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>Disposable small equipment/glassware</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 &#956;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 &#956;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>Chemicals</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>D5879_1L</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>31870-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline (PBS)</td>
			<td>Gibco, Switzerland</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute-1640 Medium (RPMI)</td>
			<td>Gibco, Switzerland</td>
			<td>11875093</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-&#945;)</td>
			<td>Immunotools</td>
			<td>11343015</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Assays used for cytotoxicity, (pro-)inflammatory response</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&#38;D, Biotechne, Switzerland</td>
			<td>DY206</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-8/CXCL8 DuoSet ELISA</td>
			<td>R&#38;D, Biotechne, Switzerland</td>
			<td>DY208</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Immunostaining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8242;,6-diamidino-2-phenylindole (DAPI), concentration 2 &#956;g/mL</td>
			<td>Sigma-Aldrich, Switzerland</td>
			<td>28718-90-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG (H+L) Alexa Fluor 647 conjugated, concentration 20 &#956;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 &#956;g/mL</td>
			<td>Agrisera, Sweden</td>
			<td>AS09 633</td>
			<td></td>
		</tr>
		<tr>
			<td>Mature Macrophage Marker Monoclonal Antibody, concentration 50 &#956;g/mL</td>
			<td>eBioScience, Thermo Fischer, Switzerland</td>
			<td>14-0115-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin rhodamine, concentration 0.264 &#181;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/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/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/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/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/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/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/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/files/ftp_upload/61090/61090fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment at JoVE.com

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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11.1K Views.  University of Fribourg. 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.

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

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...