Name: co-culture of living microbiome with microengineered human intestinal villi in a gut-on-a-chip microfluidic device
Uploaded: 2016-08-30T15:00:00.000+00:00
Duration: 10 min 51 s
Description: Watch this Scientific Journal Video about Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device at JoVE.com

We thank Sri Kosuri (Wyss Institute at Harvard University) for providing the GFP-labeled E. coli strain. This work was supported by the Defense Advanced Research Projects Agency under Cooperative Agreement Number W911NF-12-2-0036, Food and Drug Administration under contract #HHSF223201310079C, and the Wyss Institute for Biologically Inspired Engineering at Harvard University. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of the Army Research Office, Army Research Laboratory, Food and Drug Administration, or the U.S. Government. The U.S. Government is authorized to reproduce and distribute reprints for Government purposes notwithstanding any copyright notation hereon.

1. Microfabrication of a Gut-on-a-chip Device
Note: The gut-on-a-chip is a microfluidic device made by transparent, gas-permeable silicone polymer (polydimethylsiloxane, PDMS), containing two parallel microchannels (1 mm width x 150 &#181;m height x 1 cm length) separated by a flexible porous (10 &#181;m in pore diameter, 25 &#181;m in spacing pore to pore) PDMS membrane5,9. Fabricate the gut-on-a-chip (Figure 1A, left) following the steps provided.
<ol>
	<li>Microfabrication Procedure of the Gut-on-a-chip5,9.
	<ol>
		<li>Prepare uncured, degassed PDMS by mixing the PDMS prepolymer and the curing agent (15:1 w/w), and place it in the vacuum desiccator for 30 min.</li>
		<li>Pour 30 g and 3 g of uncured, degassed PDMS onto each silicon mold that has SU-8 based micropatterns of the upper and the lower microchannels of the gut-on-a-chip, respectively. Then cure at 60 &#176;C in a dry oven for at least 4 hr.</li>
		<li>Demold the upper and lower PDMS layers from each silicon mold by cutting around the edge using a surgical scalpel. Punch 6 holes (two inlets, two outlets, and two vacuum chambers) using a biopsy puncher (2.0 mm in hole diameter).</li>
		<li>Prepare the porous membrane by pouring 10 g of uncured and degassed PDMS on a silanized silicon wafer containing post arrays with circular pillars (10 &#181;m in diameter, 25 &#181;m in height) overlaying with a flat, silanized PDMS support layer (15:1, w/w; 1 cm thick). Place a 3 kg weight presser on the setup and cure the polymer at 60 &#176;C for 12 hr or longer.</li>
		<li>Demold the setup of a porous PDMS membrane adhered to the PDMS support slab from the silicon wafer by carefully lifting up the corner of the slab from the wafer, and peeling off until the porous PDMS membrane is completely detached from the wafer.</li>
		<li>Expose the channel side of an upper layer (PDMS, 15:1, w/w) and the porous membrane side to the plasma generated by a handheld corona treater for 1 min, and 3 sec, respectively.</li>
		<li>Place the plasma-treated surfaces of the porous PDMS membrane and upper microchannel layer in a conformal contact by placing each piece in parallel without any air bubbles.</li>
		<li>Incubate the whole setup at 80 &#176;C O/N for irreversible bonding of the two PDMS layers.</li>
		<li>Peel off the PDMS support layer from the assembly of an upper layer with a porous membrane by carefully lifting up the corner of the support layer, and peeling off until the support layer is completely detached from the upper layer with the membrane.</li>
		<li>Tear off the portions of this membrane located over the vacuum chambers (the two chambers located on either side of the main channel) using a fine-tip tweezers to make hollow vacuum chambers.</li>
		<li>Expose the side with the membrane attached on the top piece and the side with the channels engraved on the lower piece to plasma in a same way as described in step 1.1.6 for 1 min.</li>
		<li>Align the upper microchannel layer with a porous membrane and the lower layer with a conformal contact by placing each piece in parallel without any air bubbles under a stereoscope.</li>
		<li>Cure the whole setup in a dry oven at 80 &#176;C O/N to produce a complete scale gut-on-a-chip microfluidic device containing two hollow vacuum chambers next to the microfluidic cell channel.</li>
	</ol>
	</li>
	<li>Connect tubing to the inlet and outlet of each port linked to the upper or the lower microchannels in a gut-on-a-chip via a 90&#176; bent stainless steel blunt-end needle.</li>
	<li>Connect the tubing to the holes linked to the vacuum chambers using a 90&#176; bent stainless steel blunt-end needle.</li>
	<li>Sterilize the microchannels and tubing by flowing 70% (v/v) ethanol using a 1 ml sterilized disposable syringe.</li>
	<li>Dry out the microchannels and tubing in a 60 &#176;C dry oven O/N.</li>
</ol>
2. Growth of Microengineered Intestinal Villi in the Gut-on-a-chip Device
<ol>
	<li>Seed Intestinal Epithelial Cells (e.g. Caco-2BBE) on a Gut-on-a-chip Microdevice5,9.
	<ol>
		<li>Sterilize the device with 70% (v/v) ethanol (see step 1.4) using a 1 ml sterilized disposable syringe followed by drying in a 60 &#176;C dry oven O/N (see step 1.5) prior to the surface activation.</li>
		<li>Expose the complete setup of a gut-on-a-chip microsystem (i.e.,&#160;a body of the gut-on-a-chip connected to tubing) to UV light (253.7 nm) and ozone simultaneously for 40 min.</li>
		<li>Cool down the device at RT&#160;for 15 min under UV light in a biosafety cabinet (BSC).</li>
		<li>Introduce 100 &#181;l of the ECM coating solution- a mixture of 50 &#181;g/ml type I collagen and 300 &#181;g/ml extracellular matrix mixture diluted in the serum-free Dulbecco&#39;s Modified Eagle Medium (DMEM) - into both upper and lower microchannels using a disposable, sterile 1 ml syringe.</li>
		<li>Incubate the whole setup in a 37 &#176;C humidified 5% CO2 incubator for 1 hr.</li>
		<li>Degas 50 ml of pre-warmed (37 &#176;C) complete culture medium (DMEM, 20%, v/v, fetal bovine serum (FBS), 100 units/ml penicillin, and 100 &#181;g/ml streptomycin; aliquoted in a 3 ml disposable syringe) using a 50 ml filtration system (0.45 &#181;m in pore size) in the BSC for 1 min.
		<ol>
			<li>Pass the pre-warmed medium through the filtration system and tap gently on the filtered medium for 1 min to remove any bubbles or dissolved gas in the medium.</li>
		</ol>
		</li>
		<li>Place the aliquot of degassed complete medium in a 3 ml disposable syringe and incubate in a 37 &#176;C humidified 5% CO2 incubator for 1 hr.</li>
		<li>Connect two 3 ml disposable syringes containing degassed, pre-warmed complete culture medium to the upper and lower microchannels.</li>
		<li>Flow the degassed complete cell culture medium into the upper microchannel using a syringe pump at the volumetric flow rate of 30 &#181;l/hr (equivalent to the shear stress at 0.02 dyne/cm2) in a 37 &#176;C humidified 5% CO2 incubator O/N.</li>
	</ol>
	</li>
	<li>Formation of Microengineered Caco-2 Villi in the Gut-on-a-chip.
	<ol>
		<li>Use Caco-2BBE cells with the passage number between 50 and 65.</li>
		<li>Grow Caco-2 cells in a T75 flask containing complete culture medium in a 37 &#176;C humidified 5% CO2 incubator for 4-5 days to obtain fully confluent cells.</li>
		<li>Add 10 ml of Ca2+ and Mg2+-free PBS to fully confluent Caco-2 cells grown in a T75 flask, wash cells, then aspirate out PBS. Repeat this step twice.</li>
		<li>Add 1 ml of 0.05% trypsin/EDTA solution and incubate in a 37 &#176;C humidified 5% CO2 incubator for 5 min.</li>
		<li>Resuspend the dissociated cells with 10 ml pre-warmed complete cell culture medium (with FBS) by pipetting up and down three to five times.</li>
		<li>Spin down the cell suspension by centrifugation at 500 x g for 5 min, then remove the supernatant.</li>
		<li>Resuspend again with 1 ml pre-warmed complete medium to adjust cell density at ~1.5 x 105 cells/cm2 in the device. Use hemocytometer to estimate the cell density.</li>
		<li>Infuse 100 &#181;l of the resuspended Caco-2 cells into the upper microchannel (lumen side) by placing a 1 ml disposable syringe attached to a 25 G5/8 needle on the outlet of tubing connected to the upper microchannel.</li>
		<li>Clamp all inlets and outlets of tubing connected to the upper and lower microchannels using binder clips.</li>
		<li>Incubate the whole setup in a 37 &#176;C, humidified 5% CO2 incubator for 1 hr to allow dissociated Caco-2 cells to adhere to the surface of the porous membrane.</li>
		<li>Remove the clamps and resume the flow of culture medium only to the upper microchannel at 30 &#181;l/hr using a syringe pump until cells form an intact monolayer for 24-36 hr. Use the phase contrast or the differential interference contrast (DIC) microscopy to confirm cell-cell junctions in a cell monolayer.</li>
		<li>When cells form a monolayer, perfuse the culture medium into both upper (lumen) and lower (capillary) microchannels at the same flow rate of 30 &#181;l/hr.</li>
		<li>Application of mechanical deformations mimicking peristalsis-like motions5,9.
		<ol>
			<li>Turn on the vacuum pump equipped with the tension equipment.</li>
			<li>Connect the vacuum chambers to the vacuum controller via tubing with a stainless steel connector.</li>
			<li>Set the stretching motion of 10% mean cell strain at a frequency of 0.15 Hz with the cyclic sine function mode on the vacuum controlling software, then click &#34;Start&#34;.</li>
		</ol>
		</li>
		<li>Maintain the constant flow of culture medium (30 &#181;l/hr) in both upper and lower microchannel to the confluent Caco-2 monolayer under mechanical deformations (10%, 0.15 Hz) for ~100 hr. 
		Note: Caco-2 cells spontaneously undergo villus morphogenesis with undulated 3D projections extended towards the lumen of the epithelial microchannel5,8-10.</li>
	</ol>
	</li>
	<li>To emulate the organ-level functions of the living human intestine with lumen-capillary tissue interface in the gut-on-a-chip10, follow the steps as described.
	<ol>
		<li>Grow microengineered Caco-2 villi by repeating steps from 2.1 to 2.2.</li>
		<li>Flow the pre-warmed co-culture medium (a mixture of the complete Caco-2 cell culture medium and the complete HMVECs culture medium, 1:1 v/v) to the upper and lower microchannels at 30 &#181;l/hr.</li>
		<li>Introduce 100 &#181;l of dissociated normal human capillary microvascular endothelial cells (HMVECs; final cell density, ~5.0 x 105 cells/cm2) into the lower microchannel by the identical method described in steps from 2.2.3 to 2.2.9.</li>
		<li>Place a cured rectangular PDMS piece (0.5 cm x 1 cm x 1 cm; width x length x height; 15:1, w/w elastomer:curing agent) on top of the gut-on-a-chip device, then flip the whole device setup upside down (i.e., the upper microchannel faces to the downside, and the lower microchannel faces to the upside).</li>
		<li>Incubate the setup in a 37 &#176;C, humidified 5% CO2 incubator for 1 hr to allow dissociated HMVECs to adhere to the surface of the porous membrane in the lower microchannel.</li>
		<li>Take out a whole setup from the CO2 incubator, and flip the setup over again.</li>
		<li>Flow pre-warmed co-culture medium (a mixture of the complete Caco-2 cell culture medium and the complete HMVECs culture medium, 1:1 v/v) into both upper and lower microchannels at 30 &#181;l/hr with mechanical stretching motions (10%, 0.15 Hz) for at least three days to form cell-cell junctions of an endothelial monolayer.</li>
	</ol>
	</li>
</ol>
3. Host-gut Microbiome Co-culture in a Gut-on-a-chip Microdevice
<ol>
	<li>Perform pre-culture of the bacterial cells as follows to co-culture commensal gut microbiome on the microengineered intestinal villi.
	<ol>
		<li>Resuspend the freeze-dried probiotic bacterial powdery mixture in 10 ml of the mixture (1:1, v/v) of autoclaved Lactobacilli MRS Broth and Reinforced Clostridial Medium.</li>
		<li>Adjust the final cell density to approximately 0.2 optical density units (at 600 nm) by adding appropriate amount of medium, then aliquot 3 ml of microbial cell suspension in a 10 ml disposable sterile tube.</li>
		<li>Place tubes containing resuspended microbial cells in the anaerobic container. Add two packs of anaerobic gas generating sachet in the container. Close the container lid tightly and incubate the container setup without shaking in a 37 &#176;C humidified 5% CO2 incubator O/N.</li>
	</ol>
	</li>
	<li>Inoculation of Microbiome and Co-culture with Intestinal Epithelial Cells5,10.
	<ol>
		<li>Prepare 3 ml disposable syringes containing degassed antibiotic-free cell culture medium (i.e., DMEM with 20% FBS). See 2.1.6 for the degassing procedure of the culture medium.</li>
		<li>Take out a whole setup of the gut-on-a-chip device containing microengineered villi from the CO2 incubator, then move to the biosafety cabinet.</li>
		<li>Remove syringes connected to tubing linked to the upper and lower microchannels. Connect syringes prepared in 3.2.1 to the device, bring back to the CO2 incubator, then flow this antibiotic-free culture medium for 12 hr prior to seeding of microbiome.</li>
		<li>Spin down the pre-cultured probiotic bacterial mixture cells (see steps in 3.1) at 10,000 x g for 5 min. Aspirate out the supernatant using vacuum, then resuspend in antibiotic-free DMEM medium (final cell density, ~1.0 x 107 CFU/ml).</li>
		<li>Infuse the cell suspension into the lumen side of the microchannel containing the &#34;germ-free&#34; intestinal villi using a 1 ml disposable syringe attached to a 25 G5/8 needle. Allow the adherence of microbial cells to the apical surface of the intestinal villi for ~1.5 hr without flow by clamping to all the tubing end.</li>
		<li>Perfuse the pre-warmed antibiotic-free culture medium into both upper and lower microchannels at 40 &#181;l/hr with cyclic rhythmical deformations (10%, 0.15 Hz).</li>
		<li>For the co-culture of green fluorescence protein (GFP)-labeled E. coli (GFP E. coli; Non-pathogenic DH5-Alpha E. coli host)10,11 cells with microengineered villi, pre-cultivate GFP E. coli cells in autoclaved LB medium at 37 &#176;C under shaking condition (200 rpm) for 12 hr. Repeat the procedure from 3.2.5 to 3.2.6 to carry out co-culture of GFP E. coli cells with microengineered villi.</li>
	</ol>
	</li>
	<li>Image Cells5,8-10
	<ol>
		<li>Perform DIC, epifluorescence, or laser scanning confocal microscopy for recording microbial and epithelial morphology5,8,10.
		<ol>
			<li>For the DIC imaging, take out a setup of gut-on-a-chip microsystem from the CO2 incubator, place the device setup on the stage of a microscope, then record the cell morphology.</li>
			<li>For fluorescence imaging of villus epithelium, flow PBS for washing the cells at 100 &#181;l/hr for 10 min.
			<ol>
				<li>Fix villi with 4% (w/v) paraformaldehyde for 15 min. Then flow PBS for washing the cells at 100 &#181;l/hr for 10 min.</li>
				<li>Permeabilize the villi with 0.3% (v/v) Triton X-100 diluted in PBS containing 2% (w/v) bovine serum albumin (BSA) for 10 min. Then flow PBS for washing the cells at 100 &#181;l/hr for 10 min.</li>
				<li>Block cells with 2% (w/v) BSA solution in PBS for 1 hr. Then flow PBS for washing the cells at 100 &#181;l/hr for 10 min.</li>
				<li>Add 300 nM of 4&#39;,6-diamidino-2-phenylindole dihydrochloride (DAPI) solution diluted in PBS for the nuclear staining under light protection.</li>
				<li>Add 25 units/ml of fluorescent phalloidin (Phalloidin-CF647 conjugate) dissolved in PBS for F-actin staining under light protection.</li>
				<li>Record images of the fluorescently stained cells using a laser scanning confocal microscope. 
				Note: A 25X objective was applied with appropriate optical zoom during the confocal microscopy. In Figure 3B, approximately 525X magnification was used.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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Donald E. Ingber is a founder of Emulate Inc., holds equity in the company and chairs its Scientific Advisory Board. The other authors have no financial disclosures.

Understanding host-microbiome interactions is critical for advancing medicine; however, traditional cell culture models performed in a plastic dish or a static well plate do not support the stable co-culture of human intestinal cells with living gut microbes for more than 1-2 days because microbial cells mostly overgrow the mammalian cells in vitro. The overgrowing microbial population rapidly consumes oxygen and nutrients, subsequently producing excessive amount of metabolic wastes (e.g.,&#160;organic acids), which seriously compromise intestinal barrier functions and cause intestinal epithelial cell death. Hence, preventing the microbial overgrowth that causes the depletion of nutrients and the accumulation of microbial wastes is crucial for sustaining viable host-microbiome coexistence for an extended period (from days to weeks) in the co-culture microenvironment.
To overcome these challenges, a microphysiological gut-on-a-chip device5,8 has been developed to form fully differentiated human intestinal villi and to maintain the steady-state microenvironment of the intestinal lumen in vitro. The design and functional units (e.g., microfluidic flow cell chambers, vacuum-driven cyclic deformations) of a gut-on-a-chip microfluidic device are modified to precisely emulate the physiological, physical and chemical microenvironment for the microbial co-culture5,10. The shear stress applied in the gut-on-a-chip microfluidic culture was determined by the physiological range of luminal flow in the human intestine5,13. For the host-microbe co-culture, maintaining the &#34;steady-state&#34; of microbial population in the luminal microchannel of the gut-on-a-chip is critical in supporting chemically and nutritionally feasible conditions. In the gut-on-a-chip microsystem, it is possible to estimate the intra-luminal steady-state in the gut-on-a-chip by measuring the pH of the culture medium and the microbial cell density. Because fresh culture medium continuously flows through the microchannels, both microbial and epithelial cells do not undergo nutrient depletion when the initial seeding density of microbial cells and the volumetric flow rate of the culture medium are optimized. The conditioned medium passing through the microchannel flows out at a defined volumetric flow rate (here, 40 &#181;l/hr), so metabolic wastes (e.g., organic acids) as well as cellular secretomes (e.g., cytokines) are continuously removed from the co-culture microenvironment. Thus, the gut-on-a-chip device can be considered as a &#34;continuous bioreactor&#34; with a steady-state microenvironment necessary and sufficient to effectively wash out unbound or overgrown microbial cells from the lumen of the intestinal microchannel, which facilitates generation of a stable microbial niche within 2-3 days.
There are crucial factors that must be considered as troubleshooting for the successful co-culture of microbial cells on the intestinal villi. First, it is necessary to determine the optimized incubation time required for the microbial cells to attach to the surface of villi because microbial adhesion can vary between microbial species. For instance, probiotic bacteria, such as Lactobacillus rhamnosus GG5, generally require ~1.0-1.5 hr to attach, but some other microbial species may require shorter (e.g., pathogenic microbes) or longer times, depending on their adhesion kinetics14. Second, the initial seeding density of the microbial cells should be identified because excessive microbial cell numbers may lead to the outgrowth of bacteria in the early stage of co-culture, which can interfere with the achievement of a stable steady-state. To optimize this seeding density, it is recommended to characterize the growth profile of the target microbial strain in the antibiotic-free cell culture medium at various seeding densities. Finally, adjustment of the volumetric flow rate may be required depending on the microbial species. For example, increased flow rates will be required if the microbial cells proliferate very rapidly. It was experimentally confirmed that flow rates up to 300 &#181;l/hr (0.2 dyne/cm2) do not compromise the barrier function and cell morphology of the microengineered villi (data not shown). Although Lactobacillus rhamnosus GG5, over-the-counter probiotic mixture, VSL#3, non-pathogenic lab strain E. coli as well as the pathogenic enteroinvasive E. coli strain10 were successfully applied for the long-term co-culture in the gut-on-a-chip, it is still required to test a variety of microbial species from commensal gut microbiota to pathogenic infectious microorganisms. The cultivability of microbial cells in vitro should be contemplated in the presence or the absence of host cells in the context of symbiosis and evolution, where the test of cultivating the unculturable gut microbiome is an intriguing challenge. Finally, a robust protocol for the co-culture of both aerobic and anaerobic microbes in the gut-on-a-chip is a critical unmet need to be discovered in the future.
There are steps that can be taken to further improve the model, such as the use of primary intestinal epithelial or undifferentiated stem cells from individual human subjects who have specific gastrointestinal diseases such as Crohn&#39;s disease or colorectal cancer; or induced pluripotent stem cells (iPSCs). However, with the burgeoning interest in the role of human microbiome in orchestrating human health and disease, our host-microbiome co-culture method has an enormous significance and potential to be applied to mimic other host-microbiome ecosystems found in the human body (e.g., oral cavity15, skin16, or urogenital tract17). Finally, this co-culture method may innovate the conventional drug development process by improving the predictability in terms of how the gut microbiome influences on the bioavailability, efficacy, and toxicity of new drug compounds. Taken together, the gut-on-a-chip microphysiological system can provide a robust platform to establish a stable steady-state intestinal microenvironment that can be used to reconstitute host-microbiome ecosystem.

The human intestine harbors a stunningly diverse array of microbial species (&#60;1,000 species) and a tremendous number of microbial cells (10 times more than the human host cells) and genes (100 times more than the human genome)1. These human microbiomes play a key role in metabolizing nutrients and xenobiotics, regulating immune responses, and maintaining intestinal homeostasis2. Not surprisingly, given these diverse functions, the commensal gut microbiome extensively modulates health and disease3. Thus, understanding the role of gut microbiome and host-microbe interactions are of great importance to promote gastrointestinal (GI) health and explore new therapeutics for intestinal disorders4. However, existing in vitro intestine models (e.g., static cultures) restrict host-microbiome co-culture to a short period of time (&#60;1 day) because microbial cells overgrow and compromise intestinal barrier function5. Surrogate animal models (e.g., germ-free6 or genetically engineered mice7) are also not commonly used to study host-gut microbiome crosstalk because the colonization and stable maintenance of human gut microbiome are difficult.
To overcome these challenges, we recently developed a biomimetic human &#34;Gut-on-a-Chip&#34; microphysiological system (Figure 1A, left) to emulate the host-gut microbiome interactions that occur in the living human intestine5,8. The gut-on-a-chip microdevice contains two parallel microfluidic channels separated by a flexible, porous, extracellular matrix (ECM)-coated membrane lined by human intestinal epithelial Caco-2 cells, mimicking the intestinal lumen-capillary tissue interface (Figure 1A, right)9. Vacuum-driven cyclic rhythmical deformations induce physiological mechanical deformations that mimic changes normally induced by peristalsis (Figure 1A, right). Interestingly, when Caco-2 cells are grown in the gut-on-a-chip for more than 100 hr, they spontaneously form three-dimensional (3D) intestinal villi with tight junctions, apical brush borders, proliferative cells limited to basal crypts, mucus production, increased drug metabolizing activity (e.g., cytochrome P450 3A4, CYP3A4), and enhanced glucose reuptake8. In this &#39;germ-free&#39; microenvironment, it was possible to co-culture the probiotic Lactobacillus rhamnosus GG or a therapeutic formation of a probiotic bacterial mixture with host epithelial cells for up to two weeks5,10.
In this study, we describe the detailed protocol to perform host-gut microbiome co-culture in the gut-on-a-chip device for an extended period. In addition, we discuss critical issues and potential challenges to be considered for a broad application of this host-microbiome co-culture protocol.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM) containing 25 mM glucose and 25 mM HEPES</td><td>Gibco</td><td>10564-011</td><td>Warm it up at 37 &amp;deg;C in a water bath.</td></tr><tr><td>Difco Lactobacilli MRS broth</td><td>BD</td><td>288120</td><td>Run autoclave at 121 &amp;deg;C for 15 min.</td></tr><tr><td>Poly(dimethylsiloxane)</td><td>Dow Corning</td><td>3097358-1004</td><td>15:1 (w/w), PDMS : cureing agent</td></tr><tr><td>Caco-2BBE human colorectal carcinoma line</td><td>Harvard Digestive Disease Center</td><td></td><td>Human colorectal adenocarcinoma&amp;nbsp;</td></tr><tr><td>Heat-inactivated FBS</td><td>Gibco</td><td>10082-147</td><td>20% (v/v) in DMEM</td></tr><tr><td>Penicillin-streptomycin-glutamine</td><td>Gibco</td><td>10378-016</td><td>1/100 dilution in DMEM</td></tr><tr><td>4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride</td><td>Molecular Probes</td><td>D1306</td><td>Nuclei staining</td></tr><tr><td>Phalloidin-CF647 conjugate (25 units/ml)</td><td>Biotium</td><td>00041</td><td>F-actin staining</td></tr><tr><td>Flexcell FX-5000 tension system</td><td>Flexcell International Corporation</td><td>FX5K</td><td>Peristalsis-like stretcing motion (10% cell strain, 0.15 Hz frequency)</td></tr><tr><td>Inverted epifluorescence microscope</td><td>Zeiss</td><td>Axio Observer Z1</td><td>Imaging, DIC</td></tr><tr><td>Scanning confocal microscope</td><td>Leica</td><td>DMI6000</td><td>Imaging, Fluorescence</td></tr><tr><td>UVO Cleaner</td><td>Jelight Company Inc</td><td>342</td><td>Surface activation of the gut-chip</td></tr><tr><td>Type I collagen&amp;nbsp;</td><td>Gibco</td><td>A10483-01</td><td>Extracellular matrix component for cell culture into the chip</td></tr><tr><td>Matrigel</td><td>BD</td><td>354234</td><td>Extracellular matrix component for cell culture into the chip</td></tr><tr><td>1 ml disposable syringe</td><td>BD</td><td>309628</td><td>Cell and media injection stuff</td></tr><tr><td>25 G 5/8 needle</td><td>BD</td><td>329651</td><td>Cell and media injection stuff</td></tr><tr><td>Syringe pump</td><td>Braintree Scientific Inc.</td><td>BS-8000</td><td>Injection equipment into the chip</td></tr><tr><td>VSL#3</td><td>Sigma-Tau Pharmaceuticals</td><td>7-45749-01782-6</td><td>A formulation of 8 different commensal gut microbes</td></tr><tr><td>Reinforced Clostridial Medium</td><td>BD</td><td>218081</td><td>Anaerobic bacteria culture medium</td></tr><tr><td>GasPak EZ Anaerobe Container System with Indicator</td><td>BD</td><td>260001</td><td>Anaerobic gas generating sachet&amp;nbsp;</td></tr><tr><td>4% paraformaldehyde</td><td>Electron Microscopy Science</td><td>157-4-100</td><td>Fixing the cells for staining</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>Permeabilizing the cells</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7030</td><td>Blocking agent for staining of the cells</td></tr><tr><td>Corona treater</td><td>Electro-Technic Products</td><td>BD-20AC</td><td>Plasma generator for fabrication of the chip</td></tr><tr><td>Steriflip&amp;nbsp;</td><td>Millipore</td><td>SE1M003M00</td><td>Degasing the complete culture medium</td></tr><tr><td>Disposable hemocytometer</td><td>iNCYTO</td><td>DHC-N01</td><td>For manual cell counting</td></tr></tbody></table>

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.

To emulate the human intestinal host-microbiome ecosystem in vitro, it is necessary to develop an experimental protocol to reconstitute the stable long-term co-culture of gut bacteria and human intestinal epithelial cells under physiological conditions such as peristalsis-like mechanics and fluid flow. Here, we utilize a biomimetic gut-on-a-chip microdevice (Figure 1A) to co-culture living microbial cells in direct contact with living human villi for periods of a week or more in vitro. The intestinal epithelial cells spontaneously form well differentiated villi when cultured on one side of a porous ECM-coated membrane in one channel of a 2-channel microfluidic device in the presence of physiologically relevant fluid flow and cyclic mechanical deformations5. The microengineered villi replicate structures and functions of human small intestinal villi, including formation of columnar epithelial cells lined by an apical brush border, basal proliferative crypts with migration and differentiation of daughter cells progressing from the crypt to villus tip, high levels of mucus production, enhanced drug metabolizing activity, and increased glucose reuptake8. Living endothelial cells can be cultured on the opposite side of the same membrane to recreate the tissue-tissue interface of the intestinal wall, and immune cells can be cultured in the system as well10. To validate the protective function of intestinal epithelial cells, tight junction barrier of intestinal villi formed in a gut-on-a-chip is intermittently quantitated by measuring the transepithelial electrical resistance (TEER)5,10,12. Routine monitoring of TEER value is required to estimate the integrity of a cell monolayer or intestinal villi at every juncture (e.g., prior to adding bacteria into the lumen).
To reestablish the host-microbe ecosystem, living microbial cells resuspended in the antibiotic-free cell culture medium (cell density, ~1.0 x 107 CFU/ml) are inoculated into the lumen of the epithelial microchannel (Figure 1B, &#34;Inoculation&#34;). After microbial cells adhere on the apical surface of villi in the absence of flow for ~1.5 hr (Figure 1B, &#34;Attachment&#34;), physiological flow (40 &#181;l/hr) is resumed through both channels with cyclic mechanical deformations (10% strain at 0.15 Hz) to remove unbound gut bacteria and supply nutrients to both bacterial and villus epithelial cells (Figure 1B, &#34;Co-culture&#34;). This co-culture protocol allows the stable colonization of multiple species of probiotic bacteria in the intervillus space, with viable bacterial microcolonies being maintained for up to two weeks in co-culture (Figures 2A, 2B). In this study, a commercial probiotic formulation that contains a mixed population of 8 different facultative or obligate anaerobic, probiotic strains of Bifidobacterium breve, B. longum, B. infantis, Lactobacillus acidophilus, L. plantarum, L. paracasei, L. bulgaricus, and Streptococcus thermophiles is used.
This co-culture method can be broadly applied to emulate the human intestinal host-microbe ecosystem. For instance, non-pathogenic GFP E. coli can be co-cultured on the surface of intestinal villi grown in a gut-on-a-chip device. Based on the same protocol described above, adhered GFP E. coli cells on the villi grow from single cells at 1 hr to multiple microcolonies with 3 days (Figure 3A, 1 hr vs. 72 hr) that locate in the intervillus spaces (Figure 3B, 1 hr vs. 72 hr) when cultured under trickling flow (40 &#181;l/hr) in the presence of peristalsis-like deformations (10%, 0.15 Hz).
<img alt="Figure 1" src="/files/ftp_upload/54344/54344fig1.jpg" /> 
Figure 1. The human Gut-on-a-Chip microphysiological system for host-gut microbiome co-culture. (A) A photograph (left) and a schematic (right) of a 2-channel, gut-on-a-chip microfluidic device. Arrows indicate the direction of culture medium flow (blue, top microchannel; red, bottom microchannel). (B) Schematic diagrams of the procedure of host-gut microbiome co-culture in the gut-on-a-chip. After intestinal epithelial cells form villi (~100 hr), microbial cells resuspended in the cell culture medium are introduced to the lumen microchannel (Inoculation), then the flow of culture medium is suspended for ~1.5 hr (Attachment). After microbial cells adhere to the apical surface of the intestinal villi, flow is resumed (Co-culture). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54344/54344fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54344/54344fig2.jpg" /> 
Figure 2. Co-culture of multiple probiotic bacteria with intestinal villi in the gut-on-a-chip. (A) A differential interference contrast micrograph shows a microcolony of probiotic bacterial cells growing between the villi in the gut chip. (B) A higher power magnification view of A (a black dotted square) showing the microcolony of probiotic bacterial cells (a red arrow). V, villi; Scale bar = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54344/54344fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54344/54344fig3.jpg" /> 
Figure 3. Co-culture of non-pathogenic, GFP-labeled E. coli with intestinal villi in the gut-on-a-chip. (A) Overlaid fluorescence and DIC microscopic images taken at 1 and 72 hr, displaying the colonization of GFP E. coli on the intestinal villi grown in the gut-on-a-chip. (B) High power magnification views of overlaid fluorescence confocal micrographs taken at 1 and 72 hr showing the growth of GFP-labeled E. coli from a single cell (a red arrow) to a microcolony (a white arrow). Blue, nuclei; magenta, F-actin; Scale bar = 30 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54344/54344fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device at JoVE.com

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

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 ...

co-culture-living-microbiome-with-microengineered-human-intestinal

Nanyang Technological University

Research

JoVE Journal

Bioengineering

22.3K Views.  The University of Texas at Austin.  We beschrijven een in vitro protocol co-cultuur darm microbioom en darmvlokken voor een langere periode met een menselijke darm-on-a-chip microphysiological systeem. 

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

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Biochemistry

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Biology

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...

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 ...

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

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

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

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

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

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

Medicine

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

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

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

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

An Ex Vivo Gut Organ Culture System to Study Host-Microbiota Interactions

Source: Azriel, S., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62779">An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions.</a> J. Vis. Exp. (2021).
In this video, we describe the methodology for setting up an ex vivo gut organ culture system to study the interactions between mouse intestinal tissue fragments and specific gut bacteria. The device consists of two pairs of input-output ports, the first connecting the intestinal tissue lumen and supplying the specific bacterial suspension into it, and the second continuously supplying media to the device&#39;s wells to maintain ex vivo tissue viability.

Source: Azriel, S., et al. An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions. J. Vis. Exp. (2021).
In this video, we ...

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

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