H.K. and A.S.M. acknowledge funding from the Leibniz Science-Campus InfectoOptics Jena, financed by the funding line Strategic Networking of the Leibniz Association. M.A. and A.S.M. were supported by the IGF project IMPROVE funded by the Federal Ministry for Economic Affairs and Energy on the basis of a resolution of the German Bundestag. A.S.M further acknowledges financial support by the Cluster of Excellence Balance of the Microverse under Germany&#39;s Excellence Strategy - EXC 2051 - Project-ID 690 390713860.

HUVEC cells are isolated from umbilical cords and used up to passage 4. Primary monocytes are isolated from healthy donors from whole blood. The study was approved by the ethics committee of the Jena University Hospital, Jena, Germany (3939-12/13). According to the Declaration of Helsinki, all individuals donating cells for the study gave their informed consent.
1. Day 1: Preparation of the biochip
<ol>
	<li>The biochips are available in different sizes and models. For exper...

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

<ol>
	<li>Mettelman, R. C., Allen, E. K., Thomas, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+immune+responses+to+infection+and+vaccination+in+the+respiratory+tract.">Mucosal immune responses to infection and vaccination in the respiratory tract.</a> Immunity. 55 (5), 749-780 (2022).</li><li>Artzy-Schnirman, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+in+vitro+lung-on-chip+platforms+for+inhalation+assays:+From+prospect+to+pipeline.">Advanced in vitro lung-on-chip platforms for inhalation assays: From prospect to pipeline.</a> Eur J Pharma Biopharma. 144, 11-17 (2019).</li><li>Huh, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituting+organ-level+lung+functions+on+a+chip.">Reconstituting organ-level lung functions on a chip.</a> Science. 328 (5986), 1662-1668 (2010).</li><li>Benam, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+airway-on-a-chip+enables+analysis+of+human+lung+inflammation+and+drug+responses+in+vitro.">Small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro.</a> Nat Meth. 13 (2), 151-157 (2016).</li><li>Ronaldson-Bouchard, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multi-organ+chip+with+matured+tissue+niches+linked+by+vascular+flow.">A multi-organ chip with matured tissue niches linked by vascular flow.</a> Nat Biomed Eng. 6 (4), 351 (2022).</li><li>Deinhardt-Emmer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-infection+with+staphylococcus+aureus+after+primary+influenza+virus+infection+leads+to+damage+of+the+endothelium+in+a+human+alveolus-on-a-chip+model.">Co-infection with staphylococcus aureus after primary influenza virus infection leads to damage of the endothelium in a human alveolus-on-a-chip model.</a> Biofabrication. 12 (2), 025012 (2020).</li><li>Schicke, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+lung+infection+results+in+down-regulation+of+surfactant+protein-a+mainly+caused+by+pro-inflammatory+macrophages.">Staphylococcus aureus lung infection results in down-regulation of surfactant protein-a mainly caused by pro-inflammatory macrophages.</a> Microorganisms. 8 (4), 577 (2020).</li><li>King, S. D., Chen, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+on+surfactant+protein+a:+Cellular+function+in+lung+and+kidney+disease+development.">Recent progress on surfactant protein a: Cellular function in lung and kidney disease development.</a> Am J Physiol Cell Physiol. 319 (2), C316-C320 (2020).</li><li>Hoang, T. N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasive+aspergillosis-on-chip:+A+quantitative+treatment+study+of+human+aspergillus+fumigatus+infection.">Invasive aspergillosis-on-chip: A quantitative treatment study of human aspergillus fumigatus infection.</a> Biomaterials. 283, 121420 (2022).</li><li>Yuksel, H., Ocalan, M., Yilmaz, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-cadherin:+An+important+functional+molecule+at+respiratory+barrier+between+defence+and+dysfunction.">E-cadherin: An important functional molecule at respiratory barrier between defence and dysfunction.</a> Front Physiol. 12, 720227 (2021).</li><li>Van Roy, F., Berx, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell-cell+adhesion+molecule+e-cadherin.">The cell-cell adhesion molecule e-cadherin.</a> Cell Mol Life Sci. 65 (23), 3756-3788 (2008).</li></ol>

The alveolus-on-chip model represents a multilayered tissue model of the human alveolus, integrating essential cell types of the lower respiratory tract, including lung epithelial cells, endothelial cells, and macrophages, cultured in an organotypic arrangement at an ALI with medium perfusion of the endothelial lining. Cells of different layers express specific cell marker proteins such as E-cadherin, a calcium-dependent adhesion molecule of lung epithelial cells, which is central in establishing intercellular epithelial...

The human lung has a remarkable role in respiration and immune defense, with complex interactions between the immune responses of the alveolar mucosa1. The ability of the alveoli to create an efficient immune response is vital for preventing lung infections and securing pulmonary health. Since the lungs are constantly exposed to a wide range of potential risks, including bacteria, viruses, fungi, allergies, and particulate matter,&#160;understanding the complexities of alveolar mucosal immune responses is critical for discovering the mechanisms behind respiratory infections, inflammatory disorders and treating pulmonary diseases1.
To study infection and inflammation-related processes of the respiratory tract in vitro, models that could faithfully mimic the alveolar milieu and the immune responses are required. 2D cell culture and animal modules have been used for decades as essential tools for biomedical research on lung immune response. However, they often have limitations in their translational potential to human situations. Lung-on-chip models can contribute to filling the gap between traditional in vitro and in vivo models and provide a novel approach to studying human-specific immune responses2,3. Lung-on-chip models can mimic the air-liquid interface, which is required for lung cells to recapitulate physiological conditions of the respiratory tract and to develop a more accurate and robust tissue model. This culture technique enables a precise examination of cell differentiation, functioning, and responses to drugs or disease-related stimuli in vitro2.
In this study, we present a microfluidic-based model of the human alveolus as an effective tool to recapitulate the human alveolar milieu by applying perfusion to mimic blood flow and biomechanical stimulation of endothelial cells and incorporating an air-liquid interface with epithelial cells exposed towards an air phase4. We have developed a microfluidic perfused alveolus-on-chip that mimics the physical structure and biological interactions of the human alveolus, with a particular focus on the air-liquid interface. This interface plays a crucial role in the differentiation of respiratory epithelial cells, which is essential for accurately modeling the pulmonary environment. The model uses 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 positioned between the cell layers. This setup replicates the intricate cellular arrangement of the alveolus and is critical for accurately simulating the air-liquid interface, which is a significant factor in the physiological function of lung tissue.
The rationale behind the model development extends to integrating both circulating and tissue-resident immune cells. This approach is designed to accurately mimic the inflammatory host response to human respiratory infections, providing a dynamic environment to study pathogen-host interactions. The presence of macrophages allows for the examination of immediate immune responses and their interaction with pathogens, reflecting the first line of defense against respiratory infections. Furthermore, the design of the biochip platform facilitates convenient and precise manipulation of both biophysical and biochemical cues, which is crucial for replicating alveolus function in vitro. This flexibility is instrumental in dissecting the contributing factors to human infections, enabling researchers to adjust conditions to reflect various disease states or to test potential therapeutic interventions. The compatibility of the platform with multiple readout technologies, including advanced microscopy, microbiological analyses, and biochemical effluent analysis, enhances its utility. These capabilities allow for a comprehensive assessment of the tissue response to infections, including evaluating cellular behavior, pathogen proliferation, and the effectiveness of immune responses.
We present a detailed protocol and techniques to create and utilize a human alveolus-on-chip model focused on replicating the air-liquid interface and integrating immune cells to study human infections in vitro.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Consumables</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cellcounting chamber slides (Countess)</td>
			<td>Invitrogen</td>
			<td>C10283</td>
		</tr>
		<tr>
			<td>Cell culture Multiwell Plates, 24 Well, steril</td>
			<td>Greiner Bio-One</td>
			<td>662 160</td>
		</tr>
		<tr>
			<td>Cell culture Multiwell Plates, 6 Well, steril</td>
			<td>Greiner Bio-One</td>
			<td>657 160</td>
		</tr>
		<tr>
			<td>Coverslips (24x40mm; #1.5)</td>
			<td>Menzel-Gl&#228;ser</td>
			<td>15747592</td>
		</tr>
		<tr>
			<td>Eco wipes</td>
			<td>Dr. Schuhmacher</td>
			<td>00-915-REW10003-01</td>
		</tr>
		<tr>
			<td>Eppies 2.0</td>
			<td>Sarstedt</td>
			<td>72.691</td>
		</tr>
		<tr>
			<td>Eppis 0.5</td>
			<td>Sarstedt</td>
			<td>72.699</td>
		</tr>
		<tr>
			<td>Eppis 1.5</td>
			<td>Sarstedt</td>
			<td>72.690.001</td>
		</tr>
		<tr>
			<td>Falcons 15mL</td>
			<td>Greiner Bio-One</td>
			<td>188 271-TRI</td>
		</tr>
		<tr>
			<td>Falcons 50mL</td>
			<td>Greiner Bio-One</td>
			<td>227 261-TRI</td>
		</tr>
		<tr>
			<td>Gauze swab</td>
			<td>Noba</td>
			<td>PZN 2417767</td>
		</tr>
		<tr>
			<td>Gloves Nitril 3000</td>
			<td>Meditrade</td>
			<td>1280</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Menzel-Gl&#228;ser</td>
			<td>AAAA000001##12E</td>
		</tr>
		<tr>
			<td>Multiwell Plates 24 Well, sterile</td>
			<td>Greiner Bio-One</td>
			<td>662 160</td>
		</tr>
		<tr>
			<td>Pasteur pipettes (glass) 150mm</td>
			<td>Assistent</td>
			<td>40567001</td>
		</tr>
		<tr>
			<td>Pasteur pipettes (glass) 230mm</td>
			<td>Assistent</td>
			<td>40567002</td>
		</tr>
		<tr>
			<td>Round-bottom tubes (PS, 5mL)</td>
			<td>Falcon</td>
			<td>352052</td>
		</tr>
		<tr>
			<td>Safety-Multifly-Set, 20G, 200mm</td>
			<td>Sarstedt</td>
			<td>85.1637.235</td>
		</tr>
		<tr>
			<td>Scalpels</td>
			<td>Dahlhausen</td>
			<td>11.000.00.715</td>
		</tr>
		<tr>
			<td>Serological pipettes 10mL</td>
			<td>Greiner Bio-One</td>
			<td>607 160-TRI</td>
		</tr>
		<tr>
			<td>Serological pipettes 25mL</td>
			<td>Greiner Bio-One</td>
			<td>760 160-TRI</td>
		</tr>
		<tr>
			<td>Serological pipettes 2mL</td>
			<td>Greiner Bio-One</td>
			<td>710 160-TRI</td>
		</tr>
		<tr>
			<td>Serological pipettes 50mL</td>
			<td>Greiner Bio-One</td>
			<td>768 160-TRI</td>
		</tr>
		<tr>
			<td>Serological pipettes 5mL</td>
			<td>Greiner Bio-One</td>
			<td>606 160-TRI</td>
		</tr>
		<tr>
			<td>S-Monovette, 7,5ml Z-Gel</td>
			<td>Sarstedt</td>
			<td>1.1602</td>
		</tr>
		<tr>
			<td>S-Monovette, 9,0ml K3E</td>
			<td>Sarstedt</td>
			<td>02.1066.001</td>
		</tr>
		<tr>
			<td>Softasept N</td>
			<td>Braun</td>
			<td>3887138</td>
		</tr>
		<tr>
			<td>T25 flask</td>
			<td>Greiner Bio-One</td>
			<td>690 960</td>
		</tr>
		<tr>
			<td>Tips sterile 10&#181;L</td>
			<td>Greiner Bio-One</td>
			<td>771 261</td>
		</tr>
		<tr>
			<td>Tips sterile 1250&#181;L</td>
			<td>Greiner Bio-One</td>
			<td>750 261</td>
		</tr>
		<tr>
			<td>Tips sterile 300&#181;L</td>
			<td>Greiner Bio-One</td>
			<td>738 261</td>
		</tr>
		<tr>
			<td>Tips unsterile 10&#181;L</td>
			<td>Greiner Bio-One</td>
			<td>765 290</td>
		</tr>
		<tr>
			<td>Tips unsterile 1000&#181;L</td>
			<td>Greiner Bio-One</td>
			<td>739 291</td>
		</tr>
		<tr>
			<td>Tips unsterile 200&#181;L</td>
			<td>Greiner Bio-One</td>
			<td>686 290</td>
		</tr>
		<tr>
			<td>Tweezers (Pr&#228;zisionspinzette DUMONT abgewinkelt Inox08, 5/45, 0,06 mm)</td>
			<td>Roth</td>
			<td>K343.1</td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Descosept AF</td>
			<td>Dr. Schuhmacher</td>
			<td>N-20338</td>
		</tr>
		<tr>
			<td>Ethanol 96%</td>
			<td>Nordbrand-Nordhausen</td>
			<td>410</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate (FITC)-dextran (3-5kDa)</td>
			<td>Sigma Aldrich</td>
			<td>FD4-100MG</td>
		</tr>
		<tr>
			<td>Fluorescent Mounting Medium</td>
			<td>Dako</td>
			<td>S3023</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>20847.295</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Fluka</td>
			<td>47036</td>
		</tr>
		<tr>
			<td>Tergazyme</td>
			<td>Alconox</td>
			<td>1304-1</td>
		</tr>
		<tr>
			<td>Cell culture</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>Sigma-Aldrich</td>
			<td>C5533-5MG</td>
		</tr>
		<tr>
			<td>Dexametason</td>
			<td>Sigma-Aldrich</td>
			<td>D4902</td>
		</tr>
		<tr>
			<td>DPBS (-/-)</td>
			<td>Lonza</td>
			<td>BE17-516F</td>
		</tr>
		<tr>
			<td>DPBS (+/+)</td>
			<td>Lonza</td>
			<td>BE17-513F</td>
		</tr>
		<tr>
			<td>EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>E788S</td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium</td>
			<td>Promocell</td>
			<td>C-22020</td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium supplement mix</td>
			<td>Promocell</td>
			<td>C-39225</td>
		</tr>
		<tr>
			<td>Fetal bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>E2129-10g</td>
		</tr>
		<tr>
			<td>H441</td>
			<td>ATCC</td>
			<td></td>
		</tr>
		<tr>
			<td>Human recombinant GM-CSF</td>
			<td>Peprotech</td>
			<td>300-30</td>
		</tr>
		<tr>
			<td>Lidocain</td>
			<td>Sigma-Aldrich</td>
			<td>L5647-15G</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140-122 /-163</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Gibco</td>
			<td>72400047</td>
		</tr>
		<tr>
			<td>Trypane blue stain 0.4%</td>
			<td>Invitrogen</td>
			<td>T10282</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
		</tr>
		<tr>
			<td>Primary antibodies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cadherin-5 / VE-Cadherin (goat)</td>
			<td>BD</td>
			<td>610252</td>
		</tr>
		<tr>
			<td>CD68 (rabbit)</td>
			<td>CellSignaling</td>
			<td>76437</td>
		</tr>
		<tr>
			<td>E-Cadherin (goat)</td>
			<td>R&#38;D</td>
			<td>AF748</td>
		</tr>
		<tr>
			<td>SP-A (mouse)</td>
			<td>Abcam</td>
			<td>ab51891</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AF488 (donkey anti mouse)</td>
			<td>Invitrogen</td>
			<td>R37114</td>
		</tr>
		<tr>
			<td>AF647 (donkey anti mouse)</td>
			<td>invitrogen</td>
			<td>A31571</td>
		</tr>
		<tr>
			<td>AF647 (donkey anti rabbit)</td>
			<td>Invitrogen</td>
			<td>A31573</td>
		</tr>
		<tr>
			<td>Cy3 (donkey anti goat)</td>
			<td>jackson research</td>
			<td>705-165-147</td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dilactate)</td>
			<td>Invitrogen</td>
			<td>D3571</td>
		</tr>
		<tr>
			<td>Microfluidic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chip</td>
			<td>Dynamic 42</td>
			<td>BC002</td>
		</tr>
		<tr>
			<td>Male Luer Lock (small)</td>
			<td>ChipShop</td>
			<td>09-0503-0270-09</td>
		</tr>
		<tr>
			<td>Male mini luer plugs, row of four,PP, green</td>
			<td>Microfluidic chipshop</td>
			<td>09-0558-0336-11</td>
		</tr>
		<tr>
			<td>Male mini luer plugs, row of four,PP, opaque</td>
			<td>Microfluidic chipshop</td>
			<td>09-0556-0336-09</td>
		</tr>
		<tr>
			<td>Male mini luer plugs, row of four,PP, red</td>
			<td>Microfluidic chipshop</td>
			<td>09-0557-0336-10</td>
		</tr>
		<tr>
			<td>Plugs</td>
			<td>Cole Parmer</td>
			<td>GZ-45555-56</td>
		</tr>
		<tr>
			<td>Reservoir 4.5mL</td>
			<td>ChipShop</td>
			<td>16-0613-0233-09</td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td>Dynamic 42</td>
			<td>ST001</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Tuttnauer</td>
			<td>5075 ELV</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Heracell</td>
			<td>150i</td>
		</tr>
		<tr>
			<td>Countess automated cell counter</td>
			<td>Invitrogen</td>
			<td>C10227</td>
		</tr>
		<tr>
			<td>Flowcytometer</td>
			<td>BD</td>
			<td>FACS Canto II</td>
		</tr>
		<tr>
			<td>Freezer (-20 &#176;C)</td>
			<td>Liebherr</td>
			<td>LCexv 4010</td>
		</tr>
		<tr>
			<td>Freezer (-80 &#176;C)</td>
			<td>Heraeus</td>
			<td>Herafreeze HFU 686</td>
		</tr>
		<tr>
			<td>Fridge</td>
			<td>Liebherr</td>
			<td>LCexv 4010</td>
		</tr>
		<tr>
			<td>Heraeus Multifuge</td>
			<td>Thermo Scientific</td>
			<td>X3R</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>DM IL LED</td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Heidolph</td>
			<td>Reax2000</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>REGLO Digital MS-4/12</td>
			<td>ISM597D</td>
		</tr>
		<tr>
			<td>Pipettes 10&#181;L</td>
			<td>Eppendorf Research plus</td>
			<td>3123000020</td>
		</tr>
		<tr>
			<td>Pipettes 100&#181;L</td>
			<td>Eppendorf Research plus</td>
			<td>3123000047</td>
		</tr>
		<tr>
			<td>Pipettes 1000&#181;L</td>
			<td>Eppendorf Research plus</td>
			<td>3123000063</td>
		</tr>
		<tr>
			<td>Pipettes 2.5&#181;L</td>
			<td>Eppendorf Research plus</td>
			<td>3123000012</td>
		</tr>
		<tr>
			<td>Pipettes 20&#181;L</td>
			<td>Eppendorf Research plus</td>
			<td>3123000039</td>
		</tr>
		<tr>
			<td>Pipettes 200&#181;L</td>
			<td>Eppendorf Research plus</td>
			<td>3123000055</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Sartorius</td>
			<td>6101</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Sartorius</td>
			<td>TE1245</td>
		</tr>
		<tr>
			<td>Sterile bench</td>
			<td>Kojair</td>
			<td>Biowizard SL-130</td>
		</tr>
		<tr>
			<td>Waterbath</td>
			<td>Julabo</td>
			<td>SW-20C</td>
		</tr>
		<tr>
			<td>Fluorescence Microscope Setup</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Apotome.2</td>
			<td>Zeiss</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumination device</td>
			<td>Zeiss</td>
			<td>HXP 120 C</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss</td>
			<td>Axio Observer 5</td>
		</tr>
		<tr>
			<td>Optical Sectioning</td>
			<td>Zeiss</td>
			<td>ApoTome</td>
		</tr>
		<tr>
			<td>Power Supply Microscope</td>
			<td>Zeiss</td>
			<td>Eplax Vp232</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ZEN Blue Edition</td>
			<td>Zeiss</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

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

We develop physiologically relevant in vitro models to answer mechanistic question in human infections. Our focus is on the host immune response. When we use this model to dissect the complex interaction of the pathogen to the host to identify molecular and cellular targets for therapeutic options in human infections.Working on a chip model like lung-on-chip model, balanced by logical complexity with specific research needs, offering insights into the human-host response regulation. This system mimic in vivo cellular composition and 3D structure, providing more defined conditions and high throughput than the animal experiments. In lung-on-chip technology, one of the primary challenges is selecting the right cell types that mimics lung's complex functionality for infection study results.Moreover, extending the timeframe of the experiment beyond traditional in vitro models is crucial. Leveraging is the cheap ability to maintain cell viability with continuous nutrient flow for longer observation, the experiment is also necessary. We developed the microfluidic base model of human alveolus as an effective tool to mimic human alveolar environment.It was achieved by applying perfusion to mimic blood flow, and also by a mechanical stimulation to the endothelial cells. And also integrating air-liquid interface for the epithelial cells by exposing them to the air. The next step is to extend this model to a more advanced platform, such as induced pluripotent stem cell-based model.By doing so, we aim to bring personalized medicine closer to application, particularly in the context of antiviral drug testing, and as a tool for biomedical research and pharmaceutical development.

An examination of morphological alterations and the expression of marker proteins could be performed using immunofluorescence staining. After co-culturing for 14 days, the vascular and epithelial sides are analyzed for expression of respective cell markers. This method is useful for studying the interactions and integrity of vascular and epithelial components, which is essential for disease modeling as a functional biological readout related to infection. Immunofluorescence staining could be supported by quantifying anal...

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 ...

immunocompetent-alveolus-on-chip-model-for-studying-alveolar-mucosal