Name: Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device (Video) | JoVE Journal
Uploaded: 2016-08-30T15:00:00.000+00:00
Duration: 10 min 51 s
Description: Learn to co-culture living gut microbiome with microengineered human intestinal villi using a gut-on-a-chip microfluidic device. This method enables researchers to study host-microbe interactions and gastrointestinal disease mechanisms in vitro.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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>

<ol><li>Turnbaugh, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+obesity-associated+gut+microbiome+with+increased+capacity+for+energy+harvest%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">An obesity-associated gut microbiome with increased capacity for energy harvest.</a> Nature. 444, 1027-1031 (2006).</li>
<li>Tremaroli, V., Backhed, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+interactions+between+the+gut+microbiota+and+host+metabolism%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Functional interactions between the gut microbiota and host metabolism.</a> Nature. 489, 242-249 (2012).</li>
<li>Sekirov, I., Russell, S. L., Antunes, L. C. M., Brett Finlay, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gut+Microbiota+in+Health+and+Disease%5BTitle%5D)+AND+%22Physiol.+Rev%22%5BJournal%5D)">Gut Microbiota in Health and Disease.</a> Physiol. Rev. 90, 859-904 (2010).</li>
<li>Turnbaugh, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+human+microbiome+project%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The human microbiome project.</a> Nature. 449, 804-810 (2007).</li>
<li>Kim, H. J., Huh, D., Hamilton, G., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+gut-on-a-chip+inhabited+by+microbial+flora+that+experiences+intestinal+peristalsis-like+motions+and+flow%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow.</a> Lab Chip. 12, 2165-2174 (2012).</li>
<li>Round, J. L., Mazmanian, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+gut+microbiota+shapes+intestinal+immune+responses+during+health+and+disease%5BTitle%5D)+AND+%22Nat+Rev+Immunol%22%5BJournal%5D)">The gut microbiota shapes intestinal immune responses during health and disease.</a> Nat Rev Immunol. 9, 313-323 (2009).</li>
<li>Garrett, W. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Communicable+ulcerative+colitis+induced+by+T-bet+deficiency+in+the+innate+immune+system%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Communicable ulcerative colitis induced by T-bet deficiency in the innate immune system.</a> Cell. 131, 33-45 (2007).</li>
<li>Kim, H. J., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gut-on-a-Chip+microenvironment+induces+human+intestinal+cells+to+undergo+villus+differentiation%5BTitle%5D)+AND+%22Integr+Biol%22%5BJournal%5D)">Gut-on-a-Chip microenvironment induces human intestinal cells to undergo villus differentiation.</a> Integr Biol. 5, 1130-1140 (2013).</li>
<li>Huh, D., Kim, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfabrication+of+human+organs-on-chips%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Microfabrication of human organs-on-chips.</a> Nat Protoc. 8, 2135-2157 (2013).</li>
<li>Kim, H. J., Li, H., Collin, J. J., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contributions+of+microbiome+and+mechanical+deformation+to+intestinal+bacterial+overgrowth+and+inflammation+in+a+human+gut-on-a-chip%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci%22%5BJournal%5D)">Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human gut-on-a-chip.</a> Proc. Natl. Acad. Sci. 113, E7-E15 (2016).</li>
<li>Miller, W. G., Lindow, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+improved+GFP+cloning+cassette+designed+for+prokaryotic+transcriptional+fusions%5BTitle%5D)+AND+%22Gene%22%5BJournal%5D)">An improved GFP cloning cassette designed for prokaryotic transcriptional fusions.</a> Gene. 191, 149-153 (1997).</li>
<li>Odijk, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measuring+direct+current+trans-epithelial+electrical+resistance+in+organ-on-a-chip+microsystems%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">Measuring direct current trans-epithelial electrical resistance in organ-on-a-chip microsystems.</a> Lab Chip. 15, 745-752 (2015).</li>
<li>Lentle, R. G., Janssen, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Physical+characteristics+of+digesta+and+their+influence+on+flow+and+mixing+in+the+mammalian+intestine%3A+a+review%5BTitle%5D)+AND+%22J+Comp+Physiol+B%22%5BJournal%5D)">Physical characteristics of digesta and their influence on flow and mixing in the mammalian intestine: a review.</a> J Comp Physiol B. 178, 673-690 (2008).</li>
<li>Granato, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+surface-associated+lipoteichoic+acid+acts+as+an+adhesion+factor+for+attachment+of+Lactobacillus+johnsonii+La1+to+human+enterocyte-like+Caco-2+cells%5BTitle%5D)+AND+%22Appl+Environ+Microbiol%22%5BJournal%5D)">Cell surface-associated lipoteichoic acid acts as an adhesion factor for attachment of Lactobacillus johnsonii La1 to human enterocyte-like Caco-2 cells.</a> Appl Environ Microbiol. 65, 1071-1077 (1999).</li>
<li>Dewhirst, F. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+human+oral+microbiome%5BTitle%5D)+AND+%22J+Bacteriol%22%5BJournal%5D)">The human oral microbiome.</a> J Bacteriol. 192, 5002-5017 (2010).</li>
<li>Grice, E. A., Segre, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+skin+microbiome%5BTitle%5D)+AND+%22Nat+Rev+Microbiol%22%5BJournal%5D)">The skin microbiome.</a> Nat Rev Microbiol. 9, 244-253 (2011).</li>
<li>Hay, P. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Abnormal+bacterial+colonisation+of+the+genital+tract+and+subsequent+preterm+delivery+and+late+miscarriage%5BTitle%5D)+AND+%22Br+Med+J%22%5BJournal%5D)">Abnormal bacterial colonisation of the genital tract and subsequent preterm delivery and late miscarriage.</a> Br Med J. 308, 295-298 (1994).</li>
</ol>

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 ...

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

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 vill...

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 ...

This overall goal of this protocol is to demonstrate the villus growth of human intestinal cells under peristalsis-like motions and flow in a gut-on-a-chip microfluidic device, as well as to show how to co-culture these cells with living gut microbiome. So this method can help the key questions in the field of the gastroenterology, clinical microbiology, and the pharmaceutical sciences to identify the disease mechanism and to validate the efficacy and toxicity of the new drug compounds. The main advantage of this technique is that the researchers can establish the viable and functional post-microbiome ecosystem of the human living intestine in vitro using a microphysiological system.The implications of this technique can extend towards diseases such as ulcerative colitis or Crohn's disease, since the stable co-culture of the host cells with the microbiome can emulate the complexities of these diseases. Though this method can provide an insight into the complex interactions between the gut microbiome and the immune system in the intestine, it can also be applied to other organ systems where there are host microbe interactions, such as the skin, your genital tract, or the oral cavity. To begin, prepare the gut-on-a-chip microfluidic device as described in the accompanying text protocol.The device consists of a transparent gas-permeable silicon polymer containing two parallel microchannels, separated by a flexible, porous PDMS membrane. On each side of the channels are vacuum chambers, which can be used to apply cyclic mechanical strain. Sterilize the device by using a one-milliliter syringe to flow 70%ethanol through the channels.Then, dry the device in a 60 degree Celsius dry oven overnight. The next day, expose the gut-on-a-chip microsystem, along with its connected tubing, to UV light and ozone simultaneously for 40 minutes. Cool down the device while remaining under the UV light of a biosafety cabinet for 15 minutes.Next, load 100 microliters of an extracellular matrix coding solution into a one milliliter syringe, and dispense it into the upper microchannel of the device. Repeat this process to coat the lower microchannel as well. Place the whole setup in a 37 degree Celsius humidified 5%CO2 incubator for one hour.During the incubation, de-gas pre-warmed complete culture medium by passing it through a 0.45 micron filter in a 50-milliliter filtration system for one minute. Gently tap on the filtered medium for one minute to remove any bubbles or dissolved gas in the medium. Then, transfer the de-gassed, complete medium into two three-milliliter disposable syringes, and incubate the syringes for one hour.Next, connect the syringes to the upper and lower microchannels of the gut-on-a-chip, and wash out the coding solution by flowing complete medium into the upper and lower microchannels with a gentle pulse. Then, use a syringe pump to flow the de-gassed, complete-cell culture medium into the upper microchannel of the device, while incubating the chip overnight at 37 degrees Celsius and 5%CO2. Harvest a fully confluent T75 flask containing Caco-2 intestinal epithelial cells by first rinsing the cells with 10 milliliters of calcium and magnesium-free PBS.Then, aspirate the PBS and repeat the rinse two more times. Next, add one milliliter of 0.05%Trypsin-EDTA solution and incubate the flask for five minutes at 37 degrees Celsius. Once detached, add 10 milliliters of pre-warmed complete-cell culture medium, and pipette the cell suspension up and down three to five times before transferring it to a centrifuge tube.Spin down the cell suspension. Then, remove the supernatant, and re-suspend the cells at 150, 000 cells per centimeters squared in pre-warmed complete medium. Draw up 500 microliters of the cell suspension into a one-milliliter disposable syringe, equipped with a 25-gauge, 5/8-inch needle.Then, dispense 100 microliters of the cell suspension into the upper microchannel through the outlet tubing. Next, clamp all of the inlets and outlets that are connected to the upper and lower microchannels using binder clips. Incubate the whole setup for one hour to allow the intestinal epithelial cells to adhere to the surface of the porous membrane.Then, remove the clamps and connect the upper channel tubing to a culture medium filled syringe that has been placed on a syringe pump. Resume the flow of culture medium to the upper microchannel at 30 microliters per hour until the cells from an intact monolayer. Once the cells form a monolayer, connect the lower channel to the second syringe and perfuse the culture medium into both the upper lumen and lower capillary microchannels at the same flow rate of 30 microliters per hour.To create peristalsis-like motion in the chip, first turn on the vacuum pump. Connect the vacuum chambers of the device to the vacuum controller via tubing with a stainless steel connector. Then, set the controller so that it provides a cyclic sign stretching motion that is 10%of the mean cell strain at a frequenzy of 0.15 Hertz, then click start.Keep flowing culture medium to both the upper and lower microchannels in the presence of mechanical deformations for at least 80 hours to induce villus growth. Prepare a 50/50 mixture of Autoclaved Lactobacilli MRS Broth and Reinforced Clostridial Medium. Then, re-suspend a freeze-dried probiotic bacterial mixture in 10 milliliters of the mixture.Measure the optical density of the mixture at 600 nanometers and adjust the final cell density to approximately 0.2 optical density units. Aliquot three milliliters of the adjusted microbial cell suspension into disposable 10-milliliter sterile tubes. Place the aliquots into an anaerobic container and add two packs of anaerobic gas-generating sachets into the container.Close the container lid tightly and incubate the container setup without shaking in an incubator overnight. Next, prepare de-gassed antibiotic-free cell culture medium and load it into three-milliliter disposable syringes. Transfer the gut-on-a-chip device containing micro-engineered villi into the biosafety cabinet.Remove the syringes that are connected to the upper and lower microchannels of the device and replace them with syringes containing the de-gassed antibiotic-free cell culture medium. Once finished, place the device back into the CO2 incubator. Then, flow the antibiotic-free culture medium into the device for 12 hours prior to seating the microbiome.Next, spin down the pre-cultured probiotic bacterial cell mixture. Aspirate the supernatant, then re-suspend the bacteria in the antibiotic-free DMEM. Load approximately 100 microliters of this cell suspension into a one-milliliter syringe attached to a 25-gauge, 5/8-inch needle.Infuse the bacteria into the lumen side of the microchannel containing the previously germ-free intestinal villi. Allow the microbial cells to adhere to the apical surface of the intestinal villi for one and a half hours without flow by clamping the tubing. Then, perfuse the pre-warmed antibiotic-free culture medium into both the upper and lower microchannels at 40 microliters per hour with cyclic rhythmical deformations.This co-culture protocol allows for the stable colonization of multiple species of probiotic bacteria in the intervillous space. Viable bacterial micro-colonies can be maintained for up to two weeks in co-culture. Shown here is a different co-culture consisting of both GFP labeled E.coli and intestinal villi.The high-powered magnification clearly shows the location of bacterial micro-colonies at the 72-hour time point. While attempting this procedure, it is important to remember to gently and slowly inject the culture medium into the microfluidic channels to avoid damaging the epithelial cell monolayer in the early stages of the culture. Based on this method, a co-culture method with the human immune cells can also give some clues in terms of how the microbiome can crosstalk to the immune system and influences on the homeostasis optimum intestinal health.After watching this video, you should have a good understanding of how to reconstitute the micro-environment of the human intestine and to demonstrate that co-culture between the microbiome and the human intestinal villi in vitro. Working with pathogenic bacteria and human cells are extremely dangerous, so don't forget to work inside biosafety cabinet.

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

Research

JoVE Journal

Bioengineering

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.

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

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's wells to maintain ex vivo tissue viability.

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

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

Here, we present a protocol for culturing the gut microbiota of the colon in vitro, using a series of bioreactors that simulate the physiological conditions of the gastro intestinal tract.

Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota

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

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

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

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

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.

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

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

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

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.

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

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.

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

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

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

Learn to co-culture living gut microbiome with microengineered human intestinal villi using a gut-on-a-chip microfluidic device. This method enables researchers to study host-microbe interactions and gastrointestinal disease mechanisms in vitro.

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

Explore More Videos