Air-Liquid Interface (ALI) Organoid Culture
Materials Required
Principle
ALI organoid culture places organoid-derived epithelial cells or tissue fragments on a porous support or collagen-based matrix so that basal surfaces receive medium while the apical surface is exposed to air; in lung organoid-derived ALI cultures, this supports airway epithelial differentiation, barrier formation, mucus production, beating cilia, and pseudostratified epithelial architecture[1][5][6]. Gastrointestinal ALI organoid systems similarly support long-term 3D epithelial growth with stromal/mesenchymal components and multilineage differentiation[3][4]. The main readouts are morphology, barrier integrity, epithelial differentiation, and experimental response readouts. Lung ALI protocols used bright-field microscopy, TEER monitoring, immunofluorescence for ciliated, goblet, club, and basal-cell markers, flow cytometry, viral titration, RNA-seq, and spatial transcriptomic readouts after SARS-CoV-2 infection[1][2].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use cryopreserved or fresh human lung tissue, dissociated lung epithelial cell suspensions, extracellular matrix hydrogel such as Cultrex for organoid dome generation, organoid expansion medium, ALI differentiation medium, OCT embedding compound, FBS/DMSO cryopreservation medium, and culture media placed only in the basal compartment after air-lift[1].
• For gastrointestinal ALI organoids, published protocols used minced primary gastrointestinal tissues embedded in collagen gel to maintain epithelial and stromal/mesenchymal components[3][4].
• Reported characterization markers include PAN-CK for epithelial cells, fibronectin for residual extracellular matrix, DAPI for nuclei, acetylated α-tubulin for ciliated cells, MUC5AC for goblet cells, SCGB1A1 for club cells, CK5/KRT5 for basal cells, phalloidin for actin structure, and SARS-CoV-2 nucleoprotein or spike antibodies for infection readouts[1][2].
• Reported equipment includes tissue-processing tools, biosafety culture equipment, cell-culture inserts or permeable supports, incubators, bright-field microscope, TEER measurement system, cryomolds, liquid nitrogen-compatible freezing setup, cryostat, flow cytometer, RNA extraction/sequencing workflow, plaque-assay workflow, and fluorescence or spatial-imaging platforms[1][2][5][6].
• For gastrointestinal ALI organoids, published protocols used minced primary gastrointestinal tissues embedded in collagen gel to maintain epithelial and stromal/mesenchymal components[3][4].
• Reported characterization markers include PAN-CK for epithelial cells, fibronectin for residual extracellular matrix, DAPI for nuclei, acetylated α-tubulin for ciliated cells, MUC5AC for goblet cells, SCGB1A1 for club cells, CK5/KRT5 for basal cells, phalloidin for actin structure, and SARS-CoV-2 nucleoprotein or spike antibodies for infection readouts[1][2].
• Reported equipment includes tissue-processing tools, biosafety culture equipment, cell-culture inserts or permeable supports, incubators, bright-field microscope, TEER measurement system, cryomolds, liquid nitrogen-compatible freezing setup, cryostat, flow cytometer, RNA extraction/sequencing workflow, plaque-assay workflow, and fluorescence or spatial-imaging platforms[1][2][5][6].
Experimental Procedure
• Process lung tissue by region when tissue anatomy is being tracked, cut tissue into smaller pieces, snap-freeze pieces in OCT for histology or cryopreserve viable tissue using 10% FBS and DMSO, then thaw and dissociate viable frozen tissue to obtain a single-cell lung suspension containing airway progenitors[1].
• Resuspend the lung cell suspension in Cultrex, dispense as domes, and culture in organoid expansion medium until lung epithelial organoids are generated and passaged[1].
• For gastrointestinal ALI organoid culture, published methods describe embedding minced gastrointestinal tissue fragments containing epithelial and mesenchymal/stromal components in collagen gel, then culturing the gel at an air-liquid interface[3][4].
• For lung organoid-derived ALI culture, dissociate organoids, seed cells onto inserts, expand submerged cultures until 100% confluence, continue submerged differentiation to promote tight junction and barrier integrity, and monitor TEER until the reported air-lift criterion of >500 Ω·cm2 is reached[1].
• Remove apical medium to create the air-liquid interface while maintaining basal medium, then culture for at least 4 weeks and monitor for beating cilia and mucus production; reported images showed seeding-stage cultures at 3-4 days, confluence at approximately 12-14 days, and differentiated cultures around 34 days post-air-lift[1].
• For airway epithelial ALI differentiation more generally, well-differentiated airway epithelial culture protocols and NHBE optimization studies support 4-week ALI differentiation as a period sufficient to obtain pseudostratified cultures with ciliated and goblet-cell features under the reported conditions[5][6].
• Assess ALI maturation by bright-field microscopy, TEER measurement, and immunofluorescence for differentiated airway lineages; reported lung ALI cultures measured TEER three times per week starting at confluence, used three measurements per time point, and analyzed cultures from four donors[1].
• For infection-response experiments, published lung ALI studies used mock-infected controls, SARS-CoV-2 infection, harvests from 1-6 days post-infection, flow cytometry for viable infected cells, plaque assay from apical supernatant, RNA-seq, and spatial ROI analysis using CK5-positive and CK5-negative regions[1][2].
• Resuspend the lung cell suspension in Cultrex, dispense as domes, and culture in organoid expansion medium until lung epithelial organoids are generated and passaged[1].
• For gastrointestinal ALI organoid culture, published methods describe embedding minced gastrointestinal tissue fragments containing epithelial and mesenchymal/stromal components in collagen gel, then culturing the gel at an air-liquid interface[3][4].
• For lung organoid-derived ALI culture, dissociate organoids, seed cells onto inserts, expand submerged cultures until 100% confluence, continue submerged differentiation to promote tight junction and barrier integrity, and monitor TEER until the reported air-lift criterion of >500 Ω·cm2 is reached[1].
• Remove apical medium to create the air-liquid interface while maintaining basal medium, then culture for at least 4 weeks and monitor for beating cilia and mucus production; reported images showed seeding-stage cultures at 3-4 days, confluence at approximately 12-14 days, and differentiated cultures around 34 days post-air-lift[1].
• For airway epithelial ALI differentiation more generally, well-differentiated airway epithelial culture protocols and NHBE optimization studies support 4-week ALI differentiation as a period sufficient to obtain pseudostratified cultures with ciliated and goblet-cell features under the reported conditions[5][6].
• Assess ALI maturation by bright-field microscopy, TEER measurement, and immunofluorescence for differentiated airway lineages; reported lung ALI cultures measured TEER three times per week starting at confluence, used three measurements per time point, and analyzed cultures from four donors[1].
• For infection-response experiments, published lung ALI studies used mock-infected controls, SARS-CoV-2 infection, harvests from 1-6 days post-infection, flow cytometry for viable infected cells, plaque assay from apical supernatant, RNA-seq, and spatial ROI analysis using CK5-positive and CK5-negative regions[1][2].
Troubleshooting
Problem: ALI culture is not ready for air-lift.
• Possible Cause: Barrier formation is insufficient.• Literature-supported Solution: Continue submerged differentiation and monitor TEER; the reported lung organoid-derived ALI protocol transitioned cultures to air-lift after TEER goals were achieved, using >500 Ω·cm2 as the stated criterion[1].
Problem: Differentiated lung ALI cultures lack expected cilia or mucus.
• Possible Cause: Culture has not completed the reported differentiation interval.• Literature-supported Solution: Maintain post-air-lift cultures for at least 4 weeks and monitor for beating cilia and mucus production[1][5][6].
Problem: Residual extracellular matrix signal persists in early lung organoids.
• Possible Cause: Remaining matrix from tissue dissociation is present.• Literature-supported Solution: Use serial passaging and confirm epithelial enrichment, because fibronectin signal was reported to disappear after four passages in the lung organoid protocol[1].
Problem: Cryosections of ALI inserts are poor or the epithelial layer is damaged during embedding.
• Possible Cause: Insert mesh handling or OCT positioning damages or curves the culture.• Literature-supported Solution: Cut the insert mesh carefully, pull out the mesh without damaging the cell layer, place it flat in OCT, avoid positioning too close to the cryomold surface, snap-freeze, and mark the ALI location before cryostat trimming[1].
References:
- [1]. Castaneda DC, Jangra S, Yurieva M, Martinek J, Callender M, Coxe M, Choi A, García-Bernalt Diego J, Wu TC, Marches F, et al. Protocol for establishing primary human lung organoid-derived air-liquid interface cultures from cryopreserved human lung tissue. STAR Protoc. 2023;4(4):102735. [Content Brief]
- [2]. Castaneda DC, Jangra S, Yurieva M, Martinek J, Callender M, Coxe M, Choi A, García-Bernalt Diego J, Lin J, Wu TC, et al. Spatiotemporally organized immunomodulatory response to SARS-CoV-2 virus in primary human broncho-alveolar epithelia. iScience. 2023;26(8):107374. [Content Brief]
- [3]. Ootani A, Li X, Sangiorgi E, Ho QT, Ueno H, Toda S, Sugihara H, Fujimoto K, Weissman IL, Capecchi MR, et al. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nat Med. 2009;15(6):701-706. [Content Brief]
- [4]. Li X, et al. An air-liquid interface culture system for 3D organoid culture of diverse primary gastrointestinal tissues. Methods Mol Biol. 2016;1422:33-40. [Content Brief]
- [5]. Fulcher ML, et al. Well-differentiated human airway epithelial cell cultures. Methods Mol Med. 2005;107:183-206. [Content Brief]
- [6]. Rayner RE, et al. Optimization of Normal Human Bronchial Epithelial (NHBE) Cell 3D Cultures for in vitro Lung Model Studies. Sci Rep. 2019;9(1):500. [Content Brief]
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