The authors would like to thank Dr. Miguel Spuch-Calvar for the coculture scheme in Figure 3 and to Dr. Bedia Begum Karakocak for critical reading. This study was supported by the PATROLS project, European Union&#8217;s Horizon 2020 Research and Innovation Programme under grant agreement No 760813, and by the Adolphe Merkle Foundation. B.D. thanks the Peter und Traudl Engelhorn foundation for financial support.

The authors have nothing to disclose.

Emerging production of novel materials, including chemicals and drugs, gradually increases the need for predictive in vitro models. To comply with the three principles of replacement, reduction, and refinement of animal testing32, in vitro cell models have become powerful tools regarding the replacement and reduction aspect for elucidating mechanisms of a drug&#8217;s or material&#8217;s action8,9,10,11. Presented here is a detailed protocol of assembling the multicellular model using immune cells that are either freshly isolated or thawed from previously frozen monocytes. Also described is the cultivation of the model at ALI. Finally, the protocol illustrates an example of exposure to proinflammatory stimuli and compares the response of the two models containing either fresh or frozen monocytes.
Various studies have been performed to confirm and justify the added value of the enhanced complexity of the models grown and exposed under ALI conditions compared to conventional submerged exposure7,22,31. Observation of the higher proinflammatory response in cocultures compared to monocultures of epithelial cells confirm a previous study. The study used the presented coculture model (stimulated with LPS) and showed a higher response at gene expression levels of TNF and IL1B compared to the A549 monoculture equivalent model7. On the other hand, both models showed higher variations within measured proinflammatory mediator release values compared to A549 monocultures. This can be explained by the use of immune cells from different donors (buffy coats) within biological repetitions (i.e., one repetition, one donor), as previously shown7. If desired, the variations among the replicates can be overcome by 1) using thawed PBMs from the same donor or 2) pooling PBMs from different donors prior to freezing the cells, then subsequent usage of the same pool in each repetition. Including more biological repetitions is also recommended.
The cell-freezing technique can be considered as a critical step; however, it is a common laboratory procedure for preserving cells for phenotypic and functional analysis. Various studies have demonstrated that the quality of frozen PBMs is vital to their survival, and an appropriate freezing technique is key to the success of subsequent assays with the same cells28,32. Modification of the protocol can be performed by freezing PBMs, which provides flexibility in the experimental setup, as the availability of buffy coats is usually limited. Another advantage of using frozen PBMs (in several vials) over freshly isolated ones is that they can be used in subsequent experiments even after 1 year. This decreases the potential issue of donor-to-donor variability if this is a desired or required parameter in an experimental.
Outcomes of an interlaboratory comparison performed after up to 13 months show that PBMs, when properly stored in a liquid nitrogen tank, can be used over a long period without any effect on cell viability or cell recovery33. Longer storage times (over 1 year) may be possible upon careful validation of cell viability and cell responsiveness before performing an experiment. Also, the temperature in the liquid nitrogen tank must remain stable at all times. The main factor affecting the viability of cryopreserved PBMs was found to be DMSO concentration, with an optimal concentration of 10%&#8211;20 % (v/v)28. To minimize potentially harmful effects of freezing, different sources of proteins, FBS or BSA (with a broad range of concentration from 40% up to 100 %34) are often added to the freezing medium as natural protective components that can increase cell survival.
Due to the high cytotoxic potential of DMSO, it is recommended first to disperse PBMs in FBS, then add DMSO to PBMs already dispersed in FBS. Notably, although higher FBS concentrations (&#62;40%) did not show any improvement in cell viability, at the same time, they did not cause harm to the cells28. Nevertheless, freezing monocytes is a possible approach to overcoming issues of limited buffy coat availability. However, if the use of MDDCs and MDMs from fresh PBMs is desired, the immune cells can be differentiated and used 5&#8211;8 days after isolation7,16,17,35,36,37. If experimental planning allows, at least 6 days of differentiation in both MDDCs and MDMs is recommended. However, consistency among different repetitions in the same experiment, along with routine inspections of their specific surface marker expressions, are crucial. The responsiveness to a proinflammatory stimulus, such as LPS, after the differentiation time should also be regularly checked.
Many investigations using the A549 cell line have been performed at ALI, either as a monoculture or combined with other cell types (macrophages, dendritic cells, or fibroblasts) into 3D coculture model22,24,29,38. Using this 3D coculture model, the cytotoxicity, oxidative stress, or proinflammatory effects&#160;of (nano-)materials have been investigated for up to 72&#160;h1,17,21,24,29. The model&#8217;s resemblance to in vivo tissue has previously been investigated based on confocal laser scanning imaging of the model16. When assembling the model, it is important to consider both cell proliferation (which can affect A549 in the model presented here) as well as performance of the primary (not proliferating) immune cells (here, MDDCs and MDMs). It is also important to consider that not all the CD14 positive monocytes differentiate into MDDCs and MDMs, and that the cells can be present in both attached and suspended forms. Based on the nature of the coculture assembly (here, both cell types need to attach to the existing epithelial layer), it is recommended to use only the adherent sub-populations of both immune cell types. Additionally, routine analyses of monocytes, MDDC and MDM monoculture responsiveness to LPS, and expression of specific surface markers (CD14, CD163, CD86, CD93, or CD206, data not shown) have suggested that 6 and 7 days of differentiation are the optimum timepoints.
Although a realistic number of alveolar epithelial cells in human lungs corresponds to ~160,000 cells/cm2, the number of A549 cells counted in the model is ~1,000,000 cells/cm2 after 9 days cultured on the insert16,18. Thus, this in vitro model&#8217;s limitations need to be considered. First, the density of epithelial cells was established based on their ability to form a confluent layer on the growing membrane. It is also important to mention that the A549 represents an epithelial type II cell with a cuboidal form, contrary to epithelial type I cells, which are flat and outspread. On the other hand, the required number of immune cells was established based on the literature and presented in this protocol as cell number/surface area39,40,41. The cell density of MDDCs in the range of 400 cells/mm2 (4 cells/cm2)16 is comparable to the steady-state cell density of 500&#8211;750 cells/mm2 (5- 7 cells/cm2) reported from in vivo studies39. The density of MDMs in this model is within the same range of in vivo situation in the human alveolar region40.
Mature macrophage marker staining (25F9) was observed both in the apical side (where MDMs are present) as well as basal side (i.e., at the site of dendritic cells). Translocation of immune cells through the membrane inserts pores is possible and has also been observed using this model16, which may explain the observed differences in staining intensities. However, another possible explanation is that the mature macrophage marker can also be expressed on dendritic cells, but the expression is highly donor-specific42. Also, the intensity of the 25F9&#160;expression is much higher in MDMs (Figure 7, Figure 8). Both proinflammatory stimuli (LPS and TNF-&#945;) affected the integrity of the pulmonary epithelial barrier in both cocultures (Figure 7, Figure 8). This was expected based on previous publications43,44 showing that proinflammatory cytokines and bacterial products disrupt the integrity of epithelial barriers.
The 3D multicellular model of the human alveolar epithelium, established and characterized previously17, has served as a powerful and useful tool for assessing biological responses (i.e., acute proinflammatory reactions, oxidative stress response, particle distribution, and cellular communication) in vitro21,24,25,45. The results confirm the responsivness of coculture models to proinflammatory stimuli (here, LPS and TNF-&#945;). The response was slightly increased when using immune cells from fresh PBMs; however, there was no statistically significant difference between cocultures using fresh vs. thawed PBMs. Furthermore, the proinflammatory reactions of both coculture models were higher than those of epithelial cell monocultures cultivated under the same (ALI) conditions. In summary, the protocol describes the assembly of a 3D human alveolar epithelial tissue coculture model using either fresh or thawed PBMs for differentiation into MDMs and MDDCs. It is shown that both models are highly responsive to proinflammatory stimuli; therefore, they can serve as powerful tools for potential hazard and toxicity assessments.

In vitro lung cell cultures offer cost-effective, robust, and well-controlled platforms to assess the hazards of aerosols1. As a model cell system for human alveolar pneumocytes, the epithelial A549 cell line isolated from a pulmonary adenocarcinoma is often used2. These cells represent squamous type II epithelial cells from the alveolar region3 and are a widely used lung cell line for hazard and toxicity assessment1,4,5,6,7,8,9,10. The A549 cell line possesses relevant features of alveolar epithelial type II cells, such as the presence of characteristic lamellar bodies containing densely packed phospholipids3.
It has been shown that when the cells are cultured at an air-liquid interface (ALI), surfactant is released on the apical side of air-exposed epithelial cells, reducing surface tension11,12,13. This feature is particularly important in nanomaterial respiratory hazard and toxicity investigations. Once inhaled nanomaterials/toxicants are deposited in the alveolar region, they first interact with pulmonary surfactant and are displaced by wetting forces into the aqueous hypophase, where the interaction with pulmonary cells takes place14,15. Even though A549 cells form a monolayer (which can overgrow into multilayers at later timepoints when cultivated at ALI) and produce surfactant, a drawback is their insufficient tight junction formation, resulting in low transepithelial electrical resistance values, but still presenting&#160;a functional barrier against intercellular (nano)particles translocation16,17,18.
In the lungs, there is a variety of immune cell populations, including phagocytic and professional antigen-presenting cells (i.e., macrophages and dendritic cells) that directly communicate through cell-cell contact or intercellular signaling to control and maintain homeostasis. Macrophages and dendritic cells are critical innate immune effectors and initiators of the adaptive immune response19. Dendritic cells residing within or beneath the epithelium can form protrusions across the epithelium to the lumen to catch antigens. Alveolar macrophages are located on the apical surface of the epithelium and act as sentinel cells, representing the first cellular defense against foreign material as well as bacterial, viral and fungal infections. Their phenotypic plasticity allows rapidly induction of proinflammatory reactions in response to such stimuli as well as shifting into triggering anti-inflammatory (i.e., inhibitory) reactions20.
To simulate the human alveolar epithelial tissue barrier, we established a triple coculture model with A549 cells supplemented with human blood monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs) on the apical and basal sides, respectively17. Cultivation of this model at ALI has been previously reported16, even up to 72 h post-exposure21. Acute immune responses to carbon nanotubes exposures was significantly enhanced in cell culture exposed to the ALI as compared to submerged conditions22. The coculture model, cultured and exposed to different materials at ALI, has been previously used to investigate cytotoxicity, oxidative stress and inflammatory responses upon exposures to zinc oxide,23 graphene-related materials24, gold nanoparticles25,26, carbon nanotubes21, and volcanic ash and diesel exhaust particles27.
Furthermore, the important role of macrophages and dendritic cells as immune effector cells in an in vitro human lung model has been confirmed. In particular, an increased proinflammatory response in the model was observed only in the presence of immune cells in comparison to monoculture systems7. Potential drawbacks of using primary monocyte-derived immune cells are the limited accessibility of PBMs as well as donor-to-donor variation. As a solution to these potential drawbacks, presented here is a protocol introducing cryopreservation of freshly isolated PBMs28 for the cell culture model assembly. The aim of this study is to demonstrate the 3D human alveolar epithelial tissue model assembly, including the isolation of PBMs from human buffy coats. The responsiveness to proinflammatory stimuli is compared to the model composed of MDMs and MDDCs differentiated from fresh PBMs or differentiated from frozen/thawed PBMs.
Working with untested human blood samples involves specific care to prevent the potential transmission of infectious diseases, such as HIV (human immunodeficiency virus), hepatitis B, and hepatitis C. Therefore, the use of personal protective measures such as gloves, gowns, masks, and eye protection are crucial and must be in accordance with good laboratory practice principles. These protections reduce the risk of exposing the skin or mucous membranes to potentially infectious fluids. Additionally, for those involved in handling buffy coats and PBMs, vaccination against the hepatitis B virus is mandatory, and blood titer levels of antibodies anti-hepatitis B must be above 100 IU/L (country-specific legislative requirements need to be addressed). In addition, all work must be performed in biosafety level 2 laboratories (country-specific legislative requirements need to be addressed). Standard health and safety precautions associated with working in a laboratory environment and handling mammalian cell culture, including handling of waste, should be adopted when conducting the entire protocol.

<table><tbody><tr><td>Benchmark microplate reader</td><td>does not have to be specific, for example BioRad, Cressier, Switzerland</td><td></td><td></td></tr><tr><td>Cell culture Incubator</td><td>does not have to be specific</td><td></td><td></td></tr><tr><td>Cell freezing container (for example Mr. Frosty)</td><td>does not have to be specific</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>does not have to be specific</td><td></td><td></td></tr><tr><td>Confocal laser scanning microscope</td><td>does not have to be specific, for example</td><td>Zeiss LSM 710 meta</td><td></td></tr><tr><td>Heamatocytometer, or automatic cell counter</td><td>does not have to be specific</td><td></td><td></td></tr><tr><td>Laminar bio-safety hood class II</td><td>does not have to be specific</td><td></td><td></td></tr><tr><td>MultiStand Macs (Macs Cell Separator)</td><td>Miltenyi, Germany</td><td>130-042-303</td><td></td></tr><tr><td>pH meter</td><td>does not have to be specific</td><td></td><td></td></tr><tr><td>Phase contrast inverted light microscope</td><td>does not have to be specific</td><td></td><td></td></tr><tr><td>Pipette boy, pipettors (different volumes)</td><td>do not have to be specific</td><td></td><td></td></tr><tr><td>Scissors</td><td>do not have to be specific</td><td></td><td></td></tr><tr><td>Vacuum pump</td><td>does not have to be specific</td><td></td><td></td></tr><tr><td>Water bath</td><td>does not have to be specific</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Disposable small equipment/glassware&lt;/strong&gt;</td><td></td><td>Catalogue Number</td><td></td></tr><tr><td>15 mL and 50 mL conical centrifuge tubes</td><td>does not have to be specific</td><td></td><td></td></tr><tr><td>6- and 12-well cell culture plates, flat bottom, low evaporation lid, sterile</td><td>Falcon, Switzerland</td><td>353046 and 353043</td><td></td></tr><tr><td>Cell culture inserts, transparent PET membrane, 12-well, 3 &amp;mu;m pore size</td><td>Falcon, Switzerland</td><td>353181</td><td></td></tr><tr><td>Cell scrapper</td><td>does not have to be specific, for example VWR, Switzerland</td><td>353085</td><td></td></tr><tr><td>Cryovials</td><td>do not have to be specific</td><td></td><td></td></tr><tr><td>Glass autoclaved Petri Dishes</td><td>do not have to be specific</td><td></td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi, Germany</td><td>130-042-401</td><td></td></tr><tr><td>Sterile filtration cup for vacuum filtration, 0.2 &amp;mu;m pore size</td><td>does not have to be specific, for example VWR, Switzerland</td><td>10040-446</td><td></td></tr><tr><td>Sterile Lab Bottle compatible with Filtration cup (min. 100 mL)</td><td>does not have to be specific</td><td></td><td></td></tr><tr><td>Sterile pipettes</td><td>do not have to be specific</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bovine serum albumine (BSA)</td><td>Sigma-Aldrich, Switzerland</td><td>A7030-100g</td><td></td></tr><tr><td>CD14+ MicroBeads human - magnetic beads</td><td>Miltenyi, Germany</td><td>130-097-052</td><td></td></tr><tr><td>Deattachnig agent Trypsin-EDTA, 0.05%, phenol red</td><td>Gibco, Switzerland</td><td>25300054</td><td></td></tr><tr><td>Density gradient medium Lymphoprep</td><td>Alere Technologies AS, Norway</td><td>1114547</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma Aldrich, Switzerland</td><td>D2438</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich, Switzerland</td><td>E6758-100g</td><td></td></tr><tr><td>Fetal bovine serum (heat inactivated)</td><td>Gibco, Switzerland</td><td>10270-106</td><td></td></tr><tr><td>Human granulocyte-macrophage colony-stimulating factor (GM-CSF), premium grade</td><td>Miltenyi, Germany</td><td>130-093-864</td><td></td></tr><tr><td>Human Interleukin 4 (IL-4), premium grade</td><td>Miltenyi, Germany</td><td>130-095-373</td><td></td></tr><tr><td>Human macrophage colony-stimulating factor (M-CSF), premium grade</td><td>Miltenyi, Germany</td><td>130-096-485</td><td></td></tr><tr><td>L-glutamine</td><td>Gibco, Switzerland</td><td>25030-024</td><td></td></tr><tr><td>Lipopolysaccharid (LPS) from Escherichia coli</td><td>Sigma-Aldrich, Switzerland</td><td>4524-5mg</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich, Switzerland</td><td>158127</td><td></td></tr><tr><td>Penicilin-Streptomycin</td><td>Gibco, Switzerland</td><td>15140-122</td><td></td></tr><tr><td>Phosphate Buffer Saline (PBS)</td><td>Gibco, Switzerland</td><td>10010-015</td><td></td></tr><tr><td>Roswell Park Memorial Institute-1640 Medium (RPMI)</td><td>Gibco, Switzerland</td><td>42401-018</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich, Switzerland</td><td>T8787</td><td></td></tr><tr><td>Trypan blue solution (0.4%)</td><td>Sigma Aldrich, Switzerland</td><td></td><td></td></tr><tr><td>Tumor necrosis factor alpha (TNF-&amp;alpha;)</td><td>Immunotools</td><td>11343015</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Assays used for cytotoxicity, (pro-)inflammatory response&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cytotoxicity Detection Kit (LDH)</td><td>Roche, Switzerland</td><td>11644793001</td><td></td></tr><tr><td>Human IL-6 DuoSet ELISA</td><td>R&amp;amp;D, Biotechne, Switzerland</td><td>DY206</td><td></td></tr><tr><td>Human IL-8/CXCL8 DuoSet ELISA</td><td>R&amp;amp;D, Biotechne, Switzerland</td><td>DY208</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Immunostaining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>4&amp;prime;,6-diamidino-2-phenylindole (DAPI), concentration 2 &amp;mu;g/mL</td><td>Sigma-Aldrich, Switzerland</td><td>10236276001</td><td></td></tr><tr><td>Goat anti-mouse IgG (H+L) Alexa Fluor 647 conjugated, concentration 20 &amp;mu;g/mL</td><td>Abcam, UK</td><td>ab150115</td><td></td></tr><tr><td>Goat anti-rabbit IgG antibody (H+L) Dylight 488 conjugated, concentration 10 &amp;mu;g/mL</td><td>Agrisera, Sweden</td><td>AS09 633</td><td></td></tr><tr><td>Mature Macrophage Marker Monoclonal Antibody, concentration 50 &amp;mu;g/mL</td><td>eBioScience, Thermo Fischer, Switzerland</td><td>14-0115-82</td><td></td></tr><tr><td>Phalloidin rhodamine, concentration 0.264 &amp;micro;M</td><td>Molecular Probes, Life Technologies, Switzerland</td><td>R415</td><td></td></tr><tr><td>Recombinant Anti-CD83 antibody, 1:50 dillution</td><td>Abcam, UK</td><td>ab244204</td><td></td></tr></tbody></table>

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.

Human lung coculture models, composed of alveolar epithelial cells and immune cells, were assembled either from fresh or frozen MDDCs and MDMs progenitors (here, human peripheral blood-derived monocytes). As presented in Figure 1, A549 cells were seeded 3 days after the first section involving monocyte isolation/thawing. After 6 days of differentiation, the differentiated MDMs appeared round-shaped, whereas MDDCs formed a more elongated shape with observable protrusions. They also appeared as agglomerates, especially when differentiated from fresh monocytes (Figure 2, Figure 3). Epithelial cells formed a dense cell layer of cells after 3 days of growth on membrane inserts (Figure 4), when the cocultures were assembled. After 24 h of assembling and an additional 24 h of being subjected to ALI conditions, the cocultures were prepared for exposures.
Responsiveness of the 3D cell culture models was investigated upon exposure to known proinflammatory stimuli using a pseudo-ALI approach, as described previously29. The proinflammatory stimuli, LPS and TNF-&#945;, were added in low volumes (100 &#956;L) onto the apical surface of the air-exposed cell model. In parallel, the absence of membrane rupture as a measure of cytotoxicity was assessed via LDH assay. A significant increase in LDH release in CCM of the basal compartment was observed upon exposure to the positive control for membrane rupture, a detergent Triton-X 100 (Figure 5). These results proved responsiveness of the model to a cytotoxic substance, whereas no increase in LDH release was observed upon apical stimulation with TNF-&#945; or LPS.
A possible reason for the different measured values of LDH in the samples assembled with either fresh or previously frozen PBMs can be attributed to the sample storage. Samples from fresh PBMs were stored for a longer time at -80 &#176;C; therefore, the activity of LDH enzyme may drop. Notably, LDH is stable for only up to 4 days in CCM; thus, it is recommended to perform the assay the latest 2 days after collecting the supernatants. Alternatively, it is possible to freeze down the supernatants directly after collection. However, it is important to consider that freezing can decrease the enzymatic activity of LDH.
Secretion of proinflammatory mediators (here, TNF-&#945; and interleukins 6 [IL-6] and 8 [IL-8]) into the basal CCM was quantified via ELISA. Statistically significant (p &#60; 0.05, one-way ANOVA) increases in the release of IL-6 and IL-8 were observed in both LPS- and TNF-&#945;- treated samples compared to respective untreated cells, as well as in the cell culture models assembled from either PBMs source (Figure 6). Although concentrations (pg/mL) of all the tested cytokines in the basal CCM were higher in the cocultures composed of fresh PBMs, differences among the two cocultures and monocultures were not statistically significant (p &#62; 0.05) (Figure 6). To confirm the added value of coculture models with respect to a 2D epithelial cell culture, A549 monocultures were also exposed to LPS or TNF-&#945;. As expected, the release of all investigated mediators from A549 monocultures was lower compared to both coculture models; although, the difference between them was not statistically significant (p &#62; 0.05, one-way ANOVA).
Cellular morphology of the 3D human alveolar epithelial tissue barrier was assessed via confocal laser scanning microscopy (LSM). To visualize the composition of each model, macrophages within the coculture models (MDMs) were stained with mature macrophage marker 25F9. MDDCs were stained with CD83, which is an important marker for activated dendritic cells30. Regarding cellular morphology, no difference was observed between the coculture models using MDMs and MDDCs from fresh PBMs compared to those using thawed PBMs. In LPS- and TNF-&#945;-exposed cocultures, both composed of fresh and frozen immune cells, a disrupted epithelial layer in LSM images was observed, which was not the case in untreated cells (Figure 7, Figure 8).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61090/61090fig01.jpg" /> 
Figure 1: Schematic timeline of the protocol.&#160;Presentation of the 3D coculture model preparation, assembly, and application (exposure to a tested substance). ALI = air-liquid interface, MDDCs = monocyte-derived dendritic cells, MDMs = monocyte-derived macrophages, PBMs = peripheral blood monocytes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61090/61090fig01large.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/61090/61090fig02.jpg" /> 
Figure 2: MDMs and MDDCs differentiated from fresh PBMs.&#160;Phase-contrast microscopy image of differentiated (A) MDMs and (B) MDDCs from fresh PBMs (6 days after cell isolation). MDMs are round-shaped, while MDDCs are often observed as agglomerates. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61090/61090fig02large.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/61090/61090fig03.jpg" /> 
Figure 3: MDMs and MDDCs differentiated from frozen PBMs.&#160;Phase-contrast microscopy image of differentiated (A) MDMs and (B) MDDCs from thawed PBMs (6 days after thawing). MDMs are round-shaped, but some elongated cells can be observed. MDDCs also appear round-shaped with protrusions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61090/61090fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61090/61090fig04.jpg" /> 
Figure 4: Epithelial cells growth on membrane inserts.&#160;Phase-contrast microscopy image of confluent A549 growing on a membrane insert 4 days after seeding, forming a dense layer of cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61090/61090fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61090/61090fig05.jpg" /> 
Figure 5: Cytotoxicity results investigated via membrane rupture-based (LDH) assay.&#160;The data is presented as a fold increase over untreated cells (mean &#177; SD, n = 3, asterisk denotes statistically significant increase compared to untreated cells, **p &#60; 0.01, ****p &#60; 0.0001). In green models MDMs and MDDCs from fresh PBMs are represented, and in purple models assembled from thawed PBMs are represented. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61090/61090fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61090/61090fig06.jpg" /> 
Figure 6: Proinflammatory reactions in the cocultures and monocultures.&#160;The proinflammatory mediators (TNF-&#945;, IL-6, and IL-8) release in the cocultures upon 24 h challenge with LPS or TNF-&#945;. The data is presented as relative to untreated cells (mean &#177; SD, n = 3, **p &#60; 0.01, ***p &#60; 0.001, ****p &#60; 0.0001). In green models MDMs and MDDCs from fresh PBMs are represented, and in purple models assembled from thawed PBMs are represented. Grey represents A549 monocultures. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61090/61090fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61090/61090fig07.jpg" /> 
Figure 7: Morphology of cocultures composed of fresh immune cells.&#160;LSM images of apical and basal sides of the coculture model with xz projections of apical sides of the model using MDMs and MDDCs from fresh PBMs. Cyan represents nuclei (DAPI), magenta represents cytoskeleton (rhodamine-phalloidin), white represents MDMs (25F9), and green represents MDDCs (CD 83). The white arrow denotes MDM, whereas the green arrow denotes MDDC. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61090/61090fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61090/61090fig08.jpg" /> 
Figure 8: Morphology of cocultures composed of frozen immune cells.&#160;LSM images of the apical side of the coculture model with corresponding xz projections, and basal side of the model using MDMs and MDDCs from thawed PBMs. Cyan represents nuclei (DAPI), magenta represents cytoskeleton (rhodamine-phalloidin), white represents MDMs (25F9), and green represents MDDCs (CD 83). The white arrow denotes MDM, whereas the green arrow denotes MDDC. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61090/61090fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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 ...

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11.2K Views.  University of Fribourg. Deze individuele menselijke audio model maakt gebruik van relevante immuuncellen als een krachtig instrument om acute immuunrespons van stoffen, waaronder nanomaterialen en kennismedicijnen te voorspellen. Deze medische combineert menselijke epitheelcellen en menselijke primaire immuuncellen om die specifieke reacties te beoordelen. Het gebruik van verse en bevroren monocyten biedt flexibiliteit voor het experimentplan.Tal van studies hebben aangetoond dat dit model inzicht kan geven i...

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

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Bioengineering

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 ...

Developmental Biology

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Immunology and Infection

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

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.

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.

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

Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to gases, particles or complex atmospheres (e.g., cigarette smoke), thus providing realistic physiological exposure of the apical surface of the human alveolar region to air. In contrast to sequential exposure models with linear aerosol guidance, the modular design of the radial flow system meets all requirements for the continuous generation and transport of the test atmosphere to the cells, a homogenous distribution and deposition of the particles and the continuous removal of the atmosphere. This exposure method is primarily designed for the exposure of cells to airborne particles, but can be adapted to the exposure of liquid aerosols and highly toxic and aggressive gases depending on the aerosol generation method and the material of the exposure modules.
Within the framework of a recently completed validation study, this exposure system was proven as a transferable, reproducible and predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles, thereby potentially reducing or replacing animal experiments that would normally provide this toxicological assessment.

We present a robust, transferable and predictive in vitro exposure system for the screening and monitoring of airborne particles concerning their acute pulmonary cytotoxicity by exposing cultivated human lung cells at the air-liquid interface (ALI).

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to ...

Medicine

Establishing a Lung-on-Chip Model for Respiratory Infection Studies

Immunocompetent Alveolus-on-Chip Model for Studying Alveolar Mucosal Immune Responses

We introduce an advanced immunocompetent lung-on-chip model designed to replicate the human alveolar structure and function. This innovative model employs a microfluidic-perfused biochip that supports an air-liquid interface mimicking the environment in the human alveoli. Tissue engineering is used to integrate key cellular components, including endothelial cells, macrophages, and epithelial cells, to create a representative tissue model of the alveolus. The model facilitates in-depth examinations of the mucosal immune responses to various pathogens, including viruses, bacteria, and fungi, thereby advancing our understanding of lung immunity. The primary goal of this protocol is to provide details for establishing this alveolus-on-chip model as a robust in vitro platform for infection studies, enabling researchers to closely observe and analyze the complex interactions between pathogens and the host's immune system within the pulmonary environment. This is achieved through the application of microfluidic-based techniques to simulate key physiological conditions of the human alveoli, including blood flow and biomechanical stimulation of endothelial cells, alongside maintaining an air-liquid interface crucial for the realistic exposure of epithelial cells to air. The model system is compatible with a range of standardized assays, such as immunofluorescence staining, cytokine profiling, and colony-forming unit (CFU)/plaque analysis, allowing for comprehensive insights into immune dynamics during infection. The Alveolus-on-chip is composed of essential cell types, including human distal lung epithelial cells (H441) and human umbilical vein endothelial cells (HUVECs) separated by porous polyethylene terephthalate (PET) membranes, with primary monocyte-derived macrophages strategically positioned between the epithelial and endothelial layers. The tissue model enhances the ability to dissect and analyze the nuanced factors involved in pulmonary immune responses in vitro. As a valuable tool, it should contribute to the advancement of lung research, providing a more accurate and dynamic in vitro model for studying the pathogenesis of respiratory infections and testing potential therapeutic interventions.

Author Spotlight: Developing a Microfluidic Lung-on-Chip Model for In-Depth Study of Human Immune Response and Infection Mechanisms

Lung-on-chip models surpass traditional 2D cultures by mimicking the air-liquid interface and endothelial cell perfusion, simulating blood flow and nutrient exchange crucial for lung physiology studies. This enhances lung research relevance, offering a dynamic, physiologically accurate environment to advance the understanding and treatment of respiratory infections.

We introduce an advanced immunocompetent lung-on-chip model designed to replicate the human alveolar structure and function. This innovative model employs ...

Isolation of Immune and Non&mdash;Immune Cells from Mouse Lungs for Flow Cytometry

Isolation of Live Myeloid and Epithelial Cell Populations from the Mouse Lung

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development of disease. Studies utilizing mouse models of respiratory infection, allergy, fibrosis, and cancer have been critical to reveal mechanisms of disease progression and identify therapeutic targets. However, most studies focused on the mouse lung prioritize the isolation of either immune cells or epithelial cells, rather than both populations concurrently. Here, we describe a method for preparing a comprehensive single-cell suspension of both immune and non-immune populations suitable for flow cytometry and fluorescence-activated cell sorting. These populations include epithelial cells, endothelial cells, fibroblasts, and a variety of myeloid cell subsets. This protocol entails bronchoalveolar lavage and subsequent inflation of the lungs with dispase. Lungs are then digested in a liberase mixture. This method of processing liberates a variety of diverse cell types and results in a single-cell suspension that does not require manual dissociation against a filter, promoting cell survival and yielding high numbers of live cells for downstream analyses. In this protocol, we also define gating schemes for epithelial and myeloid cell subsets in both na&#239;ve and influenza-infected lungs. Simultaneous isolation of live immune and non-immune cells is key for interrogating intercellular crosstalk and gaining a deeper understanding of lung biology in health and disease.

This protocol details the isolation of live immune and non-immune populations from the mouse lung at a steady state and following influenza infection. It also provides gating strategies for identifying epithelial and myeloid cell subsets.

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development ...

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 ...

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

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