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. I., Bohl, J., Del Tredici, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+&#945;-synuclein+immunoreactive+inclusions+in+Meissner's+and+Auerbach's+plexuses+in+cases+staged+for+Parkinson's+disease-related+brain+pathology.">Gastric &#945;-synuclein immunoreactive inclusions in Meissner's and Auerbach's plexuses in cases staged for Parkinson's disease-related brain pathology.</a> Neuroscience Letters. 396 (1), 67-72 (2006).</li><li>Schmit, K. J., 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=Dietary+fibre+deprivation+and+bacterial+curli+exposure+shift+gut+microbiome+and+exacerbate+Parkinson's+disease-like+pathologies+in+an+alpha-synuclein-overexpressing+mouse.">Dietary fibre deprivation and bacterial curli exposure shift gut microbiome and exacerbate Parkinson's disease-like pathologies in an alpha-synuclein-overexpressing mouse.</a> bioRxiv. , (2022).</li><li>Fritz, J. V., Desai, M. S., Shah, P., Schneider, J. G., 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=From+meta-omics+to+causality:+experimental+models+for+human+microbiome+research.">From meta-omics to causality: experimental models for human microbiome research.</a> Microbiome. 1 (1), 14 (2013).</li><li>Fattahi, F., 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=Deriving+human+ENS+lineages+for+cell+therapy+and+drug+discovery+in+Hirschsprung+disease.">Deriving human ENS lineages for cell therapy and drug discovery in Hirschsprung disease.</a> Nature. 531 (7592), 105-109 (2016).</li><li>Wu, Q., 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-a-chip:+Recent+breakthroughs+and+future+prospects.">Organ-on-a-chip: Recent breakthroughs and future prospects.</a> BioMedical Engineering Online. 19 (1), 9 (2020).</li><li>May, S., Evans, S., Parry, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids,+organs-on-chips+and+other+systems,+and+microbiota.">Organoids, organs-on-chips and other systems, and microbiota.</a> Emerging Topics in Life Sciences. 1 (4), 385-400 (2017).</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> Nature Communications. 7, 11535 (2016).</li><li>Greenhalgh, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+in+vitro+and+in+silico+modeling+delineates+the+molecular+effects+of+a+synbiotic+regimen+on+colorectal-cancer-derived+cells.">Integrated in vitro and in silico modeling delineates the molecular effects of a synbiotic regimen on colorectal-cancer-derived cells.</a> Cell Reports. 27 (5), 1621-1632 (2019).</li><li>Mao, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+metabolic+links+between+the+murine+microbiome+and+memory.">Genetic and metabolic links between the murine microbiome and memory.</a> Microbiome. 8 (1), 53 (2020).</li><li>Moysidou, C. M., Owens, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+modelling+the+human+microbiome-gut-brain+axis+in+vitro.">Advances in modelling the human microbiome-gut-brain axis in vitro.</a> Biochemical Society Transactions. 49 (1), 187-201 (2021).</li><li>Kim, 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=Transneuronal+propagation+of+pathologic+&#945;-synuclein+from+the+gut+to+the+brain+models+Parkinson's+disease.">Transneuronal propagation of pathologic &#945;-synuclein from the gut to the brain models Parkinson's disease.</a> Neuron. 103 (4), 627-641 (2019).</li><li>Hugenholtz, F., de Vos, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+human+intestinal+microbiota+research:+a+critical+evaluation.">Mouse models for human intestinal microbiota research: a critical evaluation.</a> Cellular and Molecular Life Sciences. 75 (1), 149-160 (2018).</li><li>Marshall, J. J., Mason, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+vs+man:+Organoid+models+of+brain+development+&amp;+disease.">Mouse vs man: Organoid models of brain development &amp; disease.</a> Brain Research. 1724, 146427 (2019).</li><li>Goers, L., Freemont, P., Polizzi, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-culture+systems+and+technologies:+taking+synthetic+biology+to+the+next+level.">Co-culture systems and technologies: taking synthetic biology to the next level.</a> Journal of the Royal Society Interface. 11 (96), 20140065 (2014).</li><li>Sedrani, C., 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=Toward+hypothesis-driven,+personalized+microbiome+screening.">Toward hypothesis-driven, personalized microbiome screening.</a> Cell Reports Methods. 2 (1), 100139 (2022).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#228;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 &#181;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 &#38; 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&#39;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&#38;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>Limosilactobacillus reuteri</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&#228;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&#38;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/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/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/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/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/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/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/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/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

Research

JoVE Journal

Biology

2.2K 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.

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

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 ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition.
This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient.
The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...