The work was financially supported by the Collaborative Research Center PolyTarget 1278 (project number 316213987) to V.D.W. and A.S.M. A.F. and A.S.M. further acknowledge financial support by the Cluster of Excellence &#34;Balance of the Microverse&#34; under Germany&#39;s Excellence Strategy - EXC 2051 - Project-ID 690 390713860. We want to acknowledge Astrid Tannert and the Jena Biophotonic and Imaging Laboratory (JBIL) for providing us access to their confocal laser scanning microscope ZEISS LSM980. Figure 1C&#160;and Figure 2 were created with Biorender.com.

This protocol requires access to ~20 mL of fresh blood per biochip from healthy donors to isolate primary human monocytes. All donors gave written, informed consent to participate in this study, which was approved by the ethics committee of the University Hospital Jena (permission number 2018-1052-BO). For details about the materials, refer to the Table of Materials. For details about the composition of all solutions and media, refer to Table 1.
1. Gener...

Enhanced Intestine-on-Chip Platform for Immunological Insight

<ol>
	<li>Alonso-Roman, R., 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=Organ-on-chip+models+for+infectious+disease+research.">Organ-on-chip models for infectious disease research.</a> Nat Microbiol. 9 (4), 891-904 (2024).</li><li>Fahrner, R., Groger, M., Settmacher, U., Mosig, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+integration+of+natural+killer+cells+in+a+microfluidically+perfused+liver+on-a-chip+model.">Functional integration of natural killer cells in a microfluidically perfused liver on-a-chip model.</a> BMC Res Notes. 16 (1), 285 (2023).</li><li>Raasch, 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=Microfluidically+supported+biochip+design+for+culture+of+endothelial+cell+layers+with+improved+perfusion+conditions.">Microfluidically supported biochip design for culture of endothelial cell layers with improved perfusion conditions.</a> Biofabrication. 7 (1), 015013 (2015).</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>Kaden, T., 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=Generation+&amp;+characterization+of+expandable+human+liver+sinusoidal+endothelial+cells+and+their+application+to+assess+hepatotoxicity+in+an+advanced+in+vitro+liver+model.">Generation &amp; characterization of expandable human liver sinusoidal endothelial cells and their application to assess hepatotoxicity in an advanced in vitro liver model.</a> Toxicology. 483, 153374 (2023).</li><li>Maurer, 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=A+three-dimensional+immunocompetent+intestine-on-chip+model+as+in+vitro+platform+for+functional+and+microbial+interaction+studies.">A three-dimensional immunocompetent intestine-on-chip model as in vitro platform for functional and microbial interaction studies.</a> Biomaterials. 220, 119396 (2019).</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>Kaden, T., 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=Modeling+of+intravenous+caspofungin+administration+using+an+intestine-on-chip+reveals+altered+Candida+albicans+microcolonies+and+pathogenicity.">Modeling of intravenous caspofungin administration using an intestine-on-chip reveals altered Candida albicans microcolonies and pathogenicity.</a> Biomaterials. 307, 122525 (2024).</li><li>Shah, P., 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+microfluidics-based+in+vitro+model+of+the+gastrointestinal+human-microbe+interface.">A microfluidics-based in vitro model of the gastrointestinal human-microbe interface.</a> Nat Commun. 7, 11535 (2016).</li><li>Jaffe, E. A., Nachman, R. L., Becker, C. G., Minick, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+human+endothelial+cells+derived+from+umbilical+veins.+Identification+by+morphologic+and+immunologic+criteria.">Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria.</a> J Clin Invest. 52 (11), 2745-2756 (1973).</li><li>Mosig, 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=Different+functions+of+monocyte+subsets+in+familial+hypercholesterolemia:+Potential+function+of+cd14++cd16++monocytes+in+detoxification+of+oxidized+ldl.">Different functions of monocyte subsets in familial hypercholesterolemia: Potential function of cd14+ cd16+ monocytes in detoxification of oxidized ldl.</a> FASEB J. 23 (3), 866-874 (2009).</li><li>Peterson, M., Mooseker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+enterocyte-like+brush+border+cytoskeieton+of+the+c2bbe+clones+of+the+human+intestinal+cell+line,+caco-2.">Characterization of the enterocyte-like brush border cytoskeieton of the c2bbe clones of the human intestinal cell line, caco-2.</a> J Cell Sci. 102, 581-600 (1992).</li><li>Shin, W., Hinojosa, C. D., Ingber, D. E., Kim, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+intestinal+morphogenesis+controlled+by+transepithelial+morphogen+gradient+and+flow-dependent+physical+cues+in+a+microengineered+gut-on-a-chip.">Human intestinal morphogenesis controlled by transepithelial morphogen gradient and flow-dependent physical cues in a microengineered gut-on-a-chip.</a> iScience. 15, 391-406 (2019).</li><li>Kim, H. J., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut-on-a-chip+microenvironment+induces+human+intestinal+cells+to+undergo+villus+differentiation.">Gut-on-a-chip microenvironment induces human intestinal cells to undergo villus differentiation.</a> Integr Biol (Camb). 5 (9), 1130-1140 (2013).</li><li>Kim, H. J., Huh, D., Hamilton, G., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+gut-on-a-chip+inhabited+by+microbial+flora+that+experiences+intestinal+peristalsis-like+motions+and+flow.">Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow.</a> Lab Chip. 12 (12), 2165-2174 (2012).</li><li>Karra, N., Fernandes, J., James, J., Swindle, E. J., Morgan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+membrane+properties+on+cell+growth+in+an+'airway+barrier+on+a+chip'.">The effect of membrane properties on cell growth in an 'airway barrier on a chip'.</a> Organs-on-a-Chip. 5, 10025 (2023).</li></ol>

The presented protocol details the necessary steps for generating an immunocompetent intestine-on-chip model. We described specific techniques and possible readout methods such as immunofluorescence microscopy, cytokine and metabolite analysis, flow cytometry, protein and genetic analysis, and permeability measurement.
The described model consists of primary HUVECs, monocyte-derived macrophages, and monocyte-derived dendritic cells co-cultured with a 3D layer of intestinal epithelial cells rep...

Organ-on-Chip (OoC) systems represent an emerging technique of 3D cell culture that is capable of bridging the gap between conventional 2D cell culture and animal models. OoC platforms typically consist of one or more compartments containing tissue-specific cells grown on a wide range of scaffolds such as membranes or hydrogels1. The models are capable of mimicking one or more defined organotypic functions. Pumps enable continuous microfluidic perfusion of cell culture medium for removal of cellular waste products, supply with nutrition and growth factors for improved cellular differentiation, and recreating essential in vivo conditions. With the integration of immune cells, OoC systems can mimic human immune response in vitro2. To date, a wide range of organs and functional units have been presented1. These systems include models of the vasculature3, lung4, liver2,5, and intestine6 that can be facilitated for drug testing5,7 and infection studies6,8.
We here present a human intestine-on-chip model integrating human epithelial cells forming an organotypic 3D topography of villus-like and crypt-like structures combined with an endothelial lining and tissue-resident macrophages. The model is cultured in a microfluidically perfused biochip in the format of a microscopic slide.&#160;Each biochip consists of two separate microfluidic cavities. Each cavity is divided by a porous polyethylene terephthalate (PET) membrane into an upper and lower chamber. The membrane itself also serves as the scaffold for the cells to grow on each side. The pores of the membrane enable cellular crosstalk and cell migration between cell layers. Each chamber can be accessed by two female luer lock-sized ports. Optionally, an additional mini-luer lock-sized port can provide access to the upper or lower chamber (Figure 1).
The OoC platform offers a number of readouts that can be obtained from a single experiment. The intestine-on-chip is tailored towards combining perfused 3D cell culture, effluent analysis, and fluorescence microscopy to assess cell marker expression, metabolization rates, immune response, microbial colonization and infection, and barrier function3,6,8. The model includes tissue-resident immune cells and direct contact of living microorganisms with the host tissue, which is a benefit compared to other published models9. Further, epithelial cells self-organize into three-dimensional structures that provide a physiologically relevant interface for the colonization with a living microbiota6.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well plate black, clear bottom</td>
			<td>Thermo Fisher</td>
			<td>10000631</td>
			<td>Consumables</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Roth</td>
			<td>3738.4</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 AffiniPure, donkey, anti-mouse IgG (H+L)</td>
			<td>Jackson Immuno Research</td>
			<td>715-545-150</td>
			<td>Secondary Antibody Vascular Staining and Epithelial Staining</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 AffiniPure, donkey, anti-rabbit IgG (H+L)</td>
			<td>Jackson Immuno Research</td>
			<td>711-605-152</td>
			<td>Secondary Antibody Epithelial Staining</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647, donkey, anti-rabbit IgG (H+L)</td>
			<td>Thermo Fisher Scientific, Invitrogen</td>
			<td>A31573</td>
			<td>Secondary Antibody Vascular Staining</td>
		</tr>
		<tr>
			<td>Axiocam ERc5s camera</td>
			<td>Zeiss</td>
			<td>426540-9901-000</td>
			<td>Technical equipment</td>
		</tr>
		<tr>
			<td>Basal Medium MV, phenol red-free</td>
			<td>Promocell</td>
			<td>C-22225</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>Biochip</td>
			<td>Dynamic 42</td>
			<td>BC002</td>
			<td>Microfluidic consumables</td>
		</tr>
		<tr>
			<td>BSA fraction V</td>
			<td>Gibco</td>
			<td>15260-037</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>C2BBe1 (clone of Caco-2)</td>
			<td>ATCC</td>
			<td>CRL-2102</td>
			<td>Epithelial Cell Source</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>C2432</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Heracell</td>
			<td>150i</td>
			<td>Technical equipment</td>
		</tr>
		<tr>
			<td>Collagen IV from human placenta</td>
			<td>Sigma-Aldrich</td>
			<td>C5533</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>Coverslips (24 x 40 mm; #1.5)</td>
			<td>Menzel-Gl&#228;ser</td>
			<td>15747592</td>
			<td>Consumables</td>
		</tr>
		<tr>
			<td>Cy3 AffiniPure, donkey, anti-goat IgG (H+L)</td>
			<td>Jackson Immuno Research</td>
			<td>705-165-147</td>
			<td>Secondary Antibody Vascular Staining</td>
		</tr>
		<tr>
			<td>Cy3 AffiniPure, donkey, anti-rat IgG (H+L)</td>
			<td>Jackson Immuno Research</td>
			<td>712-165-150</td>
			<td>Secondary Antibody Epithelial Staining</td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidin-2-phenylindol, Dilactate)</td>
			<td>Thermo Fisher Scientific, Invitrogen</td>
			<td>D3571</td>
			<td>Vascular and Epithelial Staining</td>
		</tr>
		<tr>
			<td>Descosept PUR</td>
			<td>Dr.Schuhmacher</td>
			<td>00-323-100</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Gibco</td>
			<td>41965-062</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>DMEM high glucose w/o phenol red</td>
			<td>Gibco</td>
			<td>31053028</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>DPBS (-/-)</td>
			<td>Gibco</td>
			<td>14190-169</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>DPBS (+/+)</td>
			<td>Gibco</td>
			<td>14040-133</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>EDTA solution</td>
			<td>Invitrogen</td>
			<td>15575-038</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium</td>
			<td>Promocell</td>
			<td>C-22020</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium supplement mix</td>
			<td>Promocell</td>
			<td>C-39225</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>Ethanol 96%, undenatured</td>
			<td>Nordbrand-Nordhausen</td>
			<td>410</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Fetal bovine Serum</td>
			<td>invitrogen</td>
			<td>10270106</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate (FITC)-dextran (3-5 kDa)</td>
			<td>Sigma Aldrich</td>
			<td>FD4-100MG</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Fluorescent Mounting Medium</td>
			<td>Dako</td>
			<td>S3023</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Gentamycin (10mg/mL)</td>
			<td>Sigma Aldrich</td>
			<td>G1272</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement (100x)</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>Histopaque</td>
			<td>Sigma-Aldrich</td>
			<td>10771</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>Hoechst (bisBenzimid) H33342</td>
			<td>Sigma-Aldrich</td>
			<td>14533</td>
			<td>Epithelial Staining</td>
		</tr>
		<tr>
			<td>Holotransferrin (5mg/mL) Transferrin, Holo, Human Plasma</td>
			<td>Millipore</td>
			<td>616397</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>Human recombinant GM-CSF</td>
			<td>Peprotech</td>
			<td>300-30</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>Human recombinant M-CSF</td>
			<td>Peprotech</td>
			<td>300-25</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>Illumination device</td>
			<td>Zeiss</td>
			<td>HXP 120 C</td>
			<td>Fluorescence Microscope Setup</td>
		</tr>
		<tr>
			<td>Laser Scanning Microscope</td>
			<td>Zeiss</td>
			<td>CLSM980</td>
			<td>Fluorescence Microscope Setup</td>
		</tr>
		<tr>
			<td>Lidocain hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>L5647</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>Lipopolysaccharide (LPS)</td>
			<td>Sigma</td>
			<td>L2630</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>Loftex Wipes</td>
			<td>Loftex</td>
			<td>1250115</td>
			<td>Consumables</td>
		</tr>
		<tr>
			<td>Low attachment tubes (PS, 5 mL)</td>
			<td>Falcon</td>
			<td>352052</td>
			<td>Consumables</td>
		</tr>
		<tr>
			<td>Luer adapter for the top cap (M)</td>
			<td>Mo Bi Tec</td>
			<td>M3003</td>
			<td>Microfluidic consumables</td>
		</tr>
		<tr>
			<td>Male mini luer plugs, row of four,PP, opaque</td>
			<td>Microfluidic chipshop</td>
			<td>09-0556-0336-09</td>
			<td>Microfluidic consumables</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution</td>
			<td>Gibco</td>
			<td>11140</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Roth</td>
			<td>8388.2</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss</td>
			<td>Axio Observer 5</td>
			<td>Fluorescence Microscope Setup</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Menzel</td>
			<td>MZ-0002</td>
			<td>Consumables</td>
		</tr>
		<tr>
			<td>Monoclonal, mouse, anti-human CD68 Antibody (KP1)</td>
			<td>Thermo Fisher Scientific, Invitrogen</td>
			<td>14-0688-82</td>
			<td>Primary Antibody Vascular Staining</td>
		</tr>
		<tr>
			<td>Monoclonal, rat, anti-human E-Cadherin antibody (DECMA-1)</td>
			<td>Sigma-Aldrich, Millipore</td>
			<td>MABT26</td>
			<td>Primary Antibody Epithelial Staining</td>
		</tr>
		<tr>
			<td>Multiskan Go plate reader</td>
			<td>Thermo Fisher</td>
			<td>51119300</td>
			<td>Technical equipment</td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Biozol</td>
			<td>LIN-END9010-10</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Optical Sectioning</td>
			<td>Zeiss</td>
			<td>ApoTome</td>
			<td>Fluorescence Microscope Setup</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>Plugs</td>
			<td>Cole Parmer</td>
			<td>GZ-45555-56</td>
			<td>Microfluidic consumables</td>
		</tr>
		<tr>
			<td>Polyclonal, goat, anti-human VE-Cadherin Antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF938</td>
			<td>Primary Antibody Vascular Staining</td>
		</tr>
		<tr>
			<td>Polyclonal, rabbit, anti-human Von Willebrand Factor Antibody</td>
			<td>Dako</td>
			<td>A0082</td>
			<td>Primary Antibody Vascular Staining</td>
		</tr>
		<tr>
			<td>Polyclonal, rabbit, anti-human ZO-1 antibody</td>
			<td>Thermo Fisher Scientific, Invitrogen</td>
			<td>61-7300</td>
			<td>Primary Antibody Epithelial Staining</td>
		</tr>
		<tr>
			<td>Power Supply Microscope</td>
			<td>Zeiss</td>
			<td>Eplax Vp232</td>
			<td>Fluorescence Microscope Setup</td>
		</tr>
		<tr>
			<td>Primovert microscope</td>
			<td>Zeiss</td>
			<td>415510-1101-000</td>
			<td>Technical equipment</td>
		</tr>
		<tr>
			<td>Reglo ICC peristaltic pump</td>
			<td>Ismatec</td>
			<td>ISM4412</td>
			<td>Technical equipment</td>
		</tr>
		<tr>
			<td>SAHA (Vorinostat)</td>
			<td>Sigma Aldrich</td>
			<td>SML0061-25MG</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Fluka</td>
			<td>47036</td>
			<td>Chemicals</td>
		</tr>
		<tr>
			<td>S-Monovette, 7.5 mL Z-Gel</td>
			<td>Sarstedt</td>
			<td>01.1602</td>
			<td>Consumables</td>
		</tr>
		<tr>
			<td>S-Monovette, 9.0 mL K3E</td>
			<td>Sarstedt</td>
			<td>02.1066.001</td>
			<td>Consumables</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11360-088</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>Tank 4.5 mL</td>
			<td>ChipShop</td>
			<td>10000079</td>
			<td>Microfluidic consumables</td>
		</tr>
		<tr>
			<td>Trypane blue stain 0.4%</td>
			<td>Invitrogen</td>
			<td>T10282</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>11538876</td>
			<td>Cell culture consumables</td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td>Dynamic 42</td>
			<td>ST001</td>
			<td>Microfluidic consumables</td>
		</tr>
		<tr>
			<td>Tweezers (Pr&#228;zisionspinzette DUMONT abgewinkelt Inox08, 5/45, 0,06 mm)</td>
			<td>Roth</td>
			<td>K343.1</td>
			<td>Consumables</td>
		</tr>
		<tr>
			<td>Wheat Germ Agglutinin (WGA)</td>
			<td>Thermo Fisher Scientific, Invitrogen</td>
			<td>W32464</td>
			<td>Epithelial Staining</td>
		</tr>
		<tr>
			<td>X-VIVO 15</td>
			<td>Lonza</td>
			<td>BE02-060F</td>
			<td>Cell culture consumables, Hematopoietic cell medium</td>
		</tr>
		<tr>
			<td>Zellkultur Multiwell Platten, 24 Well, sterile</td>
			<td>Greiner Bio-One</td>
			<td>662 160</td>
			<td>Consumables</td>
		</tr>
		<tr>
			<td>Zellkultur Multiwell Platten, 6 Well, sterile</td>
			<td>Greiner Bio-One</td>
			<td>657 160</td>
			<td>Consumables</td>
		</tr>
		<tr>
			<td>Zen Blue Software</td>
			<td>Zeiss</td>
			<td>Version 3.7</td>
			<td>Microscopy Software</td>
		</tr>
	</tbody>
</table>

immunocompetent intestine-on-chip model for analyzing gut mucosal immune responses

An advanced intestine-on-chip model recreating epithelial 3D organotypic villus-like and crypt-like structures has been developed. The immunocompetent model includes Human Umbilical Vein Endothelial Cells (HUVEC), Caco-2 intestinal epithelial cells, tissue-resident macrophages, and dendritic cells, which self-organize within the tissue, mirroring characteristics of the human intestinal mucosa. A unique aspect of this platform is its capacity to integrate circulating human primary immune cells, enhancing physiological relevance. The model is designed to investigate the intestinal immune system&#39;s response to bacterial and fungal colonization and infection. Due to its enlarged cavity size, the model offers diverse functional readouts such as permeation assays, cytokine release, and immune cell infiltration, and is compatible with immunofluorescence measurement of 3D structures formed by the epithelial cell layer. It hereby provides comprehensive insights into cell differentiation and function. The intestine-on-chip platform has demonstrated its potential in elucidating complex interactions between surrogates of a living microbiota and human host tissue within a microphysiological perfused biochip platform.

Author Spotlight: Developing Immunocompetent Organ-on-Chip Models for Infectious Disease Research

Immunocompetent Intestine-on-Chip Model for Analyzing Gut Mucosal Immune Responses

Our research focuses on the development and use of immunocompetent organ-on-chip platforms. By creating these physiologically relevant, humanized in vitro models, we are able to study the complex host-pathogen interaction. The knowledge we create can be used to manipulate and identify molecular targets for the therapy of infectious diseases.One key challenge is maintaining a living microbiota under homeostatic conditions alongside host tissue in vitro. This includes replicating the organotypic 3D topography of crypt and villus structures and their microbial colonization. Intestine-on-chip platforms address this by using flow to shape the organotypic structures and prevent bacterial overgrowth.This ensures stable culture conditions. Compared to other available intestine-on-chip platforms, our model enables the detailed study of the human immune response to pathogens due to the inclusion of primary tissue-resident immune cells. Additionally, the model size enables multiple readouts in one single experiment, reducing the overall cost and number of experiments.Our laboratory is focusing on using induced pluripotent stem cells to develop autologous systems that enable personalized medicine.

These representative results show the distinct tissue layers of the intestine-on-chip model. They are immunofluorescent stained as described in protocol section 11. The images were taken with an epifluorescence or confocal fluorescence microscope as z-stacks and processed to an orthogonal projection. See the Table of Materials for details about the microscopical setup and software. Figure 5 shows the vascular layer, a barrier-forming endothelial monolayer, consisting of HUVE...

Our detailed protocol outlines the creation and use of the advanced intestine-on-chip model, which simulates human intestinal mucosa with 3D structures and various cell types, enabling in-depth analysis of immune responses and cellular functions in response to microbial colonization.

An advanced intestine-on-chip model recreating epithelial 3D organotypic villus-like and crypt-like structures has been developed. The immunocompetent ...

immunocompetent-intestine-on-chip-model-for-analyzing-gut-mucosal

Research

JoVE Journal

Immunology and Infection

Harvesting and Seeding of Human Umbilical Vein Endothelial Cells (HUVECs) for Human Intestine-on-Chip Model

Microfluidic Perfusion of HUVEC and C2BBe1 Cultured Biochips for Analyzing Immune Responses to Microbial Colonization

671 Views.  Jena University Hospital.  Our detailed protocol outlines the creation and use of the advanced intestine-on-chip model, which simulates human intestinal mucosa with 3D structures and various cell types, enabling in-depth analysis of immune responses and cellular functions in response to microbial colonization.

Enhanced Intestine-on-Chip Platform for Immunological Insight

Summary