iPSC-Derived Epithelial, Mesenchymal, Endothelial, and Immune Cell Co-Culture to Model Airway Barrier Integrity in Lung Health and Disease

Summary

This article describes the generation of a complex, multi-cellular airway barrier model composed of induced pluripotent stem cell (iPSC)-derived lung epithelium, mesenchyme, endothelial cells, and macrophages in an air-liquid interface culture.

Abstract

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the nasopharynx to the smallest alveolar sac. Interactions between these cells are crucial in lung development and disease, acting as a barrier against harmful chemicals and pathogens. Current in vitro co-culture models utilize immortalized cell lines with different biological backgrounds, which may not accurately represent the cellular milieu or interactions of the lung. We differentiated human iPSCs into 3D lung organoids (containing both epithelium and mesenchyme), endothelial cells, and macrophages. These were co-cultured in an air-liquid interface (ALI) format to form an epithelial/mesenchymal apical barrier invested with macrophages and a basolateral endothelial barrier (iAirway). iPSC-derived iAirways showed a reduction in barrier integrity in response to infection with respiratory viruses and cigarette toxins. This multi-lineage lung co-culture system provides a platform for studying cellular interactions, signaling pathways, and molecular mechanisms underlying lung development, homeostasis, and disease progression. iAirways closely mimic human physiology and cellular interactions, can be generated from patient-derived iPSC's, and can be customized to include different cell types of the airway. Overall, iPSC-derived iAirway models offer a versatile and powerful tool for studying barrier integrity to better understand genetic drivers for disease, pathogen response, immune regulation, and drug discovery or repurposing in vitro, with the potential to advance our understanding and treatment of airway diseases.

Introduction

The blood-air barrier in the large airways includes the trachea, bronchi, and bronchioles. It plays a crucial role in maintaining respiratory health and is made up of the airway epithelium, basement membrane, blood vessels and endothelial cells, and immune cells. The primary epithelial cells in the airway encompass basal cells, club cells, ciliated cells, and goblet cells. Basal cells, acting as the stem cells of the airway epithelium, are multipotent progenitors with high proliferative and self-renewal capabilities, giving rise to mature airway epithelial cells¹. Club cells are non-ciliated, secretory cells that contribute to the maintenance of the airway lining by secreting protective proteins and surfactants². Goblet cells, located in the lumen and in the submucosal glands, secrete mucins to trap debris and safeguard the airway³. Ciliated cells are integral to the mucociliary escalator mechanism, preventing the accumulation of harmful microorganisms⁴. The basement membrane is made up of an extracellular matrix, which provides structural support⁵. The trachea and the rest of the airway are surrounded by a rich network of blood vessels, which are lined with endothelial cells that play a vital role in supporting tracheal function by supplying nutrients and oxygen, removing waste, regulating inflammation, and contributing to tissue repair and angiogenesis⁶. Finally, airway macrophages are tissue-specific immune cells, essential for protecting the respiratory system from infections, clearing inhaled particles, and maintaining a balanced immune response⁷.

The coordinated actions of epithelial, mesenchymal, endothelial cells, and macrophage cells are critical for an effective immune response to pathogens in the airway⁸. Epithelial cells form the first line of defense against viral infections by acting as a physical barrier, with tight junctions restricting the passage of harmful substances. The coordinated action of ciliated cells and goblet cells helps to trap and remove inhaled particles, pathogens, and debris⁴. Additionally, airway epithelial cells produce cytokines and chemokines to recruit immune cells⁹. Endothelial cells maintain vascular integrity, preventing the spread of viral particles through the bloodstream, upregulate adhesion molecules (VCAM-1) to facilitate immune cell adhesion and produce pro-inflammatory cytokines to recruit immune cells from the bloodstream to the site of infection¹⁰. Airway macrophages engulf and digest viral particles, infected cells, and debris, present viral antigens to T cells, and produce cytokines to activate and recruit other immune cells, along with type I interferons to inhibit viral replication¹¹. The coordinated actions of epithelial, mesenchymal, endothelial, and macrophage cells create a robust and dynamic defense system that protects the airway from viral infections and maintains respiratory health.

Understanding the dynamic interactions among various cell types in the human lung is crucial for comprehending the lung's response to viral infections, inflammatory diseases, and drug delivery. In vitro co-cultures allow for the study of cell-cell signaling between the epithelium, endothelial cells, and innate immune cells¹². We developed the first authentic multi-cell type lung model derived from patient-specific hiPSCs¹³. This incorporates both epithelial and mesenchymal cell populations, formed in a 3D orientation. Subsequently, the lung progenitor cells can be differentiated into an "airway organoid"¹⁴, cultured onto sterile cell culture inserts, and exposed to an air-liquid interface (ALI), replicating the conditions of the human airway^15,16,17. iPSC-derived endothelial cells are cultured on the basolateral side of the membrane, mimicking their orientation in the human airway, situated below the epithelial/mesenchymal layer in the basement membrane. Finally, iPSC-derived macrophages are added to the apical side of the membrane, interacting with epithelial cells and awaiting activation signals (Figure 1A). This model accurately reproduces the biology and function of the airway. We posit that hiPSC-derived, patient-specific, authentic multi-cell type iAirway cultures are best suited to elucidate the intrinsic, acute response of the airway barrier and pathogens, including viral infections. For instance, this model can be used to (1) study viral entry and replication, (2) investigate the initial immune response by epithelial and tissue-specific immune cells, (3) examine barrier integrity and function, (4) test the efficacy of therapeutic agents, and (5) study cellular and molecular mechanisms of pathogenesis in a patient-specific model.

This article describes a detailed protocol for preparing multi-cellular lung co-cultures to study cellular responses to viral infections.

Protocol

This study protocol was approved by the Institutional Review Board of UCSD's Human Research Protections Program (181180). This protocol uses small molecules and growth factors to direct the differentiation of pluripotent stem cells into airway cells, endothelial cells, and macrophages. These cells are then co-cultured onto cell culture inserts and polarized in an air-liquid interface. The details of the reagents, consumables, and equipment used are listed in the Table of Materials. The media and buffer compositions are provided in Supplementary File 1.

1. Generation of iPSC-derived airway organoids (Day 1 - 30)

NOTE: This protocol outlines the steps required to generate iPSC-derived airway organoids (Figure 1B), following the methodology described in Leibel et al.¹³. The process includes induction of definitive endoderm (Days 1-3), generation of anterior foregut endoderm (Days 4-6), and differentiation into lung progenitors (Days 7-16). Detailed methodology can be found in the prior publication¹³. The following steps detail the generation of airway organoids from lung progenitors.

1. Extracting and dissociating lung progenitor spheroids from ECM polymer (Day 17)
   1. Thaw extracellular matrix (ECM) polymer (8-9 mg/mL) on ice.
   2. Aspirate spent media, using vacuum aspirator, from lung organoids embedded in ECM.
      NOTE: For subsequent aspiration steps, use a vacuum aspirator unless noted otherwise.
   3. Add 1 mL of 2 U/mL dispase supplemented with 10 µM of ROCK1 inhibitor (ROCKi) to the cells and use a P1000 pipette to manually resuspend the lung progenitors in ECM. Incubate for 30 min at 37 °C, resuspending the mixture every 15 min to enhance dispase dissociation efficacy through mechanical processing.
   4. 15 min prior to use, place 45 mL of room temperature PBS into a -20 °C freezer to chill. Ensure PBS is colder than 4 °C for optimal ECM depolymerization in the subsequent steps.
   5. After 30 min of dispase incubation, transfer the organoid and dispase solution into a 15 mL conical tube.
   6. Add chilled PBS (2 mL per 1 mL of protease) to wash and collect residual organoid and ECM material from the plate. Resuspend organoids using a P1000 pipette and centrifuge at 400 x g for 5 min.
      NOTE: All centrifugation steps are performed at room temperature unless otherwise specified in this protocol.
   7. After centrifugation, a cloudy ECM pellet with organoids should be visible. Carefully aspirate the supernatant via pipette, avoiding the ECM pellet.
   8. Perform a second PBS wash with chilled PBS, resuspend using a P1000 pipette, and centrifuge at 400 x g for 5 min. Aspirate the supernatant via pipette, leaving ~100 µL of residual solution.
   9. Add 2 mL of trypsin-like protease to the organoids in the 15 mL conical tube. Resuspend using a P1000 pipette. Incubate for 10-12 min at 37 °C, inverting or flicking the tube to resuspend the mixture halfway through the incubation.
      NOTE: Perform 10-12 min of Trypsin-like protease dissociation to pass organoids as aggregates.
   10. After 12 min, stop the trypsin-like protease reaction by adding 2 mL of 2% FBS in base media (Stop Media, see Supplementary File 1). Resuspend the solution with a P1000 pipette and centrifuge at 400 x g for 5 min.
   11. Aspirate the supernatant and resuspend organoids in Stop Media supplemented with 10 µM of ROCKi. Take a 10 µL sample for cell counting using a hemocytometer and trypan blue. Keep organoids on ice during the count.
   12. Calculate the volume needed to obtain 100,000 cells per well. Aliquot cell aggregates into a 1.5 mL microcentrifuge tube and centrifuge for 5 min at 400 x g. Remove excess supernatant, leaving 10 µL of residual media.
   13. Resuspend the cell pellet in 200 µL of cold ECM polymer (avoid bubbles and work quickly to prevent premature polymerization). Add 200 µL of the ECM and cell mixture to the bottom of each well in a 12-well plate. Allow ECM to partially polymerize at room temperature in the biosafety cabinet for 5 min.
   14. Transfer the plate to a 37 °C incubator for 30-60 min to complete ECM polymerization. Then, add 1.5 mL of airway organoid induction medium (Supplementary File 1).
   15. Change the medium every other day for 14 days until Day 30. If the medium becomes yellow within 24 h, increase the volume to 2 mL.

2. Generation of iPSC-derived endothelial cells (Day 1 - 14)

NOTE: The following procedure details the generation of endothelial cells from iPSCs (Figure 1C), adapted from Patsch et al.¹⁸. This method includes the preparation of plates, differentiation of iPSCs, endothelial cell induction, sorting, and expansion. Table 1 lists the antibodies used in this study.

1. Plating iPSCs for endothelial differentiation (Day 0)
   1. Begin the endothelial cell differentiation when hiPSCs reach 70%-80% confluency. Add 10 µM of ROCKi Y-27632 to each well an hour prior to dissociation.
   2. Aspirate the media, wash wells with 1 mL of PBS then dissociate iPSCs by adding 1 mL of cell detachment solution per well of a 12-well plate. Incubate for 20 min at 37 °C.
   3. Neutralize cell detachment solution by adding 2 mL of Stop Media to the wells. Pipette to obtain a single-cell suspension. Transfer cells to a 15 mL conical tube and centrifuge for 5 min at 300 x g.
   4. Aspirate the supernatant, resuspend iPSCs in iPSC culture media supplemented with 10 µM of ROCKi, and perform a cell count. Plate 100,000 hiPSCs per well of an ECM-coated 12-well plate in 1 mL of iPSC culture media with 10 µM of ROCKi. Incubate overnight at 37 °C.
      NOTE: Seeding density for iPSC-endothelial differentiation may need optimization per cell line. Assess differentiation efficiency using flow cytometry for CD31 on Day 6.
2. Lateral mesoderm induction (Day 1-3)
   1. Aspirate iPSC culture media from plated iPSCs and add 3 mL of N2B27 base media supplemented with 6 µM of CHIR and 25 ng/mL BMP4 per well (Supplementary File 1). Warm the media prior to adding it to the cells. Do not change the media for 3 days.
3. Endothelial cell induction (Day 4-5)
   1. On Day 4, aspirate the N2B27 media and add 2 mL of endothelial differentiation media (EDM) supplemented with 200 ng/mL VEGF165 and 2 µM of forskolin per well. Change the media on Day 5.
4. Endothelial cell sorting and replating (Day 6)
   1. On Day 5 or 6, prepare a fibronectin-coated T75 flask for fluorescence-activated cell sorting (FACS) enrichment by reconstituting 1 mg of fibronectin in sterile water to make a 100 µg/mL of fibronectin solution. Coat the T75 flask with 6 mL of the fibronectin solution and incubate at room temperature for one hour. Aspirate the fibronectin solution and wash with sterile water. Let the T75 flask dry in room temperature. Extra flasks made can be stored at 4 °C.
   2. Prepare endothelial maintenance media (EMM)¹⁸.
   3. On Day 6, enrich iPSC-derived endothelial cells via FACS using the CD31 antibody¹⁸.
   4. Add 10 µM of ROCKi Y-27632 to each well an hour prior to dissociation at 37°C. Aspirate the medium and wash with PBS. Add 1 mL of pre-warmed cell detachment solution per well of a 12-well plate and incubate for 8-10 min at 37°C.
   5. Gently pipette to ensure single-cell detachment. Transfer the cells to a 15 mL conical tube and add equal volume of Stop Media. Centrifuge for 5 min at 300 x g.
   6. Aspirate the supernatant and resuspend endothelial cells in 1 mL Stop Media supplemented with 10 µM of ROCKi.  Pass cells through 70μm filter by first adding 1mL of stop media to wet the filter, then pipette the cells through the filter. Take a 10 µL sample for cell counting using a hemocytometer and trypan blue. Keep cells on ice during the count.
   7. Perform a cell count. Prepare an aliquot of 200,000 cells as an unstained negative control. Transfer the remaining cells into a 1.5 mL Eppendorf tube and add 10 µL of CD31-APC for every 1 million cells in 100 µL of FACS buffer. Incubate for 30 min on a rotator at 4 °C.
   8. Once incubation is completed, Centrifuge the Eppendorf tube with cells for 5 min at 300 x g, wash the cells by adding 1mL of PBS. Repeat the washing and centrifugation step twice. Manually remove the supernatant between washes.
   9. Resuspend the cell pellet in 1mL of FACS buffer and 5 ug/mL DAPI solution for viability staining, 5 minutes prior to sorting. Sort per institutional guidelines.
   10. Collect FACS-sorted cells in a 15 mL conical tube with 2 mL of EMM. Centrifuge the 15mL conical tube with cells for 5 min at 300 x g. Aspirate the supernatant and resuspend endothelial cells in 10 mL of EMM supplemented with 10 µM of ROCKi Y-27632 and Penicillin Streptomycin (1%).
   11. Transfer the resuspended FACS-enriched iPSC-derived endothelial cells to the fibronectin-coated flask. Evenly distribute cells and place in a 37 °C incubator overnight.
       NOTE: Use a minimum of 500,000 cells and a maximum of 2,000,000 cells per T75 flask.
5. Expansion and cryopreservation of sorted endothelial cells (Day 7+)
   1. Change the EMM media every 2-3 days post-FACS enrichment. Once the T75 flask becomes confluent (approximately 7 days after FACS, dependent on seeding density), the iPSC-derived endothelial cells can be used for co-culture or cryopreserved for future applications.
   2. Prepare a 2x endothelial freezing solution prior to cell dissociation (80% Endo-CM2, 20% DMSO, 20 µM of ROCKi).
   3. Label cryovial tubes in an ethanol-proof pen with the respective information. Line ID, passage number, culture media, 'thaw into a 6 well', date of freeze.
   4. When the sorted iPSC derived endothelial cells are confluent, initiate dissociation.
   5. Wash T75 flask with 5ml of PBS -/-.
   6. Dissociate iPSC-derived endothelial cells with 5mL of trypsin-like protease per T75. Incubate cells at 37 °C FOR 10 min, checking the T75 periodically to verify cells have lifted.
   7. Neutralize the reaction by adding 5 mL of Stop Media. Transfer 10 mL of cells in solution to 15 mL tube. Centrifuge at 300 x g for 5 min.
   8. Aspirate the supernatant, resuspend endothelial cells in E-CBM media, and take 10 µL sample out for count via hemacytometer.
   9. After cell count, aliquot 1 million cells per 0.5 mL of EGM2 media.
      NOTE: The following step is time-sensitive. Prepare cryovials and freezing vessels to be used immediately.
   10. Aliquot equal volume of 2x freezing solution to endothelial cells. The final concentration is 90% Endo-CM2, 10% DMO, and 10 µM of ROCKi (1 mL/1 million cells).
   11. Transfer the capped vials immediately into a freezing chamber and place in -80 °C overnight, then into liquid nitrogen (-180 °C) the next day for long-term storage.

3. Generation of iPSC-derived macrophages (Day 1 - 26)

NOTE: This procedure outlines the steps to generate macrophages from iPSCs (Figure 1D), adapted from van Wilgenburg et al.¹⁹ and Pouyanfard et al.²⁰. It covers single-cell adaptation of iPSCs, embryoid body differentiation, macrophage progenitor formation, and macrophage maturation.

1. Single-cell adaption of iPSCs
   1. When iPSCs reach ~70%-90% confluency with no visible signs of differentiation, begin single-cell passage with trypsin-like protease.
   2. Aspirate spent media and rinse with PBS. Remove PBS and add trypsin-like protease (1 mL/well of a 6-well plate, 500 µL/well of a 12-well plate). Incubate at 37 °C for 2-5 min until iPSC colonies dissociate from the plate.
   3. Neutralize trypsin-like protease with an equal volume of Stop Media (1 mL/well of a 6-well plate, 500 µL/well of a 12-well plate). Centrifuge the cells at 200 x g for 5 min.
   4. Aspirate the supernatant and gently resuspend cells in iPSC culture media with 10 µM of ROCKi. Passage onto new ECM-coated plates. Repeat for 2-3 passages before cryopreserving or using the single-cell adapted iPSCs.
      NOTE: Adjust the passage ratio from 1:2 to 1:10 based on cell health and adaptation. In addition, iPSCs that have undergone at least three single-cell passages and are at low passage yield optimal macrophage differentiations.
2. Embryoid body formation (Day 0-6)
   1. When single-cell adapted iPSCs reach ~75% confluency, passage iPSCs with trypsin-like protease as per steps 3.1-3.4.
   2. Resuspend cells in Stop Media and pass through a 70 µm cell strainer into a fresh 50 mL conical tube to remove clumps. Resuspend cells and take 10 µL sample for cell count via hemacytometer.
   3. For embryoid body (EB) generation, 8,000-50,000 cells are plated per well of a 96-well ultra-low attachment (ULA) plate. Calculate the total number of cells needed to seed 60 wells of a 96-well ULA plate.
      NOTE: The seeding density of iPSCs may require optimization per cell line.
   4. Centrifuge iPSCs in 15 mL conical at 200 x g for 5 min. Aspirate the supernatant.
   5. For 480,000 iPSCs (8,000 cells/ well in 60 wells), resuspend in 6 mL (100 ul/well in 60 wells) EB induction media.
   6. Add 150 µL of PBS to the 36 outer wells of a round-bottom 96-well ULA plate.
   7. Resuspend and transfer iPSCs in EB media to a media trough. Using a multichannel pipette, add 100 µL/well of cell suspension into the center 60 wells of the 96-well ULA plate. Resuspend iPSCs in EB media intermittently to ensure even distribution of cells.
   8. Centrifuge the 96-well plate at 300 x g for 5 min at 4 °C (if available). Transfer plates to a 37 °C incubator.
   9. After 48-72 h, change 50 µL of the media (half media change). Check for cyst formation after 6 days.
      NOTE: Some iPSC lines form EBs earlier or more easily than others. Monitor EB formation and ensure that EBs are transferred at the appropriate time point (they should be developing cysts).
3. Gelatin coating and EB transfer for macrophage progenitor formation (Day 6-19)
   1. Prepare two 0.1% gelatin-coated 6-well plates. Add 1 mL of 0.1% gelatin per well and incubate at room temperature for 20 min. Aspirate the gelatin solution and allow plates to dry in the hood for 30-60 min.
   2. Transfer EBs from the 96-well plate to the gelatin-coated 6-well plates using a 2 mL serological pipette. Distribute approximately 8-10 EBs per well of a 6-well gelatin-coated plate.
   3. Carefully remove residual EB media used from the transfer. Add 2 mL of macrophage culture media 1 (Mac-CM1) per well. Incubate EB's undisturbed at 37 °C with 5% CO₂ for 5-7 days.
      NOTE: Ideally, one 96-well plate of embryoid bodies can be split into two 6-well plates. If the EBs are poorly developed, the number of EBs transferred per 6-well plate can be adjusted to optimize the iPSC-to-embryoid-body differentiation step.
4. Macrophage progenitor generation
   1. Replace 2/3 of the media twice a week, increasing the volume to 3 mL if the media color changes.
   2. Check cultures between Days 8-19 to determine if they are ready for macrophage progenitor harvesting. If EBs detach, transfer EBs to a freshly coated plate with Mac-CM1 and incubate undisturbed for 7 days.
      NOTE: The maintenance of the macrophage progenitor-forming complex is vital for the development and continuous generation of macrophage progenitors. Optimized macrophage progenitor-forming complexes will continually generate myeloid progenitors for 2-6+ months.
5. Harvesting myeloid progenitors (Day 19-26+)
   1. When there are macrophage progenitor cells in suspension, harvest the media with cells and transfer to a 50 mL conical tube. Centrifuge at 200 x g for 5 min and carefully remove the supernatant.
   2. Resuspend macrophage progenitors in Mac-CM2 and transfer to an untreated sterile culture dish/flask. Incubate at 37 °C with 5% CO₂ for 3-4 days.
      NOTE: Depending on the number of cells harvested, transfer to a 10 cm Petri dish (3-4 wells of a 6-well plate), T25 flask (1-2 wells of a 6-well plate), or T75 flask (full 6-well plate) with the appropriate volume of media. Use 5-6 mL of media for a T25 flask, 10-12 mL of media in a Petri dish, and 12-15 mL of media for a T75 flask. For subsequent harvests, myeloid progenitors and macrophage cells can be pooled from previous harvests if they are from the same line/myeloid forming complexes. Just ensure the flask is sufficient to maintain and feed the number of cells in the flask(s). Macrophages do not proliferate; they are generated from macrophage progenitors. Macrophages can be pooled and maintained for 2-6 weeks without losing expression markers.
6. Harvesting macrophages
   NOTE: After 14 days of macrophage differentiation in Mac-CM2 media, macrophage cultures are ready for co-culture or flow cytometry analysis.
   1. Collect spent media and transfer to a 50 mL conical tube. Rinse the flask/plate with PBS to remove residual media and cells.
   2. Add base media to the flask (3 mL for T25 and 5 mL for T75) and use a sterile cell scraper to detach cells from the bottom of the flask/plate. Transfer cells to a conical tube and repeat scraping if necessary.
   3. Centrifuge at 200 x g for 5 min at room temperature. Carefully remove the supernatant and resuspend cells in appropriate media or buffer for further use.

4. Co-culture of airway cells, endothelial cells, and macrophages

NOTE: This procedure describes the steps for the co-culture of airway cells, endothelial cells, and macrophages (Figure 1A) using cell culture inserts, adapted from Costa et al.¹².

1. ECM coating of cell culture inserts for co-culture (Day 0 of co-culture)
   1. Coat 3.0 µm pore polyester (PET) cell culture inserts with 4 mg/mL ECM solution. Briefly coat the apical side of the cell culture inserts inserts within the plate with 4 mg/mL of ECM solution. Pipet off residual ECM solution. Place the plate in the incubator for 1 h at 37 °C.
   2. Obtain a large Petri dish (100 mm x 20 mm or 150mm x 20mm). With clean tweezers, sterilely transfer cell culture inserts from the 12-well plate into the large Petri dish. Invert inserts so the basolateral side is upright.
   3. Coat the basolateral side with 4 mg/mL ECM solution. Pipet off residual ECM solution. Place the Petri dish with ECM-coated cell culture inserts into a 37 °C incubator to dry overnight.
2. Dissociation and plating of iPSC-derived endothelial cells (Day 1 of co-culture)
   1. Wash the T75 flask with PBS and aspirate solution. Add 5 mL of trypsin-like protease to the T75 flask and incubate at 37 °C for 8 min. Visually assess that endothelial cells have lifted from the flask.
      NOTE: Increase the time of typsin-like protease dissociation if the iPSC derived endothelial cells have not lifted form the flask.
   2. Tap flask to ensure detachment of endothelial cells and transfer cells into a 15 mL conical. Add 5 mL of Stop Media to halt dissociation. Centrifuge cells at 300 x g for 5 min.
   3. Aspirate supernatant and resuspend cell pellet in 1 mL of endothelial culture media with 10 µM of ROCKi. Obtain 10 µL of resuspended solution for cell count and place cells on ice during the count.
   4. Use 150,000 iPSC-endothelial cells per 12 mm cell culture insert in 100 µL of endothelial culture media. Adjust the respective media volume to reach the desired number of cells per insert.
   5. Obtain a Petri dish with ECM-coated 3.0 µm cell culture inserts from the incubator (see steps 4.1-4.3).
   6. Resuspend iPSC-endothelial cells and pipette 100 µL with 150,000 cells onto the inverted cell culture insert (basolateral side facing up). Repeat pipetting for each of the prepared cell culture inserts
   7. Carefully transfer cells into the incubator and leave them undisturbed for 3 h.
   8. In a 12-well plate, add 1 mL of endothelial culture media in each well (for the respective number of cell culture inserts prepared).
   9. Take out the Petri dish with endothelial cells from the incubator. Using clean tweezers, carefully transfer cell culture inserts into the plate with endothelial culture media (flipping the insert so the bottom is now facing down into the media).
   10. Visually verify on a microscope that endothelial cells had adhered. Place the plate in a 37 °C incubator overnight.
3. Dissociation and plating of iPSC-derived airway organoids (Day 2 of co-culture)
   NOTE: Refer to steps 1.1.1-1.1.15 for isolation of airway organoids from ECM polymer. Modify the duration of trypsin-like protease (step 1.1.10) dissociation to 15-20 min to obtain single-cell suspension.
   1. After 15-20 min, stop the trypsin-like protease reaction by adding 3 mL of Stop Media. Resuspend the solution with a P1000 pipette and centrifuge at 400 x g for 5 min.
   2. Aspirate the supernatant and resuspend airway organoids in 1 mL airway expansion media with 10 µM of ROCKi. Take a 10 µL sample for a hemacytometer cell count and place cells on ice during the count.
   3. Seed 300,000 iPSC-airway cells in 500 µL of airway expansion media per 12 mm cell culture insert. Adjust the respective media volume to reach the desired number of cells per prepared insert.
   4. Remove the plate with cell culture inserts with endothelial cells (seeded on the basolateral side) from the 37 °C incubator.
   5. Resuspend iPSC-airway cells and pipette 500 µL with 300,000 cells into the apical chamber of each cell culture insert. Place the plate in a 37 °C incubator for 48 h with the apical cells in liquid-liquid conditions.
4. Air-lifting co-culture (Day 4 of co-culture)
   1. Remove media from the apical side of the cell culture insert. Change basolateral media to 1:1 airway differentiation media and endothelial culture media. Return the plate to the incubator.
5. Dissociation and plating of iPSC-derived macrophages (Day 5 of co-culture)
   1. Dissociate iPSC-derived macrophages from the flask using a cell scraper (see steps 3.22-3.24). Centrifuge at 300 x g for 5 min and aspirate the supernatant.
   2. Resuspend iPSC-macrophages in 1 mL of Mac-CM2. Take a 10 µL sample for cell counting and place cells on ice during the count. The number of macrophages will mirror the number of airway cells seeded (1:1 ratio of macrophages to airway cells). 
   3. After cell count, aliquot 300,000 macrophages in 35 µL media per well of prepared cell culture inserts into a 1.5 mL microcentrifuge tube. Centrifuge at 200 x g for 5 min.
   4. After centrifugation, pipet out the supernatant. Resuspend macrophages in respective Mac-CM2 from calculations.
   5. Remove inserts with co-cultures of endothelial and airway cells from the incubator. Seed 300,000 macrophages in 35 µL of Mac-CM2 onto the apical side of the cell culture insert. Place the plate in a 37 °C incubator for 48 h.
      NOTE: To confirm adherence of iPSC-immune cells to the cell culture insert, cells can be tagged with a fluorescent probe. Confirm adherence via microscopy for fluorescent signal 48 h post macrophage plating.
6. Triple co-culture (Day 7 of co-culture)
   1. Ensure that the co-culture is ready for downstream applications such as infection studies, transepithelial electrical resistance (TEER) measurements, and barrier integrity assays.

Results

There are multiple stages at which the differentiation of iPSC-derived airway organoids, endothelial cells, immune cells, and co-cultures can be assessed as successfully completed. Differentiations can be performed in different iPSC lines, and this protocol has been tested in at least five different lines. The protocol does need to be adapted to every new iPSC line, specifically by modifying and optimizing the seeding density.

The successful yield of iPSC-derived airway organoids can be assessed via brightfield and will have four characteristics: defined boundaries/edges, translucent/cystic morphology, ability to proliferate/expand, and lack of necrotic cells. Airway organoids that appear fuzzy do not increase in size over the 2 weeks of culture, and have necrosis should not be used for co-culture experiments. Organoids with a thick luminal layer on the periphery vs. the translucent/cystic organoids can still be used for subsequent experiments. However, the translucent/cystic airway organoids are composed of predominantly basal cells, whereas the thick dual membrane organoids are composed of predominantly club cells. Because basal cells proliferate and expand easily, they are more suited for air-liquid interphase culture and polarization. Other methods for assessing the successful yield of iPSC-derived lung organoids are flow-cytometry analysis at the lung progenitor stage (Day 16 of the differentiation) for surface marker CPM (Table 1), a successful differentiation yield >50% CPM+ cells.

For the airway organoid dissociation and air-liquid interphase culture, ensure that all of the ECM polymer has been removed, or it will prevent the cells from expanding on the membrane. The cells should be confluent by Day 3 of culture, prior to air-lifting. Confirmation of cell types in the iAirways can be assessed by staining for respective cell type-specific markers: basal cells (p63+), club cells (SCGB1A1+), goblet cells (MUC5AC+), and ciliated cells (AcTub+). Characterization and hallmark lung cell type-specific markers are verified on LungMAP²¹. Mesenchyme can be detected with antibodies against vimentin or smooth muscle actin. To generate more ciliated cells, the cultures may require air-lifting for a longer period of time or the addition of DAPT.

The successful yield of iPSC-derived endothelial cells can be assessed via brightfield, showing a cobblestone-like morphology (Day 6 of the iPSC-derived endothelial differentiation). A successful iPSC-derived endothelial culture will contain >50% CD31+ cells, and enough cells should be generated so that after sorting, the yield should be more than 500,000 cells. Cells can be passaged or cryopreserved, and the CD31 marker should be tested via flow with each passage or new culture. Cells should not be passaged beyond P4. For reference, see iPSC-derived endothelial cells pre/post CD31 sort (Figure 1C). Endothelial cells will be flat with a large cell body with elongated and spindle-shaped morphology.

For macrophage differentiation, embryoid bodies should remain at the bottom of the ULA well, and only one EB should be in each well. They should show cystic changes by Day 5-7. If they do not form an EB after centrifugation, then seed the iPSCs into the 96-well ULA plate without spinning. Increasing the number of iPSCs may be necessary. The successful yield of iPSC-derived immune cells is entirely dependent on the ability of embryoid bodies (EBs) to develop into cysts (Day 7 of differentiation). iPSC-derived immune EBs that do not develop cysts will not form stroma when plated onto gelatin and will not produce myeloid progenitors. After transfer to the gelatin-coated plate, the EBs should remain attached to the bottom of the plate. Movement of the plate should be done carefully so as not to dislodge them. Plates should be changed twice a week manually, and the suspended cells should be passaged into Mac-CM2 or cryopreserved. Additionally, after harvesting the suspended myeloid progenitors, they should adhere to the non-coated plastic flask, and generate bright cells under brightfield with vacuoles, indicating macrophages. The marker for monocytes, CD14+, should be >90% in the suspended cells. The macrophage marker, CD68, should increase over time, with ~50% of macrophages expressing CD68+ after 2 weeks (Figure 1D). Adhesion efficiency should be determined for each cell line. We achieve an average of 20% adhesion efficiency. For polarization, M0 macrophages are polarized to either M1 (iPSC-M1) or M2 (iPSC-M2) macrophages for 48 h in differentiation medium II supplemented with different stimuli: 100 ng/mL LPS and 20 ng/mL IFN-g for M1 polarization or 50 ng/mL IL-4 and 20 ng/mL IL-13 for M2 polarization. The marker for M1 polarization is CD80+, and M2 polarization is CD206+.

Successful generation of the triple co-culture can be assessed via immunofluorescence for epithelial tight junction markers EPCAM, ECAD, or ZO-1, or endothelial tight junction marker CD31 (Figure 2C, Table 1). The functionality of the triple co-culture can also be assessed via trans-electrical epithelial/endothelial resistance (TEER). Well-assembled airway organoid and endothelial co-cultures will have a greater electrical resistance, indicating optimal cell communication and connection. Environmental insults, such as e-cigarettes or viral challenge, will compromise the integrity of the epithelial and endothelial barrier, resulting in reduced electrical resistance of the co-culture (Figure 3A,B) and disorganized tight junctions (Figure 3C).

Figure 1: Differentiation and characterization of iPSC-derived airway epithelium, endothelium, and immune cultures. (A) Schematic of iPSC-directed differentiations for the assembly of the iAirway. (B) iPSC-derived airway epithelium organoids characterized by brightfield (left) and flow cytometry analysis (right) for EPCAM and ECAD. (C) iPSC-derived airway endothelium characterized by brightfield (left), immunofluorescence, and flow cytometry analysis (right) for CD31. The respective brightfield after CD31 sort is shown on the right. (D) iPSC-derived macrophages are characterized by brightfield (left) and flow cytometry analysis (right) for the pan hematopoietic marker CD45, monocyte maker CD14, and macrophage marker CD68. Scale bars: 100 µm. Please click here to view a larger version of this figure.

Figure 2: Co-culture of iPSC-derived epithelial, mesenchymal, endothelial, and macrophage cells. (A) Schematic of the assembly of the iAirway. (B) Brightfield images of the respective epithelial and endothelial monoculture, the epithelial and endothelial bi-culture, and the epithelial, endothelial, and macrophage co-culture. Macrophages tagged with red cytotracker. Scale bars: 100 µm. (C) Immunofluorescent staining for tight junction marker ZO-1 on the apical side of the cell culture insert and CD31 on the basolateral side. Scale bars: 100 µm. (D) Cross-section of the cell culture insert with dissociated airway organoids on the apical side, 1-week post air-lift. The left panel with H&E staining with the black arrow shows ciliated cells. The middle panel, with immunofluorescence imaging, shows the epithelial marker E-cadherin (ECAD). The right panel, with immunofluorescence imaging, shows mesenchymal markers such as vimentin (VIM) and smooth muscle actin (SMA). Scale bars: 100 µm. Please click here to view a larger version of this figure.

Figure 3: Changes in airway barrier function of the iAirways exposed to e-cigarettes and viruses. (A) Schematic of trans-electrical epithelial/endothelial resistance (TEER) measurement tool. (B) Bar graph of TEER after 7 days of menthol e-cigarette addition to media. Mean +/- 95% confidence interval. N = 5 biological replicates and 3 technical replicates; p-value <0.001. (C) Immunofluorescent staining for tight junction protein ZO-1 before and after infection with SARS-CoV-2. Note the disordered layout of ZO-1 post-infection. Scale bars: 50 µm. Please click here to view a larger version of this figure.

Antibody	Application	Dilution
CD 31 PECAM-1(APC)	flow cytometry (FC)	10 μl in 100 ul buffer
EPCAM/CD 326 (APC)	FC	1:500
E-CAD/CD 324 (APC)	FC	5 μl in 100 ul buffer
CPM	FC	1:200
CD 45 (PE)	FC	5 μl in 100 ul buffer
CD 14 (FITC)	FC	5 μl in 100 ul buffer
CD 68 (PE)	FC	5 μl in 100 ul buffer
ZO-1	Immunofluorescence (IF)	1:300
Vimentin	IF	1:200
SMA	IF	1:100

Table 1: Antibodies used in FACS and Immunofluorescence.

Supplementary File 1: Media and buffer compositions. Please click here to download this File.

Discussion

The development and implementation of a model of the blood-air barrier in the large airways for studying viral infections and other toxins require meticulous attention to detail to ensure the successful differentiation and function of the various cell types involved. This discussion will address key factors for successful differentiation, potential challenges, alternative applications, and implications for studying human diseases.

To ensure a successful differentiation, attention to the type of cell culture insert and pore size is important. Large pores should be used to ensure cell-cell communication through the layers of ECM. Ensuring the optimal seeding density for induced pluripotent stem cell (iPSC) lines is critical. Proper density facilitates uniform differentiation and prevents the formation of unwanted cell clumps, leading to a more homogenous and functional epithelial layer. To maintain a healthy and functional epithelial layer, washing the apical surface before adding any compounds, toxins, or viruses is important. This helps remove accumulated mucus, dead cells, and other debris, thereby promoting a stable and viable culture environment and allowing access to the epithelial cells.

For the endothelial component, it is essential to ensure that the cells are properly sorted and homogeneous. This can be achieved through techniques such as FACS, which ensures that only the desired endothelial cells are used, thereby maintaining consistency and function.

For the macrophage component, despite adhering to plastic easily, macrophages do not adhere well to apical epithelial cells. Increasing the number of macrophages may be beneficial to ensure a large population of macrophages in the iAirways. Adherent macrophages are better positioned to interact with pathogens and other immune cells, thus enhancing the model's relevance. If the study of endothelial immune signaling is preferred, the macrophages can be seeded in the basolateral side of the cell culture inserts in the media to mimic the circulating monocyte population²².

The iAirway model has several other significant applications, including studying lung development of the airway. The model can be used to study the intricate processes involved in lung development, which involves multi-cellular signaling between different populations of cells, providing insights into normal and pathological conditions. To study alveolar development and disease, airway organoids can be substituted for distal lung organoids (DLOs)^23,24 This allows researchers to study the distal lung, including alveolar development and function. This is particularly useful for generating and studying alveolar macrophages, which play a key role in lung immunity.

Developing a reliable and reproducible model of the blood-air barrier in large airways comes with several challenges. Lung progenitor cells must express greater than 50% NKX2-1 for successful differentiation. If the yield is not reached, the ALI cultures may harbor more mesenchyme or be contaminated with non-lung epithelial cells. Maintaining sterility during the plating of endothelial cells is a significant challenge since they are cultured on the basolateral side of the cell culture insert. This prevents the plate lid from closing the sterile environment, so other plates must be used during the seeding of endothelial cells. Contaminants can disrupt the culture and compromise the integrity of the model. Most ALI cultures are incubated for a minimum of 3 weeks to generate most of the airway cells, including ciliated cells. Most protocols seed basal cells, and thus must allow time for signaling mechanisms to differentiate the other airway cells. The current protocol dissociates airway organoids, which already contain many of the airway cells, albeit at lower quantities compared to mature HBEC-derived ALI. Few ciliated cells are seen along with epithelial and mesenchymal cell types (Figure 2D). To generate more ciliated cells, DAPT can be added to the airway organoid cultures and/or the ALI media²⁵. This platform has also only tested the short-term viability of the apical and basolateral cells after air-lift, when the basolateral media is changed to 1:1 epithelial and endothelial media. More research needs to be done to test the viability over a span of weeks, which may increase the complexity of the cells in ALI.

The blood-air barrier model has profound implications for studying human diseases and developing therapeutic interventions. The model can be used to screen and optimize therapeutic agents, including antiviral drugs, by providing a physiologically relevant environment that mimics human airways, especially with the exposure of the airway cells to air. Gene knockout techniques can be employed to study specific mechanisms of barrier dysfunction and host-pathogen interactions. The model offers a platform to study various aspects of human respiratory diseases, from viral infections to chronic conditions like asthma and COPD. This understanding can lead to the development of more effective and targeted therapies.

In conclusion, the blood-air barrier model of the large airway (iAirway) represents a powerful tool for studying barrier dysfunction due to viral infections and other respiratory diseases. Ensuring successful differentiation of cell types, addressing developmental challenges, and exploring alternative applications will enhance the utility and accuracy of this model. Its application in therapeutic targeting and gene knockout studies underscores its potential in advancing our understanding and treatment of human respiratory diseases.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
12 well plates	Corning	3512
12-well inserts, 0.4 µm, translucent	VWR	10769-208
2-mercaptoethanol	Sigma-Aldrich	M3148
Accutase	Innovative Cell Tech	AT104
Activin A	R&D Systems	338-AC
All-trans retinoic acid (RA)	Sigma-Aldrich	R2625
ascorbic acid	Sigma	A4544
B27 without retinoic acid	ThermoFisher	12587010
BMP4	R&D Systems	314-BP/CF
Bovine serum albumin (BSA) Fraction V, 7.5% solution	Gibco	15260-037
Br-cAMP	Sigma-Aldrich	B5386
CD 14 (FITC)	BioLegend	982502
CD 31 PECAM-1(APC)	R&D System	FAB3567A
CD 45 (PE)	BD Biosciences	560975
CD 68 (PE)	BioLegend	33808
CHIR99021	Abcam	ab120890
CPM	Fujifilm	014-27501
Dexamethasone	Sigma-Aldrich	D4902
Dispase	StemCellTech	7913
DMEM/F12	Gibco	10565042
Dorsomorphin	R&D Systems	3093
E-CAD/CD 324 (APC)	BioLegend	324107
EGF	R&D Systems	236-EG
EGM2 Medium	Lonza	CC-3162
EPCAM/CD 326 (APC)	BioLegend	324212
FBS	Gibco	10082139
FGF10	R&D Systems	345-FG/CF
FGF7	R&D Systems	251-KG/CF
Fibronectin	Fisher	356008
Forskolin	Abcam	ab120058
Glutamax	Life Technologies	35050061
Ham’s F12	Invitrogen	11765-054
HEPES	Gibco	15630-080
IBMX (3-Isobtyl-1-methylxanthine)	Sigma-Aldrich	I5879
IL-3	Peprotech	200-03
Iscove’s Modified Dulbecco’s Medium (IMDM) + Glutamax	Invitrogen	31980030
Knockout Serum Replacement (KSR)	Life Technologies	10828028
Matrigel	Corning	354230
M-CSF	Peprotech	300-25
Monothioglycerol	Sigma	M6145
mTeSR plus Kit (10/case)	Stem Cell Tech	5825
N2	ThermoFisher	17502048
NEAA	Life Technologies	11140050
PBS	Gibco	10010023
Pen/strep	Lonza	17-602F
ReleSR	Stem Cell Tech	5872
RPMI1640 + Glutamax	Life Technologies	12633012
SB431542	R&D Systems	1614
SCF	PeproTech	300-07
SMA	Invitrogen	50-9760-80
STEMdiff APEL 2 Medium	STEMCELL Technologies	5275
TrypLE Express	Gibco	12605-028
VEGF165	Preprotech	100-20
Vimentin	Cell Signaling	5741S
Y-27632 (Rock Inhibitor)	R&D Systems	1254/1
ZO-1	Invitrogen	339100