This research was supported by CIRM (DISC2COVID19-12022).

Airway Model for Studying Lung Barrier Integrity, Disease, and Drug Discovery

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The authors have nothing to disclose.

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.

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&#39;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 &#34;airway organoid&#34;14, cultured onto sterile cell culture inserts, and exposed to an air-liquid interface (ALI), replicating the conditions of the human airway15,16,17. 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.

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

ipsc-derived epithelial, mesenchymal, endothelial, and immune cell co-culture to model airway barrier integrity in lung health and disease

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&#39;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.

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 &#62;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 &#62;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 &#62;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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67247/67247fig01v2.jpg" /> 
Figure 1:&#160;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 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67247/67247fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67247/67247fig02.jpg" /> 
Figure 2:&#160;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 &#181;m. (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 &#181;m. (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&#38;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 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67247/67247fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67247/67247fig03.jpg" /> 
Figure 3: Changes in airway barrier function of the iAirways exposed to e-cigarettes and viruses.&#160;(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 &#60;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 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67247/67247fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibody</td>
			<td>Application</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>CD 31 PECAM-1(APC)</td>
			<td>flow cytometry (FC)</td>
			<td>10 &#956;l in 100 ul buffer</td>
		</tr>
		<tr>
			<td>EPCAM/CD 326 (APC)</td>
			<td>FC</td>
			<td>&#160;1:500</td>
		</tr>
		<tr>
			<td>E-CAD/CD 324 (APC)</td>
			<td>FC</td>
			<td>5 &#956;l in 100 ul buffer</td>
		</tr>
		<tr>
			<td>CPM</td>
			<td>FC</td>
			<td>&#160;1:200</td>
		</tr>
		<tr>
			<td>CD 45 (PE)</td>
			<td>FC</td>
			<td>5 &#956;l in 100 ul buffer</td>
		</tr>
		<tr>
			<td>CD 14 (FITC)</td>
			<td>FC</td>
			<td>5 &#956;l in 100 ul buffer</td>
		</tr>
		<tr>
			<td>CD 68 (PE)</td>
			<td>FC</td>
			<td>5 &#956;l in 100 ul buffer</td>
		</tr>
		<tr>
			<td>ZO-1</td>
			<td>Immunofluorescence (IF)</td>
			<td>&#160;1:300</td>
		</tr>
		<tr>
			<td>Vimentin</td>
			<td>IF</td>
			<td>&#160;1:200</td>
		</tr>
		<tr>
			<td>SMA</td>
			<td>IF</td>
			<td>&#160;1:100</td>
		</tr>
	</tbody>
</table>
Table 1: Antibodies used in FACS and Immunofluorescence.
Supplementary File 1: Media and buffer compositions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67247/Supplementary File 1.zip" target="_blank">Please click here to download this File.</a>

Author Spotlight: Modeling Human Airway Remodeling and Viral Responses Using Isogenic Epithelial, Endothelial, and Immune Cells

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.

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

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the ...

ipsc-derived-epithelial-mesenchymal-endothelial-immune-cell-co

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

807 Views.  Sanford Consortium for Regenerative Medicine. Mijn onderzoek richt zich op het begrijpen van de remodellering van de menselijke luchtwegen. De menselijke luchtwegen dienen als een barrière tegen gifstoffen uit het milieu, waaronder virussen, luchtvervuiling en tabaksrook. Deze gifstoffen kunnen de luchtwegen beschadigen, waardoor ze veranderen of hermodelleren, waardoor het moeilijk wordt om te ademen.Het modelsysteem omvat niet alleen de epitheelcellen die de barrière vormen, maar ook andere ce...

Video: Auteur Spotlight: Modellering van menselijke luchtwegremodellering en virale reacties met behulp van isogene epitheel-, endotheel- en immuuncellen

Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 alveolar epithelial cells (AT2s) are the facultative progenitors of the distal lung epithelium. Dysfunction and injury of AT2s can result from and contribute to various lung diseases. Improved understanding of AT2 biology is, thus, critical for understanding lung biology and disease; however, primary human AT2s are generally difficult to isolate and limited in supply. To overcome these limitations, human induced pluripotent stem cell (iPSC)-derived type 2 alveolar epithelial cells (iAT2s) can be generated through a directed differentiation protocol that recapitulates in vivo lung development. iAT2s grow in feeder-free conditions, share a transcriptomic program with human adult primary AT2s, and execute key functions of AT2s such as production, packaging, and secretion of surfactant. This protocol details the methods for maintaining self-renewing iAT2s through serial passaging in three-dimensional (3D) culture or adapting iAT2s to air-liquid interface (ALI) culture. A single-cell suspension of iAT2s is generated before plating in 3D solubilized basement membrane matrix (hereafter referred to as &#34;matrix&#34;), where they self-assemble into monolayered epithelial spheres. iAT2s in 3D culture can be serially dissociated into single-cell suspensions to be passaged or plated in 2D ALI culture. In ALI culture, iAT2s form a polarized monolayer with the apical surface exposed to air, making this platform readily amenable to environmental exposures. Hence, this protocol generates an inexhaustible supply of iAT2s, producing upwards of 1 x 1030 cells per input cell over 15 passages while maintaining the AT2 program indicated by SFTPCtdTomato expression. The resulting cells represent a reproducible and relevant platform that can be applied to study genetic mutations, model environmental exposures, or screen drugs.

The present protocol describes human induced pluripotent stem cell-derived type 2 alveolar epithelial-like cells (iAT2s). These cells can be cultured as self-renewing spheres in 3D culture or adapted to air-liquid interface (ALI) culture.

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 ...

Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major causes of morbidity and mortality worldwide. In vitro platforms using human bronchial epithelial cells (HBECs) have been instrumental to our understanding of the airway epithelium in health and disease. Access to HBECs from individuals with rare genetic diseases or rare mutations is a bottleneck in lung research.
Induced pluripotent stem cells (iPSCs) are readily generated by "reprogramming" somatic cells and retain the unique genetic background of the individual donor. Recent advances allow for the directed differentiation of iPSCs to lung epithelial progenitor cells, alveolar type 2 cells, as well as the cells of the conducting airway epithelium via basal cells, the major airway stem cells.
Here we outline a protocol for the maintenance and expansion of iPSC-derived airway basal cells (hereafter iBCs) as well as their trilineage differentiation in air-liquid interface (ALI) cultures. iBCs are maintained and expanded as epithelial spheres suspended in droplets of extracellular matrix cultured in a primary basal cell medium supplemented with inhibitors of TGF-&#223; and BMP signaling pathways. iBCs within these epithelial spheres express key basal markers TP63 and NGFR, can be purified by fluorescence activated cell sorting (FACS), and when plated on porous membranes in standard ALI culture conditions, differentiate into a functional airway epithelium. ALI cultures derived from healthy donors are composed of basal, secretory and multiciliated cells and demonstrate epithelial barrier integrity, motile cilia, and mucus secretion. Cultures derived from individuals with CF or PCD recapitulate the dysfunctional CFTR-mediated chloride transport or immotile cilia, the respective disease-causing epithelial defects.
Here, we present a protocol for the generation of human cells that can be applied for modeling and understanding airway diseases.

Recent advances in human induced pluripotent stem cell differentiation protocols allow for the stepwise derivation of organ-specific cell types. Here, we provide detailed steps for the maintenance and expansion of iPSC-derived airway basal cells and their differentiation into a mucociliary epithelium in air-liquid interface cultures.

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major ...

Preparation and Structural Evaluation of Epithelial Cell Monolayers in a Physiologically Sized Microfluidic Culture Device

In vitro microfluidic experimentation holds great potential to reveal many insights into the microphysiological phenomena occurring in conditions such as acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). However, studies in microfluidic channels with dimensions physiologically relevant to the terminal bronchioles of the human lung currently face several challenges, especially due to difficulties in establishing appropriate cell culture conditions, including media flow rates, within a given culture environment. The presented protocol describes an image-based approach to evaluate the structure of NCI-H441 human lung epithelial cells cultured in an oxygen-impermeable microfluidic channel with dimensions physiologically relevant to the terminal bronchioles of the human lung. Using phalloidin-based filamentous-actin staining, the cytoskeletal structures of the cells are revealed by confocal laser scanning microscopy, allowing for the visualization of individual as well as layered cells. Subsequent quantification determines whether the cell culture conditions being employed are producing uniform monolayers suitable for further experimentation. The protocol describes cell culture and layer evaluation methods in microfluidic channels and traditional fixed-well environments. This includes channel construction, cell culture and requisite conditions, fixation, permeabilization and staining, confocal microscopic imaging, image processing, and data analysis.

The presented protocol describes the development and use of a phalloidin-based filamentous-actin staining technique with confocal laser scanning microscopy (CLSM) to visualize adherent cell layer structure in microfluidic dynamic-culture channels and traditional fixed-well static-culture chambers. This approach aids in evaluating cell layer confluency, monolayer formation, and layer-thickness uniformity.

In vitro microfluidic experimentation holds great potential to reveal many insights into the microphysiological phenomena occurring in conditions such as ...

Bioengineering

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

Immunology and Infection

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The significance of innate immunity signaling in viral infections has increased substantially as patients with respiratory infections who exhibit high innate T cell activation show a better disease outcome. Hence, dissecting these early innate immune interactions allows the elucidation of the processes that govern them and may facilitate the development of potential therapeutic targets and strategies for dampening or even preventing early progression of viral infections. This protocol details a versatile model that can be used to study early crosstalk, interactions, and activation of innate immune cells from factors secreted by virally infected airway epithelial cells. Using an H3N2 influenza virus (A/Aichi/2/1968) as the representative virus model, innate cell activation of co-cultured peripheral blood mononuclear cells (PBMCs) has been analyzed using flow cytometry to investigate the subsets of cells that are activated by the soluble factors released from the epithelium in response to the viral infection. The results demonstrate the gating strategy for differentiating the subsets of cells and reveal the clear differences between the activated populations of PBMCs and their crosstalk with the control and infected epithelium. The activated subsets can then be further analyzed to determine their functions as well as molecular changes specific to the cells. Findings from such a crosstalk investigation may uncover factors that are important for the activation of vital innate cell populations, which are beneficial in controlling and suppressing the progression of viral infection. Furthermore, these factors can be universally applied to different viral diseases, especially to newly emerging viruses, to dampen the impact of such viruses when they first circulate in na&#239;ve human populations.

This protocol details an investigation of the early interactions between virally infected nasal epithelial cells and innate cell activation. Individual subsets of immune cells can be distinguished based on their activation in response to viral infections. They can then be further investigated to determine their effects on early antiviral responses.

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The ...

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled substances, including infectious agents and air pollutants. The airway epithelial layer plays a central role in a large variety of acute and chronic lung diseases, and various treatments targeting this epithelium are administered by inhalation. Understanding the role of epithelium in pathogenesis and how it can be targeted for therapy requires robust and representative models. In vitro epithelial culture models are increasingly being used and offer the advantage of performing experiments in a controlled environment, exposing the cells to different kinds of stimuli, toxicants, or infectious agents. The use of primary cells instead of immortalized or tumor cell lines has the advantage that these cells differentiate in culture to a pseudostratified polarized epithelial cell layer with a better representation of the epithelium compared to cell lines.
Presented here is a robust protocol, that has been optimized over the past decades, for the isolation and culture of airway epithelial cells from lung tissue. This procedure allows successful isolation, expansion, culture, and mucociliary differentiation of primary bronchial epithelial cells (PBECs) by culturing at the air-liquid interface (ALI) and includes a protocol for biobanking. Furthermore, the characterization of these cultures using cell-specific marker genes is described. These ALI-PBEC cultures can be used for a range of applications, including exposure to whole cigarette smoke or inflammatory mediators, and co-culture/infection with viruses or bacteria. 
The protocol provided in this manuscript, illustrating the procedure in a step-by-step manner, is expected to provide a basis and/or reference for those interested in implementing or adapting such culture systems in their laboratory.

Presented here is a reproducible, affordable, and robust method for the isolation and expansion of primary bronchial epithelial cells for long-term biobanking and the generation of differentiated epithelial cells by culture at the air-liquid interface.

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled ...

Human Esophageal Organoids and Air-Liquid Interface Culture Protocols

Three-Dimensional Cell Culture Models to Investigate the Epithelial Barrier in Eosinophilic Esophagitis

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and microbes. Maintaining the integrity of the epithelial barrier is critical for preventing infections and avoiding inflammation caused by harmless food-derived antigens. This article provides simplified protocols for generating human esophageal organoids and air-liquid interface cultures from patient biopsies to study the epithelial compartment of the esophagus in the context of tissue homeostasis and disease. These protocols have been significant scientific milestones in the last decade, describing three-dimensional organ-like structures from patient-derived primary cells, organoids, and air-liquid interface cultures. They offer the possibility to investigate the function of specific cytokines, growth factors, and signaling pathways in the esophageal epithelium within a three-dimensional framework while maintaining the phenotypic and genetic properties of the donor. Organoids provide information on tissue microarchitecture by assessing the transcriptome and proteome after cytokine stimulation. In contrast, air-liquid interface cultures allow the assessment of the epithelial barrier integrity through transepithelial resistance (TEER) or macromolecule flux measurements. Combining these organoids and air-liquid interface cultures is a powerful tool to advance research in impaired esophageal epithelial barrier conditions.

Author Spotlight: Investigating the Pathophysiology of Eosinophilic Esophagitis

Here, a protocol for the culture of human esophageal organoids and air-liquid interface culture is provided. Esophageal organoids' air-liquid interface culture can be used to study the impact of cytokines on the esophageal epithelial barrier.

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor &#945;) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...

Biology

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...

In Vitro Model of Murine Airway Epithelial Cells and Chronic Smoke Exposure

In Vitro Model for Studying Differentiation and Changes of Multi-Omics on Murine Airway Epithelial Cells Stimulated with Cigarette Smoke Extract

Chronic obstructive pulmonary disease (COPD) is largely attributed to tobacco smoke exposure. Investigating how airway epithelial cells functionally adapt to tobacco smoke is crucial for understanding the pathogenesis of COPD. The present study was to set up an in vitro model using primary murine airway epithelial cells to mimic the real-life impact of tobacco smoke. Unlike established cell lines, primary cells retain more in vivo-like properties, including growth patterns, aging, and differentiation. These cells exhibit a sensitive inflammatory response and efficient differentiation, thus closely representing physiological conditions. In this model, primary murine airway epithelial cells were cultured for 28 days under an air-liquid interface with an optimal concentration of cigarette smoke extract (CSE), which led to the transformation of a monolayer of undifferentiated cells into a pseudostratified columnar epithelium, indicative of CSE acclimation. Comprehensive multi-omics analyses were then applied to elucidate the mechanisms by which CSE influences the differentiation of basal airway cells. These insights provide a deeper understanding of the cellular processes underpinning COPD progression in response to tobacco smoke exposure.

Author Spotlight: Development and Characterization of an In Vitro Model to Study Chronic Cigarette Smoke Exposure and Its Impact on Airway Epithelial Cells in COPD Research

Here, we describe an in vitro model for isolating and differentiating murine airway epithelial cells, focusing on their acclimation to chronic cigarette smoke extract (CSE). The model could be utilized to comprehensively characterize the multi-omics impact of CSE, which possibly provides insights into the cellular responses under chronic smoke exposure.

Chronic obstructive pulmonary disease (COPD) is largely attributed to tobacco smoke exposure. Investigating how airway epithelial cells functionally adapt ...

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

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