The authors would like to thank Dr. Jared Sterneckert for providing us with the cells from the K7 line. We also want to thank the long-standing collaborators Dr. Frederic Zenhausern and Matthew W. Barret from the University of Arizona for their assistance with the engineering aspects. We would also like to acknowledge Dr. Valentina Galata for her help in designing the schematic representation of neuroHuMiX. This project has received funding from the European Research Council (ERC) under the European Union&#39;s Horizon 2020 research and innovation program (grant agreement 863664). Figure 1 was partially created with Biorender.com.

<ol>
	<li>Heintz-Buschart, A., Wilmes, P. <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+microbiome:+function+matters.">Human gut microbiome: function matters.</a> Trends in Microbiology. 26 (7), 563-574 (2018).</li><li>Toor, 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=Dysbiosis+disrupts+gut+immune+homeostasis+and+promotes+gastric+diseases.">Dysbiosis disrupts gut immune homeostasis and promotes gastric diseases.</a> International Journal of Molecular Sciences. 20 (10), 2432 (2019).</li><li>Braak, H., de Vos, R. A. 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P.W. declares being listed as an inventor on patents PCT/EP2013/056607, PCT/EP2016/062024, PCT/US2017/061602, and PCT/EP2019/081424. P.W., C.S., and L.G. declare being listed as inventors on patent LU503075.

It is now established that the human gut microbiome influences the host&#39;s health and disease. Despite the knowledge suggesting the importance of our microbiome, especially in neurological disorders such as Alzheimer&#39;s or Parkinson&#39;s disease3,13, it remains largely unknown how the gut microbiome interacts with the enteric nervous system, and subsequently, with the brain.
A representative model to study the interactions between the gut microbiome and the nervous system has thus far been unavailable. Studies regarding the gut-brain axis have traditionally been performed using murine models13. Mice and humans share 85% of their genomic sequences14, but there are significant differences to consider when comparing mice to humans. Regarding the gut, it is important to note that, compared to humans, mice are exclusively herbivores. As a result, their gastrointestinal tract differs in length and characteristics, such as the &#39;gastric emptying&#39;14. Murine brains also show important differences, whereby the overall structure between mice and humans are different15. Importantly, humans have longer cell cycle times of neural progenitors15. Consequently, it is important to develop representative models that include human-derived cells, including intestinal and neuronal cells5. In this context, the development of more reproducible research viain vitro models reduces the need to use animal models and improves reproducibility.
neuroHuMiX is an advanced version of the previous HuMiX model9. HuMiX is a gut-on-a-chip model allowing proximal and representative co-cultures of epithelial and bacterial cells. Cell-cell communication is possible through the proximal co-culture and diffusion of secreted factors and metabolites via semipermeable membranes. However, to expand the utility of the initial device to study the human gut environment, the introduction of an additional cell type is required. To address this, neuroHuMiX, developed with the introduction of iPSC-derived ENs, enables a proximal co-culture of bacteria, intestinal epithelial cells, and ENs. The resulting in vitro model allows us to address questions regarding the human gut microbiome in relation to the human nervous system. Co-culturing different cell types, especially co-cultures of mammalian cells and bacteria, has several challenges, including the loss of viability, poor adhesion, and overall loss in confluence16. Here, we have demonstrated that within this device, we are able to co-culture three different cell types within the same system while keeping the cell viability high.
A critical step in the protocol is to ensure confluency of the neuronal cells-80%-90% cell confluency and viability-before inoculating into the device. Since it is not possible to assess the cell growth during the run, it is of utmost importance to ensure the cells are confluent and growing well before introducing them in the model. While this may be a limiting factor, the overall viability and confluency observed within the device is generally high.
The device is connected via tubing lines to a peristaltic pump. Each cell chamber has its specific tubing line. The tubing comprises a pump tubing that allows the use of a peristaltic pump for the perfusion of medium, as well as tubing connecting the pump tubing to the device and tubing connecting the device to the outflow/waste bottles. Sampling ports are included before and after the device, to allow the inoculation and sampling of outflow medium. Each chamber can be connected to a different medium, allowing the best culture conditions for each individual cell type. Each chamber can be opened or closed depending on the specific needs for medium supply. In the device, the neuronal chamber stays closed for most of the experiment, while the bacterial and epithelial chambers are open all the time, meaning they get fresh medium throughout the whole experimental run. To make sure the medium is flowing without interruption, it is crucial to not have any air left in the tubings, connectors, or in the device. Therefore, it is important to first let the devices run for a few minutes at the priming step. This often resolves the issue. If not, one of the other lines that are dropping can be closed for a short amount of time by closing the three-way stopcock of the outflow. This redirects the medium to the line with the air bubble, thus resolving the issue by pushing the bubble outward through the tubing.
For any cell culture experiment, the medium is a key component, where each cell type has its respective medium. In a co-culture setup, the medium needs to be compatible not only for the cell type growing in it, but also for the other cell types within the co-culture. This is no different for the device, which poses an additional challenge as we have three different compartments with three different cell types inside-bacterial, epithelial, and neuronal cells. We have, however, shown that by modifying the bacterial media-with the addition of 5% MRS to RPMI 1640 with 10% FBS-all cell types, in particular bacterial and epithelial cells, can be successfully co-cultured within the system. However, in the device, different cell types are co-cultured in proximity, and are hence not in direct contact with one another. Even though this is not fully representative of the direct contact between cells in the human gut, and therefore a limitation, the proximal and representative co-culture condition is a strength for downstream analyses. Soluble factors exchange between the different chambers and cell types; hence, the cells are still interacting with each other. Additionally, the fact that the cell types can be harvested and analyzed separately allows us to study the effect of a healthy and/or diseased microbiome on different cell types (including neuronal cells) and thereby determine/retrieve cell type-specific read-outs. Another limitation is that the morphology of the cells cannot be followed-up during the experimental run, as the device can only be opened and the cells checked at the end of each experiment.
To our knowledge, neuroHuMiX is the first gut-on-a-chip model including ENs. This is a step toward elucidating the communication between the gut microbiota and the enteric nervous system. It is a model allowing investigation of the interplay between a bacterial species, an epithelial layer, and ENs. Its design allows us to study the exchange of soluble factors secreted by the different cell types and their effect on one another. Going forward, it would be important to not only have iPSC-derived ENs, but also iPSC-derived epithelial cells inside the device, to transition the device into a personalized model. Importantly, this personalized model could be used to test pre-, pro-, and synbiotics10,11 and potentially develop personalized screening and therapeutic approaches17. Personalized neuroHuMiX could eventually shed light on the &#39;dark matter&#39; of the human gut microbiome and its interactions with the nervous system along the gut microbiome-nervous system axis, paving the way for therapeutic assessment and interventions.
We can conclude that being able to have a gut-on-a-chip including the enteric neuronal system is crucial to progressing in the study and understanding of interactions along the gut microbiome-nervous system axis. NeuroHuMiX allows us to study the effects of bacterial species on host cells and provides us with a good basis to improve the model even further in an even more physiologically representative way.

The human gut microbiome plays a crucial role in human health and disease. It has been extensively studied over the past decade and a half, and its potential role in modulating health and disease is now established1. A disruption in the microbiome leading to an unbalanced microbial community (dysbiosis) has been postulated to be involved in the pathogenesis of many chronic disorders, such as obesity, inflammatory bowel disease, and colorectal cancer, or even neurodegenerative diseases such as Parkinson&#39;s disease2,3.
Although the human gut microbiome has been associated with neurological conditions, it is still unclear how the gut microbiome communicates with and affects the enteric nervous system. As the human enteric nervous system is not easily accessible for immediate study, animal models have been used in experiments so far4. However, given the apparent differences between animal models and humans5, the development of in vitro models mimicking the human gut is of immediate interest. In this context, the burgeoning and advancing field of human induced pluripotent stem cells (iPSCs) has allowed us to obtain representative enteric neurons (ENs)6. iPSC-derived ENs allow for the study of the enteric nervous system in in vitro culture models, such as cell culture inserts, organoids, or organs-on-a-chip7,8.
The human-microbial crosstalk (HuMiX) model is a gut-on-a-chip model mimicking the human gut9. The initial HuMiX model (hereafter referred to as the initial device) accommodated epithelial cells (Caco-2) and bacterial cells10,11. However, to study the gut microbiome-nervous system link, iPSC-derived ENs6 have also been introduced in the system (Figure 1). The proximal co-culture of neuronal, epithelial, and bacterial cells allows the analysis of the different cell types individually and study of the interactions between the different cell types in an environment mimicking that of the human gut.
In recent years, advances have been made in the development of models to study organs in more physiologically representative ways by using organs-on-a-chip (e.g., gut-on-a-chip) models. These models are more representative of the human gut environment due to constant nutrient supply and waste removal, as well as real-time monitoring of, for example, oxygen levels or barrier integrity8,12. These models specifically allow study of the effects of gut bacteria on host cells. However, to be able to use organs-on-a-chip to study the interrelationships between the gut microbiome and the nervous system, neuronal cells need to be integrated into such systems. Therefore, the aim of further developing HuMiX and establishing the neuroHuMiX system (hereafter referred to as the device) was to develop a gut-on-a-chip model, which includes enteric neuronal cells in proximal co-culture with gut epithelial cells and bacteria.

<table><tbody><tr><td>2-Mercaptoethanol</td><td>Sigma Aldrich</td><td>10712</td><td></td></tr><tr><td>Aeration cannula (length: 1.10 diameter: 30 mm)</td><td>VWR (B.Braun)</td><td>BRAU4190050</td><td></td></tr><tr><td>Agar-agar</td><td>Merck Millipore</td><td>1.01614.1000</td><td></td></tr><tr><td>Aluminium Crimp</td><td>Glasger&amp;auml;tebau Ochs</td><td>102050</td><td></td></tr><tr><td>Ascorbic acid</td><td>Sigma Aldrich</td><td>A4544</td><td></td></tr><tr><td>B-27 Supplement Minus Vitamin A (50x)</td><td>Gibco</td><td>12587-010</td><td></td></tr><tr><td>Bacterial Cell Membrane, pore size: 1 &amp;micro;m</td><td>VWR (Whatman)</td><td>515-2084</td><td></td></tr><tr><td>Caco-2 cells</td><td>DSMZ</td><td>ACC169</td><td></td></tr><tr><td>Cell Counter &amp;amp; Analyzer CASY</td><td>OMNI Life Sceince</td><td></td><td></td></tr><tr><td>CHIR</td><td>Axon Mechem BV</td><td>CT99021</td><td></td></tr><tr><td>Collagen I, Rat Tail</td><td>Invitrogen</td><td>A1048301</td><td></td></tr><tr><td>Costar 6-well Clear Flat Bottom Ultra-Low Attachment Plates</td><td>Corning</td><td>3471</td><td></td></tr><tr><td>Difco Lactobacilli MRS Broth</td><td>BD Biosciences</td><td>288130</td><td></td></tr><tr><td>Discofix 3-way stopcock</td><td>B. Braun</td><td>BRAU40951111</td><td></td></tr><tr><td>DMEM/F12, no glutamine</td><td>Thermofisher Scientific</td><td>21331020</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline, D-PBS</td><td>Sigma Aldrich</td><td>14190-169</td><td></td></tr><tr><td>Essential 6 Medium</td><td>Thermofisher Scientific</td><td>A1516401</td><td></td></tr><tr><td>Essential 8 Medium</td><td>Thermofisher Scientific</td><td>A1517001</td><td></td></tr><tr><td>Female Luer Lock to Barb Connector</td><td>Qosina</td><td>11733</td><td></td></tr><tr><td>FGF2</td><td>R&amp;amp;D Systems</td><td>233-FB</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma Aldrich</td><td>F1141</td><td></td></tr><tr><td>Foetal Bovine Serum, FBS</td><td>Thermofisher Scientific</td><td>10500-064</td><td></td></tr><tr><td>GDNF</td><td>PeproTech</td><td>450-10</td><td></td></tr><tr><td>Human Cell Membrane, pore size: 50 nm</td><td>Sigma Aldrich (GE Healthcare)</td><td>WHA111703</td><td></td></tr><tr><td>HuMiX Gasket Collagen</td><td>Auer Precision</td><td>216891-003</td><td></td></tr><tr><td>HuMiX Gasket Sandwich Bottom</td><td>Auer Precision</td><td>216891-002</td><td></td></tr><tr><td>HuMiX Gasket Sandwich Top</td><td>Auer Precision</td><td>216891-001</td><td></td></tr><tr><td>iPSC</td><td>Max Planck Institute for Molecular Biomedicine</td><td>K7 line</td><td></td></tr><tr><td>L-Glutamine (200 mM)</td><td>Gibco</td><td>25030081</td><td></td></tr><tr><td>Laminin from Engelbreth-Holmswarm</td><td>Sigma Aldrich</td><td>L2020</td><td></td></tr><tr><td>LDN193189</td><td>Sigma Aldrich</td><td>SML0559</td><td></td></tr><tr><td>&lt;em&gt;Limosilactobacillus reuteri&lt;/em&gt;</td><td>ATCC</td><td>23272</td><td></td></tr><tr><td>Live/Dead BacLight Bacterial Viability kit</td><td>Thermofisher Scientific</td><td>L7012</td><td></td></tr><tr><td>Male Luer with Spin Lock to Barb</td><td>Qosina</td><td>11735</td><td></td></tr><tr><td>Marprene tubing (0.8 mm x 1.6 mm)</td><td>Watson-Marlow</td><td>902.0008.J16</td><td></td></tr><tr><td>Matrigel hESC-qualified matrix</td><td>Corning</td><td>354277</td><td></td></tr><tr><td>Mucin, from porcine stomach</td><td>Sigma Aldrich</td><td>T3924</td><td></td></tr><tr><td>N2 Supplement (100x)</td><td>Gibco</td><td>17502048</td><td></td></tr><tr><td>NEAA</td><td>Thermofisher Scientific</td><td>11140050</td><td></td></tr><tr><td>Needle (length: 120 mm; diameter: 0.80 mm)</td><td>B.Braun (color code: green)</td><td>466 5643</td><td></td></tr><tr><td>Needle (length: 40 mm; diameter: 0.70 mm)</td><td>Henke Sass Wolf (color code: black)</td><td>4710007040</td><td></td></tr><tr><td>Needle (length: 80 mm; diameter: 0.60 mm)</td><td>B.Braun (color code: blue)</td><td>466 5635</td><td></td></tr><tr><td>Neurobasal Medium</td><td>Gibco</td><td>21103049</td><td></td></tr><tr><td>PE/Cy7 anti-human CD49d antibody</td><td>Biolegend</td><td>304314</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma Aldrich</td><td>P0781</td><td></td></tr><tr><td>Peristaltic pump</td><td>Watson-Marlow</td><td>205CA</td><td></td></tr><tr><td>Poly-L-ornithine Hydrobromide</td><td>Sigma Aldrich</td><td>P3655</td><td></td></tr><tr><td>Polycarbonate lids (HuMiX)</td><td>University of Arizona</td><td>HuMiX 1.0 / 2.0</td><td></td></tr><tr><td>Retinoic Acid</td><td>Sigma Aldrich</td><td>R2625</td><td></td></tr><tr><td>RLT Buffer (RNeasy Minikit)</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>RPMI 1640 Medium</td><td>Thermofisher Scientific</td><td>72400-021</td><td></td></tr><tr><td>SB431542, ALK inhibitor</td><td>Abcam</td><td>ab120163</td><td></td></tr><tr><td>Serum bottles</td><td>Glasger&amp;auml;tebau Ochs</td><td>102091</td><td></td></tr><tr><td>Syringe</td><td>BD Biosciences</td><td>309110</td><td></td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma Aldrich</td><td>T3924</td><td></td></tr><tr><td>Y-27632 Dihydrochloride</td><td>R&amp;amp;D Systems</td><td>1254</td><td></td></tr></tbody></table>

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.

In neuroHuMiX, we co-cultured three different cell types together-bacterial, epithelial, and neuronal cells (Figure 1). To make sure the cells were all viable, we performed different assays on the different cell types. For example, we performed CFU counts on bacterial cells, cell count and cell viability assays on the epithelial cells, while the neuronal cells were assessed via microscopic analyses.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64483/64483fig01.jpg" /> 
Figure 1: Schematic representation of neuroHuMiX and its experimental setup. (A) The three chambers are held between two PC lids to keep them closed. Each chamber is filled with a specific medium for the cells grown inside. The different chambers are separated by semi-permeable membranes allowing cell communication via soluble factors passing the membranes. (B) Representation of the neuroHuMiX setup. Each chamber is connected to different media bottles. For the bacterial chamber, for the first 12.5 days, the chamber is connected to RPMI + 10% FBS, before being changed for the last 36 h to RPMI + 10% FBS + 5% MRS. Abbreviations: PC = polycarbonate; P/L/F = poly L-ornithine/laminin/fibronectin; RPMI = Roswell Park Memorial Institute cell culture medium; MRS = De Man, Rogosa, and Shapre culture medium. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64483/64483fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To determine whether the cells were appropriately attached, upon opening the devices, we assessed the formation of a cell layer on the collagen-coated membrane (Figure 6A). To make sure the cells in the device were viable, an automated cell counter count (Figure 6B) and a trypan blue exclusion assay cell count were performed (Figure 6C). The assays were performed on Caco-2 cells from three different HuMiX setups: (i) Caco-2 in culture with ENs, (ii) Caco-2 in culture with L. reuteri, and (iii) the device involving co-culture of all three cell types. Statistical testing using a one-way ANOVA did not yield any significant differences between the cell types, suggesting that the Caco-2 cells remained viable in all these initial device setups and conditions tested in this study. This underlines the fact that the bacterial density reached during the co-culture of L. reuteri and the two human cell types do not have cytotoxic effects on the human cells.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64483/64483fig06.jpg" /> 
Figure 6: Assessment of Caco-2 cells on the collagen-coated membrane. (A) Layer of Caco-2 cells on the collagen-coated membrane after opening. The arrow indicates the collagen-coated membrane, which is surrounded by a dashed circle. The Caco-2 cells were growing on the spiral shape on the membrane. Cell viability of Caco-2 cells after 14 days in HuMiX. Cell counts were obtained using (B) the automated cell counter and (C) the trypan blue exclusion assay cell count. Caco-2 cell counts were determined from different culture setups in the initial device: co-culture with enteric neurons (ENs) (black), co-culture with L. reuteri (orange), and in the device (ENs and L. reuteri) (blue). A one-way ANOVA was performed, showing there is no significant difference between the different culture setups (one-way ANOVA, p = 0.1234 [ns]; error bars indicate standard error). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64483/64483fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To be able to culture L. reuteri with mammalian cells, we first optimized and adapted the culture media for use in the device. We found that a 5% mix of MRS in RPMI 1640 (supplemented with 10% FBS) was optimally suited for the growth of L. reuteri, while not being cytotoxic for the mammalian cells used in these assays. Subsequently, a CFU count was performed to assess the growth of L. reuteri when cultured in the device for 24 h. The CFU count was assessed for two different initial device setups (Figure 7)-L. reuteri co-cultured with Caco-2 and L. reuteri in the device. In both setups, the CFU counts were significantly different from the HuMiX inoculum and the harvested cells (one-way ANOVA, p =&#160;0.0002), indicating growth of the bacterial cells inside the initial device.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64483/64483fig07.jpg" /> 
Figure 7: Limosilactobacillus reuteri CFU count of the inoculum (diluted 1:100,000) and after 24 h in HuMiX. Two different setups: Caco-2 cells in co-culture with L. reuteri and the device. A one-way ANOVA shows a significant difference (p = 0.0002 [***]) between the inoculum and the harvested cells, meaning the bacteria are growing inside HuMiX. Error bars indicate the standard error. Abbreviations: CB.HX = Caco-2 bacteria HuMiX; nHX = neuroHuMiX. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64483/64483fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To assess whether culturing the ENs within the device would alter the phenotype of the cells, the gross morphology of the ENs was observed using an inverted phase-contrast microscope. During this step, both the confluency and EN morphology were assessed. Establishment of a confluent neuronal network indicated that the cells had attached well onto the coated device&#39;s PC lid. Importantly, this highlights the notion that they grew in co-culture with Caco-2 and L. reuteri. The edge between the confluent neuronal network and the gasket-delineated spiral was clearly apparent (Figure 8).
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/64483/64483fig08.jpg" /> 
Figure 8: Enteric neurons after 14 days of culture in the device. On the left side of the image, the neurons have grown to a confluent layer on the spiral. The edge, between the neuronal layer and the space without cells, is the edge of the spiral; magnified 10x, scale bar = 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64483/64483fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64483/64483fig02.jpg" /> 
Figure 2: Lids used in the device. Images show top (left) and bottom (right) PC lids. Each side of the PC lid is 6.4 cm. Abbreviation: PC = polycarbonate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64483/64483fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64483/64483fig03.jpg" /> 
Figure 3: Epithelial chamber gasket on bottom PC lid. Top view of the epithelial chamber gasket placed on the bottom PC lid (left), and bottom view (right) showing the alignment of the epithelial chamber gasket with the inlets and outlets of the bottom PC lid. Each side of the gaskets, as well as the PC lid, measures 6.4 cm. Abbreviation: PC = polycarbonate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64483/64483fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64483/64483fig04.jpg" /> 
Figure 4: Assembly of the device. (A) Different parts for assembling HuMiX: (1) bottom PC lid; (2) gasket with collagen-coated microporous membrane, which is placed on top of (1); (3) sandwich gasket with a mucin-coated nanoporous membrane in between and placed on top of (2); (4) top PC lid placed on top of (3). Each side of the gaskets and PC lids measures 6.4 cm. (B) All parts from (A) placed together. (C,D) Assembled device-top (left) and side (right) view. B is placed into the clamping system to close the system. (C) Each side of the top clamp measures 8 cm. Abbreviation: PC = polycarbonate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64483/64483fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64483/64483fig05.jpg" /> 
Figure 5: Parts needed for tubing line and assembled tubing line for one chamber. (A) Different parts to build a tubing line: a. pump tubing line; b. three-way stopcock; c. 40 mm needle; d. 80 mm needle; e. 120 mm needle; f. long tubing line (20 cm); g. short tubing line (8 cm); h. male Luer; i. female Luer; j. adaptor. (B) Assembled tubing line for the bacterial or epithelial chamber. For the neuronal chamber, the 120 mm needle would need to be changed to an 80 mm needle. (C) Three-way stopcock valve turned to redirect the medium flow from the device to the &#39;open connector&#39; and to close the chamber. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64483/64483fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Day</td>
			<td>0</td>
			<td>2</td>
			<td>4</td>
			<td>6</td>
			<td>8</td>
			<td>10</td>
		</tr>
		<tr>
			<td rowspan="6">Media Composition</td>
			<td>100% E6</td>
			<td>100% E6</td>
			<td>75% E6</td>
			<td>50% E6</td>
			<td>25% E6</td>
			<td>100% N2</td>
		</tr>
		<tr>
			<td>+ LDN</td>
			<td>+ LDN</td>
			<td>25% N2</td>
			<td>50% N2</td>
			<td>75% N2</td>
			<td>+ LDN</td>
		</tr>
		<tr>
			<td>+ SB</td>
			<td>+ SB</td>
			<td>+ LDN</td>
			<td>+ LDN</td>
			<td>+ LDN</td>
			<td>+ SB</td>
		</tr>
		<tr>
			<td></td>
			<td>+ CHIR</td>
			<td>+ SB</td>
			<td>+ SB</td>
			<td>+ SB</td>
			<td>+ CHIR</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>+ CHIR</td>
			<td>+ CHIR</td>
			<td>+ CHIR</td>
			<td>+ RA</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>+ RA</td>
			<td>+ RA</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecule</td>
			<td>[concentration]</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LDN</td>
			<td>100 nM</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SB</td>
			<td>10 &#181;M</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR</td>
			<td>3 &#181;M</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic Acid (RA)</td>
			<td>1 &#181;M</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Media composition.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Media</td>
			<td>Components (concentrations listed in Table of materials)</td>
			<td>Volume (mL)</td>
		</tr>
		<tr>
			<td rowspan="5">N2 media (50 mL)</td>
			<td>DMEM-F12</td>
			<td>48</td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td rowspan="5">N2B27/ENS Media (50 mL)</td>
			<td>Neurobasal</td>
			<td>48</td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>B27-A</td>
			<td>0.5</td>
		</tr>
	</tbody>
</table>
Table 2: Media recipes.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sterilization Temperature (&#176;C)</td>
			<td>116</td>
		</tr>
		<tr>
			<td>Sterilization Time (min)</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Dry Time (min)</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Pulses</td>
			<td>3</td>
		</tr>
		<tr>
			<td>End Temperature (&#176;C)</td>
			<td>99</td>
		</tr>
	</tbody>
</table>
Table 3: HuMiX autoclave run. 
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Rotations per minute (rpm)</td>
			<td>Average flow rate (&#181;L/min)</td>
		</tr>
		<tr>
			<td>0.5</td>
			<td>13</td>
		</tr>
		<tr>
			<td>2</td>
			<td>79</td>
		</tr>
		<tr>
			<td>5</td>
			<td>180</td>
		</tr>
	</tbody>
</table>
Table 4: Flow rates of the peristaltic pump.

Watch this Scientific Journal Video about A Gut-on-a-Chip Model to Study the Gut Microbiome-Nervous System Axis at JoVE.com

A Gut-on-a-Chip Model to Study the 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 ...

a-gut-on-a-chip-model-to-study-the-gut-microbiome-nervous-system-axis

Nanyang Technological University

Research

JoVE Journal

Biology

2.4K Views.  University of Luxembourg. 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.

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

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Badania wizualne Wrażliwość na prędkość i kierunek ruchu w Jaszczurki

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and species-rich microbial population that assists in the degradation of lignocellulose predominantly into acetate, the key nutrient fueling termite metabolism (Odelson &amp; Breznak, 1983).  Within these microbial populations are bacteria, methanogenic archaea and, in some ("lower") termites, eukaryotic protozoa. Thus, termites are excellent research subjects for studying the interactions among microbial species and the numerous biochemical functions they perform to the benefit of their host.  The species composition of microbial populations in termite guts as well as key genes involved in various biochemical processes has been explored using molecular techniques (Kudo et al., 1998; Schmit-Wagner et al., 2003; Salmassi &amp; Leadbetter, 2003). These techniques depend on the extraction and purification of high-quality nucleic acids from the termite gut environment.  The extraction technique described in this video is a modified compilation of protocols developed for extraction and purification of nucleic acids from environmental samples (Mor  et al., 1994; Berthelet et al., 1996; Purdy et al., 1996; Salmassi &amp; Leadbetter, 2003; Ottesen et al. 2006) and it produces DNA from termite hindgut material suitable for use as template for polymerase chain reaction (PCR).

Extracting DNA from the Gut Microbes of the Termite Zootermopsis Angusticollis and Visualizing Gut Microbes

This video illustrates the technique for extracting DNA from the species of microbes resident in the termite hindgut. The preparation of a wet mount slide, which is useful for visualizing the gut microbial community is also illustrated, and a tour through the species-rich gut environment is given.

Termites are among the few animals known to have the capacity to subsist solely by consuming wood.  The termite gut tract contains a dense and ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

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, ...

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for maintaining local and systemic homeostasis; changes to gut microbiota composition (dysbiosis) associate with a wide array of human diseases. Methods for dissecting host-microbiota interactions encompass an inherent tradeoff among preservation of physiological tissue structure (when using in vivo animal models) and the level of control over the experiment factors (as in simple in vitro cell culture systems). To address this tradeoff, Yissachar et al. recently developed an intestinal organ culture system. The system preserves a naive colon tissue construction and cellular mechanisms and it also permits tight experimental control, facilitating experimentations that cannot be readily performed in vivo. It is optimal for dissecting short-term responses of various gut components (such as epithelial, immunological and neuronal elements) to luminal perturbations (including anaerobic or aerobic microbes, whole microbiota samples from mice or humans, drugs and metabolites). Here, we present a detailed description of an optimized protocol for organ culture of multiple gut fragments using a custom-made gut culture device. Host responses to luminal perturbations can be visualized by immunofluorescence staining of tissue sections or whole-mount tissue fragments, fluorescence in-situ hybridization (FISH), or time-lapse imaging. This system supports a wide array of readouts, including next-generation sequencing, flow cytometry, and various cellular and biochemical assays. Overall, this three-dimensional organ culture system supports the culture of large, intact intestinal tissues and has broad applications for high-resolution analysis and visualization of host-microbiota interactions in the local gut environment.

This article presents a unique method for analyzing host-microbiome interactions using a novel gut organ culture system for ex vivo experiments.

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for ...

Immunology and Infection

Microfluidic Vagina Chip for Studying the Vaginal Microbiome

Modeling Healthy and Dysbiotic Vaginal Microenvironments in a Human Vagina-on-a-Chip

Women's health, and particularly diseases of the female reproductive tract (FRT), have not received the attention they deserve, even though an unhealthy reproductive system may lead to life-threatening diseases, infertility, or adverse outcomes during pregnancy. One barrier in the field is that there has been a dearth of preclinical, experimental models that faithfully mimic the physiology and pathophysiology of the FRT. Current in vitro and animal models do not fully recapitulate the hormonal changes, microaerobic conditions, and interactions with the vaginal microbiome. The advent of Organ-on-a-Chip (Organ Chip) microfluidic culture technology that can mimic tissue-tissue interfaces, vascular perfusion, interstitial fluid flows, and the physical microenvironment of a major subunit of human organs can potentially serve as a solution to this problem. Recently, a human Vagina Chip that supports co-culture of human vaginal microbial consortia with primary human vaginal epithelium that is also interfaced with vaginal stroma and experiences dynamic fluid flow has been developed. This chip replicates the physiological responses of the human vagina to healthy and dysbiotic microbiomes. A detailed protocol for creating human Vagina Chips has been described in this article.

Author Spotlight: Revolutionizing Research on Vaginal Microbiome Interactions Using a Vaginal Chip

This article describes a protocol for creating a microfluidic vagina-on-a-chip (Vagina Chip) culture device that enables the study of human host interactions with a living vaginal microbiome under microaerophilic conditions. This chip can be used as a tool to investigate vaginal diseases as well as to develop and test potential therapeutic countermeasures.

Women's health, and particularly diseases of the female reproductive tract (FRT), have not received the attention they deserve, even though an unhealthy ...

Enhanced Intestine-on-Chip Platform for Immunological Insight

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

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 ...

A Necrotizing Enterocolitis Model for Mechanistic Studies and Drug Discovery

Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, which remains incompletely understood. The pathophysiology of NEC includes disruption of intestinal tight junctions, increased gut barrier permeability, epithelial cell death, microbial dysbiosis, and dysregulated inflammation. Traditional tools to study NEC include animal models, cell lines, and human or mouse intestinal organoids. While studies using those model systems have improved the field&#39;s understanding of disease pathophysiology, their ability to recapitulate the complexity of human NEC is limited. An improved in vitro model of NEC using microfluidic technology, named NEC-on-a-chip, has now been developed. The NEC-on-a-chip model consists of a microfluidic device seeded with intestinal enteroids derived from a preterm neonate, co-cultured with human endothelial cells and the microbiome from an infant with severe NEC. This model is a valuable tool for mechanistic studies into the pathophysiology of NEC and a new resource for drug discovery testing for neonatal intestinal diseases. In this manuscript, a detailed description of the NEC-on-a-chip model will be provided.

Author Spotlight: Enhancing Understanding and Treatment Strategies with the NEC-on-a-Chip Model 

This protocol describes an in vitro model of necrotizing enterocolitis (NEC), which can be used for mechanistic studies into disease pathogenesis. It features a microfluidic chip seeded with intestinal enteroids derived from the human neonatal intestine, endothelial cells, and the intestinal microbiome of a neonate with severe NEC.

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, ...

Medicine

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 ...