progenitor expansion, while FGF10 promotes distal lung bud tip progenitor maintenance and alveolar fate. FGF2 and FGF10 are also critical for PSC-derived lung organoid differentiation [1,6].
• Notch signaling: Notch signaling regulates cell fate decisions in the airway epithelium, with high Notch activity promoting basal cell and club cell fate, while Notch inhibition (using DAPT) promotes ciliated cell differentiation [5,8].
• Retinoic acid: Retinoic acid signaling promotes proximal-distal patterning and alveolar differentiation. It is essential for the specification of AT1 and AT2 cell fates in both adult and PSC-derived lung organoids [1,6].

3. Airway Organoid Culture from Adult Basal Cells

3.1 Tissue Procurement and Basal Cell Isolation

Airway basal cells are the stem cells of the proximal airway epithelium and can be isolated from bronchial brushings, nasal inferior turbinate brushings, or lung resection specimens. Basal cells express the transcription factor p63 (TP63) and the cytokeratins KRT5 and KRT14, and they have the capacity to self-renew and differentiate into ciliated cells, goblet cells, and club cells.

Isolation from Bronchial Brushings:

1. Collect bronchial brushings during bronchoscopy in DMEM/F12 + 10% FBS + antibiotics on ice.
2. Centrifuge at 300 × g for 5 minutes and resuspend the cells in PBS.
3. Count the cells and resuspend in cold Matrigel for plating. Typical yield from a single brushing is 10,000–50,000 cells [5].

Isolation from Lung Resection or Nasal Turbinate:

1. Obtain fresh airway tissue and wash extensively with PBS.
2. Incubate the tissue in 0.5 mg/mL pronase + 0.5 mg/mL DNase I in DMEM/F12 at 4°C overnight (12–16 hours) [5].
3. Scrape the epithelial layer with a scalpel to release the epithelial cells.
4. Filter the cell suspension through a 40 µm cell strainer to remove debris and tissue fragments.
5. Centrifuge at 300 × g for 5 minutes at 4°C and resuspend the pellet in cold Matrigel.

3.2 Airway Organoid Expansion Medium

Commercial Formulation (Recommended for beginners):

• PneumaCult-Ex Plus (STEMCELL Technologies, #05040) – a serum-free, defined medium optimized for the expansion of human airway basal cells as organoids [5].

Custom Formulation:

Base: DMEM/F12 (1:1 mixture)

Supplements:

• 1× B27 supplement (without vitamin A)
• 1× N2 supplement
• 2 mM GlutaMAX
• 10 mM HEPES
• 1× Penicillin/Streptomycin
• 1.25 mM N-acetylcysteine
• 5 µM A83-01 (TGF-β inhibitor)
• 10 µM SB202190 (p38 inhibitor)
• 50 ng/mL EGF
• 100 ng/mL FGF7 (recombinant human, PeproTech, 100-19) – promotes airway epithelial proliferation
• 100 ng/mL FGF10 (recombinant human, PeproTech, 100-26) – supports distal progenitor maintenance
• 500 ng/mL R-Spondin 1 (recombinant human)
• 100 ng/mL Noggin (recombinant human)
• 10 µM Y-27632 (ROCK inhibitor, first 48 hours only) [5,8]

3.3 Airway Organoid Differentiation Medium

For ciliated cell differentiation, switch the organoids to differentiation medium and culture under air-liquid interface (ALI) conditions or in suspension with modified medium:

Base: PneumaCult-ALI Medium (STEMCELL Technologies, #05001) or DMEM/F12 + 1× B27 + 1× N2

Supplements:

• 50 ng/mL EGF
• 10 µM DAPT (Tocris Bioscience, 2634) – Notch inhibitor that promotes ciliated cell differentiation
• Remove Noggin and R-Spondin 1 (these factors maintain stemness and must be withdrawn to allow differentiation) [5,8]

For goblet cell enrichment: Add IL-13 (10 ng/mL, R&D Systems, 213-IL) to the differentiation medium. IL-13 promotes mucus production and goblet cell hyperplasia, which is relevant for modeling asthma and allergic airway inflammation [9].

3.4 Culture Protocol for Airway Organoids

1. Resuspend basal cells in cold Matrigel (50 µL per well, 24-well format) at 1,000–5,000 cells per droplet.
2. Polymerize at 37°C for 15 minutes.
3. Add 500 µL airway organoid expansion medium.
4. Change medium every 3–4 days.
5. Passage every 10–14 days at a 1:3 ratio by mechanical dissociation and re-embedding in Matrigel.
6. For differentiation, transfer organoids to air-liquid interface (ALI) culture or change to differentiation medium for 14–28 days (see Section 5.1 for ALI protocol) [5,8].

4. Alveolar Organoid Culture from AT2 Cells

4.1 AT2 Cell Isolation from Human Lung Tissue

AT2 cells are the stem cells of the alveolar epithelium and can regenerate both AT2 and AT1 cells after lung injury. The isolation of AT2 cells requires careful enzymatic digestion and enrichment to obtain a pure population of viable cells.

Materials:

• Fresh human lung tissue (surgical resection or cadaveric donor tissue)
• Elastase (1–2 mg/mL, Worthington, LS002292)
• Collagenase (0.5 mg/mL) + DNase I (0.1 mg/mL)
• 100 µm and 40 µm cell strainers
• Magnetic-activated cell sorting (MACS) system
• HTII-280 antibody (Terrace Biotech, TB-29A) or EpCAM antibody for MACS

Detailed Procedure:

1. Obtain fresh human lung tissue and perfuse with PBS through the pulmonary vasculature to remove blood. If the tissue is from a surgical resection, perfuse gently via the pulmonary artery with 50–100 mL of ice-cold PBS.
2. Instill 1–2 mg/mL elastase in PBS into the airways via the trachea or main bronchus. Incubate the lung tissue at 37°C for 30–45 minutes. The elastase digests the elastic fibers in the alveolar walls, releasing the epithelial cells.
3. Mince the lung tissue into small pieces and digest further in 0.5 mg/mL collagenase + 0.5 mg/mL DNase I at 37°C for 30 minutes with gentle agitation.
4. Filter the digested suspension sequentially through 100 µm and 40 µm strainers.
5. Centrifuge at 300 × g for 5 minutes and resuspend the pellet in PBS.
6. For AT2 cell enrichment: Incubate the cell suspension with HTII-280 antibody (a surface marker specific for AT2 cells) followed by magnetic microbeads. Apply the suspension to a MACS column and collect the positive fraction (HTII-280+ cells). Alternatively, sort by flow cytometry for EpCAM+ HTII-280+ cells [4,10].
7. Resuspend the purified AT2 cells in cold Matrigel for plating.

4.2 Alveolar Organoid Medium (Alveolosphere Assay)

Base: DMEM/F12

Supplements:

• 1× B27 supplement (without vitamin A)
• 1× N2 supplement
• 2 mM GlutaMAX
• 10 mM HEPES
• 1× Penicillin/Streptomycin
• 10 µM Y-27632 (first 48 hours only)
• 50 ng/mL EGF
• 10 ng/mL FGF7 (recombinant human)
• 10 ng/mL FGF10 (recombinant human)
• 3 µM CHIR99021 (Tocris Bioscience, 4423) – Wnt activator that promotes AT2 cell proliferation [4,10]

Note: Co-culture with PDGFRα+ mesenchymal cells or human fetal lung fibroblasts (such as MRC-5 or IMR-90) at a 1:1 ratio significantly enhances alveolosphere formation and AT1 differentiation. The mesenchymal cells provide essential paracrine signals (including Wnt ligands and BMP inhibitors) that support AT2 stem cell maintenance and differentiation [4,10].

4.3 Alveolar Organoid Culture Protocol

1. Mix AT2 cells with mesenchymal cells (if using co-culture) at a 1:1 ratio. If not using co-culture, plate AT2 cells alone at a higher density (10,000–20,000 cells per droplet).
2. Resuspend the cells in a 1:1 mixture of 50% Matrigel and 50% MTEC+Plus medium (or equivalent basal medium) at 100 µL per well in 24-well transwell inserts (for ALI) or 30 µL droplets in 24-well plates [4,10].
3. For ALI culture: Add 500 µL medium to the basal compartment. After 48–72 hours, when the cells have formed a confluent monolayer, remove the apical medium to establish the air-liquid interface. Maintain the culture by changing the basal medium every 2–3 days.
4. For suspension culture: Culture the organoids in Matrigel droplets with alveolar organoid medium, changing the medium every 2–3 days.
5. Alveolospheres will form within 14–21 days. AT1 cells (identified by HOPX and AQP5 expression) typically appear after 14–21 days, while AT2 cells (identified by SFTPC and ABCA3) are present from the earliest stages [4,10].

5. PSC-Derived Lung Organoids

5.1 Directed Differentiation Protocol from iPSCs

PSC-derived lung organoids are generated by directed differentiation through the sequential stages of definitive endoderm, anterior foregut endoderm, ventral anterior foregut endoderm (VAFEC), and NKX2-1+ lung progenitors. This approach is particularly valuable for modeling developmental disorders, genetic diseases (such as cystic fibrosis and surfactant protein deficiencies), and for generating large numbers of lung cells for drug screening.

1. iPSCs are maintained in mTeSR1 or StemFlex on Matrigel-coated plates. When cells reach 70–80% confluency, begin differentiation.
2. Day 0–3: Definitive Endoderm Induction. Culture cells in RPMI 1640 + B27 (without insulin) + 100 ng/mL Activin A + 10 ng/mL BMP4 + 3 µM CHIR99021. The cells should transition to an epithelial morphology characteristic of definitive endoderm.
3. Day 4–6: Anterior Foregut Endoderm Specification. Change to DMEM/F12 + B27 + 10 ng/mL FGF10 + 100 ng/mL Noggin + 10 µM SB431542 (TGF-β inhibitor) + 2 µM retinoic acid. Noggin and SB431542 suppress BMP and TGF-β signaling, respectively, to promote anterior foregut identity.
4. Day 7+: Ventral Anterior Foregut Endoderm (VAFEC) and NKX2-1+ Lung Progenitors. Culture in DMEM/F12 + B27 + 10 ng/mL FGF10 + 10 ng/mL FGF7 + 3 µM CHIR99021 + 2 µM retinoic acid. By day 10–12, the cells should express NKX2-1 (the master transcription factor for lung development) and CPM (carboxypeptidase M, a surface marker for lung progenitors) [6,7,11].
5. Day 10–15: Embed NKX2-1+ progenitors in Matrigel droplets and culture in lung organoid maturation medium (DMEM/F12 + B27 + FGF10 + FGF7 + CHIR99021).
6. Day 15+: For airway differentiation, add DAPT (10 µM) and remove CHIR99021. For alveolar differentiation, add the DCI cocktail: dexamethasone (0.1 µM) + cAMP (0.1 mM, Sigma-Aldrich, D0260) + IBMX (0.1 mM, Sigma-Aldrich, I5879) + triiodothyronine (T3, 10 nM, Sigma-Aldrich, T6397) + FGF7 + FGF10. The DCI cocktail promotes the maturation of alveolar epithelial cells and enhances surfactant production [6,7,11].

5.2 Lung Bud Tip Organoids (LBTs)

Fetal lung bud tip cells can be isolated from 5–22 week gestational tissue and cultured in medium containing EGF, FGF7, FGF10, Noggin, SB431542, and CHIR99021. These organoids retain SOX2+SOX9+ progenitor identity and can be differentiated into airway or alveolar lineages by modulation of growth factors. LBT-derived organoids are particularly valuable for studying early lung development and branching morphogenesis [1,12].

6. Air-Liquid Interface (ALI) Culture for Differentiated Epithelium

ALI culture is the gold standard for promoting mucociliary differentiation in airway organoids and is essential for modeling airway epithelial function, host-pathogen interactions, and ciliopathies.

6.1 ALI Protocol

1. Dissociate airway organoids into single cells or small fragments using TrypLE Express (5–10 minutes at 37°C).
2. Seed 4.5 × 10^4 cells/cm² on transwell inserts (e.g., Corning 24-well inserts, 0.4 µm pore polyester membrane, #3470) that have been coated with 1–2% Geltrex (diluted in basal medium) and incubated at 37°C for 1–2 hours.
3. Add 500 µL maturation medium (PneumaCult-ALI or custom formulation) plus 10 µM Y-27632 to the basal compartment. The Y-27632 improves the attachment and survival of the cells during the initial seeding period.
4. Incubate at 37°C, 5% CO2. After 48–72 hours, check under a microscope to confirm that the cells have formed a confluent monolayer. If the monolayer is not confluent, continue culture for an additional 2–3 days.
5. Once the monolayer is confluent, carefully remove the apical medium to establish the air-liquid interface. The basal medium should remain in the lower compartment.
6. Continue to feed the basal compartment every 48–72 hours with fresh maturation medium (without Y-27632).
7. Motile cilia should begin to appear within 2–4 weeks and become fully functional by 4–6 weeks. The ciliary beat frequency (CBF) can be measured by high-speed video microscopy. Normal human bronchial epithelium exhibits a CBF of 8–15 Hz [5,8,13].

7. Quality Control and Validation

7.1 Morphological Assessment

• Airway organoids: In expansion culture, airway organoids appear as spherical structures with budding protrusions. Under ALI culture, the differentiated epithelium forms a pseudostratified columnar layer with cilia projecting into the apical space. The cilia should be visibly beating under the microscope (visible at 400× magnification). Goblet cells appear as clear, mucin-filled cells interspersed among the ciliated cells [5,8].
• Alveolar organoids: Alveolospheres appear as spherical cystic structures with thin epithelial walls. AT2 cells appear cuboidal with visible lamellar bodies (surfactant-containing vesicles) by electron microscopy. AT1 cells are thin and squamous, spreading over the surface of the alveolosphere [4,10].

7.2 Immunofluorescence Staining

Airway markers:

• p63 (TP63, basal cell marker) – p63 antibody (Abcam ab124762, 1:200)
• MUC5AC (goblet cell marker) – MUC5AC antibody (Abcam ab3649, 1:200)
• FOXJ1 (ciliated cell transcription factor) – FOXJ1 antibody (Abcam ab246301, 1:200)
• Acetylated α-tubulin (cilia marker) – Acetylated tubulin antibody (Sigma-Aldrich T7451, 1:500)
• SCGB1A1 (secretoglobin family 1A member 1, club cell marker) – CC10 antibody (Abcam ab40873, 1:200) [5,8,13]

Alveolar markers:

• SFTPC (surfactant protein C, AT2 cell marker) – SFTPC antibody (Abcam ab90716, 1:200)
• ABCA3 (ATP-binding cassette subfamily A member 3, AT2 cell marker) – ABCA3 antibody (Abcam ab24749, 1:200)
• AQP5 (aquaporin 5, AT1 cell marker) – AQP5 antibody (Abcam ab78451, 1:200)
• HOPX (HOP homeobox, AT1 cell marker) – HOPX antibody (Abcam ab198011, 1:200)
• HTII-280 (AT2 cell surface marker, for live cell staining) – HTII-280 antibody (Terrace Biotech, TB-29A) [4,10]

Lung progenitor markers:

• NKX2-1 (thyroid transcription factor 1, lung master regulator) – NKX2-1 antibody (Abcam ab76013, 1:200)
• SOX2 (airway progenitor marker) – SOX2 antibody (Cell Signaling 4900, 1:200)
• SOX9 (distal progenitor marker) – SOX9 antibody (Cell Signaling 82630, 1:200)
• CPM (carboxypeptidase M, surface marker for lung progenitors) – detected by flow cytometry [6,7,11]

7.3 Functional Assays

• Mucociliary Clearance: In ALI cultures, measure ciliary beat frequency (CBF) using high-speed video microscopy (minimum 200 frames per second). Normal human bronchial epithelium exhibits a CBF of 8–15 Hz. CBF is reduced in primary ciliary dyskinesia (PCD) and can be modulated by temperature and pharmacological agents [5,13].
• Mucus Secretion: Detect MUC5AC by ELISA in the apical wash or by Periodic Acid-Schiff (PAS) staining. Goblet cell hyperplasia can be induced by IL-13 (10 ng/mL) treatment for 7–14 days [9,13].
• Viral Infection: Lung organoids support infection by respiratory pathogens including RSV, influenza A, and SARS-CoV-2. Viral infection can be monitored by viral titer assays, immunofluorescence for viral proteins (e.g., RSV F protein, SARS-CoV-2 spike protein), and cytopathic effect. Organoids derived from cystic fibrosis patients show increased susceptibility to viral infection due to impaired mucociliary clearance [3,13].
• Barrier Function: Measure transepithelial electrical resistance (TEER) across ALI cultures. Normal airway epithelium exhibits TEER values of 300–600 Ω·cm². Barrier integrity can be disrupted by inflammatory cytokines (IL-13, TNF-α) and restored by glucocorticoids [5,13].

8. Troubleshooting Common Issues in Lung Organoid Culture

Issue: Low basal cell expansion efficiency

Potential Causes and Solutions:

• Y-27632 is omitted: Include Y-27632 (10 µM) during the initial 48 hours of seeding to prevent anoikis.
• FGF7 or EGF is inactive: Verify growth factor activity. FGF7 is particularly sensitive to freeze-thaw degradation.
• Use PneumaCult-Ex Plus: For reliable results, use the commercial medium optimized for airway basal cell expansion.
• Check for mycoplasma contamination: Mycoplasma can profoundly affect epithelial cell proliferation.
• Basal cell purity is low: If isolating from tissue, ensure that the epithelial layer is properly scraped and that the pronase digestion is complete [5,8].

Issue: Poor ciliated cell differentiation

Potential Causes and Solutions:

• Noggin and R-Spondin are still present in differentiation medium: Completely remove these stemness-maintaining factors.
• DAPT concentration is insufficient: Verify DAPT stock and use 10 µM.
• ALI culture is not established: Ciliated cell differentiation requires air exposure. Ensure that the apical medium is completely removed.
• Differentiation time is too short: Extend differentiation to 3–4 weeks. Cilia formation is a slow process that requires extended culture.
• Medium changes are too frequent: In ALI culture, changing the basal medium too frequently can disrupt the established microenvironment. Change every 48–72 hours [5,8,13].

Issue: AT2 cells fail to form alveolospheres

Potential Causes and Solutions:

• Mesenchymal cells are not included: Include PDGFRα+ mesenchymal cells or lung fibroblasts (MRC-5, IMR-90) at a 1:1 ratio. The mesenchymal signals are essential for AT2 stem cell maintenance.
• CHIR99021 is inactive: Verify CHIR99021 activity. Use 3 µM for optimal Wnt activation.
• AT2 cell viability is low: Use freshly isolated AT2 cells. AT2 cells lose viability rapidly after isolation; do not store for more than 2–4 hours before plating.
• Elastase digestion is insufficient: Optimize the elastase concentration and digestion time for the specific tissue. Over-digestion can damage AT2 cells, while under-digestion leaves too much connective tissue [4,10].

Issue: PSC-derived organoids lack lung identity

Potential Causes and Solutions:

• Verify NKX2-1 expression: Perform immunofluorescence or qPCR for NKX2-1 at day 10. If NKX2-1 is not expressed, the anterior foregut specification was unsuccessful.
• Optimize Activin A and CHIR99021 concentrations during definitive endoderm induction: Insufficient activin signaling will result in poor endoderm formation. Use 100 ng/mL Activin A and 3 µM CHIR99021.
• Ensure proper BMP4/Noggin balance: Too much BMP4 during anterior foregut specification can drive cells toward a posterior fate. Use Noggin (100 ng/mL) to suppress BMP signaling during days 4–6.
• Check iPSC quality: Poor-quality iPSCs with signs of differentiation or genetic abnormalities will not differentiate efficiently [6,7,11].

9. Conclusion

Lung organoid technology provides physiologically relevant platforms for studying respiratory biology, modeling pulmonary diseases, screening inhaled therapeutics, and investigating host-pathogen interactions. Both airway and alveolar organoids can be established from adult stem cells or PSCs, with air-liquid interface culture enabling the differentiation of functional mucociliary epithelium that closely mimics the native airway. GBiowit offers lung organoid culture solutions, including airway and alveolar organoid media, Matrigel and defined matrices, PSC-derived lung organoid kits, and specialized services for respiratory disease modeling and drug screening.

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