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.

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>

M.R. is CEO of Dynamic42 GmbH and holds equity in the company. A.S.M. is a scientific advisor to Dynamic 42 GmbH and holds equity in the company.

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.

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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&amp;auml;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>Descosept 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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&amp;amp;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&amp;auml;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.

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...

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

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.

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 ...

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

Research

JoVE Journal

Immunology and Infection

1.4K Views.  Jena University Hospital. Ons onderzoek richt zich op de ontwikkeling en het gebruik van immunocompetente organ-on-chip platforms. Door deze fysiologisch relevante, gehumaniseerde in vitro modellen te creëren, zijn we in staat om de complexe interactie tussen gastheer en ziekteverwekker te bestuderen. De kennis die we creëren, kan worden gebruikt om moleculaire doelen voor de therapie van infectieziekten te manipuleren en te identificeren.Een belangrijke uitdaging is het in stand houden van een levende microb...

Video: Auteur in de schijnwerpers: ontwikkeling van immunocompetente orgaan-op-chip-modellen voor onderzoek naar infectieziekten

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Bioengineering

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.
Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand&#160;as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.
Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure&#160;for the in vitro modeling of interferon gamma (IFN&#947;)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, ...

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

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

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

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

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

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

Establishment of 3D Morphogenesis in a Canine IBD Gut-on-a-Chip System

Three-Dimensional Morphogenesis in Canine Gut-on-a-Chip Using Intestinal Organoids Derived from Inflammatory Bowel Disease Patients

Canine intestines possess similarities in anatomy, microbiology, and physiology to those of humans, and dogs naturally develop spontaneous intestinal disorders similar to humans. Overcoming the inherent limitation of three-dimensional (3D) organoids in accessing the apical surface of the intestinal epithelium has led to the generation of two-dimensional (2D) monolayer cultures, which expose the accessible luminal surface using cells derived from the organoids. The integration of these organoids and organoid-derived monolayer cultures into a microfluidic Gut-on-a-Chip system has further evolved the technology, allowing for the development of more physiologically relevant dynamic in vitro intestinal models.
In this study, we present a protocol for generating 3D morphogenesis of canine intestinal epithelium using primary intestinal tissue samples obtained from dogs affected by inflammatory bowel disease (IBD). We also outline a protocol for generating and maintaining 2D monolayer cultures and intestine-on-a-chip systems using cells derived from the 3D intestinal organoids. The protocols presented in this study serve as a foundational framework for establishing a microfluidic Gut-on-a-Chip system specifically designed for canines. By laying the groundwork for this innovative approach, we aim to expand the application of these techniques in biomedical and translational research, aligning with the principles of the One Health Initiative. By utilizing this approach, we can develop more physiologically relevant dynamic in vitro models for studying intestinal physiology in both dogs and humans. This has significant implications for biomedical and pharmaceutical applications, as it can aid in the development of more effective treatments for intestinal diseases in both species.

Author Spotlight: Development and Application of a Canine IBD Gut-on-a-Chip Model for 3D Intestinal Morphogenesis Studies 

Integration of canine intestinal organoids and a Gut-on-a-Chip microfluidic system offers relevant translational models for human intestinal diseases. Protocols presented allow for 3D morphogenesis and dynamic in vitro modeling of the gut, aiding in the development of effective treatments for intestinal diseases in dogs and humans with One Health.

Canine intestines possess similarities in anatomy, microbiology, and physiology to those of humans, and dogs naturally develop spontaneous intestinal ...

Medicine

A Gut-on-a-Chip Model to Study the Gut Microbiome-Nervous System Axis

The human body is colonized by at least the same number of microbial cells as it is composed of human cells, and most of these microorganisms are located in the gut. Though the interplay between the gut microbiome and the host has been extensively studied, how the gut microbiome interacts with the enteric nervous system remains largely unknown. To date, a physiologically representative in vitro model to study gut microbiome-nervous system interactions does not exist. 
To fill this gap, we further developed the human-microbial crosstalk (HuMiX) gut-on-chip model by introducing induced pluripotent stem cell-derived enteric neurons into the device. The resulting model, 'neuroHuMiX', allows for the co-culture of bacterial, epithelial, and neuronal cells across microfluidic channels, separated by semi-permeable membranes. Despite separation of the different cell types, the cells can communicate with each other through soluble factors, simultaneously providing an opportunity to study each cell type separately. This setup allows for first insights into how the gut microbiome affects the enteric neuronal cells. This is a critical first step in studying and understanding the human gut microbiome-nervous system axis.

neuroHuMiX is an advanced gut-on-a-chip model to study the interactions of bacterial, epithelial, and neuronal cells under proximal and representative co-culture conditions. This model allows unravelling of the molecular mechanisms underlying the communication between the gut microbiome and the nervous system.

The human body is colonized by at least the same number of microbial cells as it is composed of human cells, and most of these microorganisms are located ...

Biology

Construction of a 3D Intestinal Mucosa Model with Paraffin Embedding

Basic Three-Dimensional (3D) Intestinal Model System with an Immune Component

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the pharmacological screening of potentially beneficial substances, and for toxicity studies on potentially harmful food components. Of relevance, there is a current demand for the development of cell-based in vitro models to substitute animal models. Here, a protocol for a basic, &#8220;healthy tissue&#8221; three-dimensional (3D) intestinal equivalent model using cell lines is presented with the dual benefit of providing both experimental simplicity (standardized and easily repeatable system) and physiological complexity (Caco-2 enterocytes with a supporting immune component of U937 monocytes and L929 fibroblasts). The protocol also includes paraffin embedding for light microscopic evaluation of fixed intestinal equivalents, thereby providing the advantage of analyzing multiple visual parameters from a single experiment. Hematoxylin and eosin (H&#38;E) stained sections showing the Caco-2 columnar cells forming a tight and regular monolayer in control treatments are used to verify the efficacy of the model as an experimental system. Using gluten as a pro-inflammatory food component, parameters analyzed from sections include reduced monolayer thickness, as well as disruption and detachment from the underlying matrix (H&#38;E), decreased tight junction protein expression as shown from occludin staining (quantifiable statistically), and immune-activation of migrating U937 cells as evidenced from the cluster of differentiation 14 (CD14) staining and CD11b-related differentiation into macrophages. As shown by using lipopolysaccharide to simulate intestinal inflammation, additional parameters that can be measured are increased mucus staining and cytokine expression (such as midkine) that can be extracted from the medium prior to fixation. The basic three-dimensional (3D) intestinal mucosa model and fixed sections can be recommended for inflammatory status and barrier integrity studies with the possibility of analyzing multiple visual quantifiable parameters.

Author Spotlight: Investigating the Effects of Compounds on Intestinal Tissue Using 3D Human Cell Line Models 

Here we describe constructing a basic three-dimensional (3D) intestinal cell line model system and a paraffin embedding protocol for light microscopic evaluation of fixed intestinal equivalents. Staining of selected proteins permits the analysis of multiple visual parameters from a single experiment for potential use in preclinical drug screening studies.

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the ...

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

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