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

Summary

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.

Abstract

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 'germ-free' 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.

Introduction

The human intestine harbors a stunningly diverse array of microbial species (<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 (<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 "Gut-on-a-Chip" microphysiological system (Figure 1A, left) to emulate the host-gut microbiome interactions that occur in the living human intestine^5,8. 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 'germ-free' 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 weeks^5,10.

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.

Protocol

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 µm height x 1 cm length) separated by a flexible porous (10 µm in pore diameter, 25 µm in spacing pore to pore) PDMS membrane^5,9. Fabricate the gut-on-a-chip (Figure 1A, left) following the steps provided.

1. Microfabrication Procedure of the Gut-on-a-chip^5,9.
   1. 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.
   2. 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 °C in a dry oven for at least 4 hr.
   3. 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).
   4. 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 µm in diameter, 25 µm 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 °C for 12 hr or longer.
   5. 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.
   6. 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.
   7. 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.
   8. Incubate the whole setup at 80 °C O/N for irreversible bonding of the two PDMS layers.
   9. 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.
   10. 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.
   11. 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.
   12. 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.
   13. Cure the whole setup in a dry oven at 80 °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.
2. 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° bent stainless steel blunt-end needle.
3. Connect the tubing to the holes linked to the vacuum chambers using a 90° bent stainless steel blunt-end needle.
4. Sterilize the microchannels and tubing by flowing 70% (v/v) ethanol using a 1 ml sterilized disposable syringe.
5. Dry out the microchannels and tubing in a 60 °C dry oven O/N.

2. Growth of Microengineered Intestinal Villi in the Gut-on-a-chip Device

1. Seed Intestinal Epithelial Cells (e.g. Caco-2BBE) on a Gut-on-a-chip Microdevice^5,9.
   1. 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 °C dry oven O/N (see step 1.5) prior to the surface activation.
   2. Expose the complete setup of a gut-on-a-chip microsystem (i.e., a body of the gut-on-a-chip connected to tubing) to UV light (253.7 nm) and ozone simultaneously for 40 min.
   3. Cool down the device at RT for 15 min under UV light in a biosafety cabinet (BSC).
   4. Introduce 100 µl of the ECM coating solution- a mixture of 50 µg/ml type I collagen and 300 µg/ml extracellular matrix mixture diluted in the serum-free Dulbecco's Modified Eagle Medium (DMEM) - into both upper and lower microchannels using a disposable, sterile 1 ml syringe.
   5. Incubate the whole setup in a 37 °C humidified 5% CO₂ incubator for 1 hr.
   6. Degas 50 ml of pre-warmed (37 °C) complete culture medium (DMEM, 20%, v/v, fetal bovine serum (FBS), 100 units/ml penicillin, and 100 µg/ml streptomycin; aliquoted in a 3 ml disposable syringe) using a 50 ml filtration system (0.45 µm in pore size) in the BSC for 1 min.
      1. 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.
   7. Place the aliquot of degassed complete medium in a 3 ml disposable syringe and incubate in a 37 °C humidified 5% CO₂ incubator for 1 hr.
   8. Connect two 3 ml disposable syringes containing degassed, pre-warmed complete culture medium to the upper and lower microchannels.
   9. Flow the degassed complete cell culture medium into the upper microchannel using a syringe pump at the volumetric flow rate of 30 µl/hr (equivalent to the shear stress at 0.02 dyne/cm²) in a 37 °C humidified 5% CO₂ incubator O/N.
2. Formation of Microengineered Caco-2 Villi in the Gut-on-a-chip.
   1. Use Caco-2BBE cells with the passage number between 50 and 65.
   2. Grow Caco-2 cells in a T75 flask containing complete culture medium in a 37 °C humidified 5% CO₂ incubator for 4-5 days to obtain fully confluent cells.
   3. 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.
   4. Add 1 ml of 0.05% trypsin/EDTA solution and incubate in a 37 °C humidified 5% CO₂ incubator for 5 min.
   5. Resuspend the dissociated cells with 10 ml pre-warmed complete cell culture medium (with FBS) by pipetting up and down three to five times.
   6. Spin down the cell suspension by centrifugation at 500 x g for 5 min, then remove the supernatant.
   7. 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.
   8. Infuse 100 µl 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.
   9. Clamp all inlets and outlets of tubing connected to the upper and lower microchannels using binder clips.
   10. Incubate the whole setup in a 37 °C, humidified 5% CO₂ incubator for 1 hr to allow dissociated Caco-2 cells to adhere to the surface of the porous membrane.
   11. Remove the clamps and resume the flow of culture medium only to the upper microchannel at 30 µl/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.
   12. When cells form a monolayer, perfuse the culture medium into both upper (lumen) and lower (capillary) microchannels at the same flow rate of 30 µl/hr.
   13. Application of mechanical deformations mimicking peristalsis-like motions^5,9.
       1. Turn on the vacuum pump equipped with the tension equipment.
       2. Connect the vacuum chambers to the vacuum controller via tubing with a stainless steel connector.
       3. 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 "Start".
   14. Maintain the constant flow of culture medium (30 µl/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 microchannel^5,8-10.
3. 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.
   1. Grow microengineered Caco-2 villi by repeating steps from 2.1 to 2.2.
   2. 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 µl/hr.
   3. Introduce 100 µl 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.
   4. 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).
   5. Incubate the setup in a 37 °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.
   6. Take out a whole setup from the CO₂ incubator, and flip the setup over again.
   7. 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 µl/hr with mechanical stretching motions (10%, 0.15 Hz) for at least three days to form cell-cell junctions of an endothelial monolayer.

3. Host-gut Microbiome Co-culture in a Gut-on-a-chip Microdevice

1. Perform pre-culture of the bacterial cells as follows to co-culture commensal gut microbiome on the microengineered intestinal villi.
   1. 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.
   2. 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.
   3. 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 °C humidified 5% CO₂ incubator O/N.
2. Inoculation of Microbiome and Co-culture with Intestinal Epithelial Cells^5,10.
   1. 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.
   2. 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.
   3. 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.
   4. 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).
   5. Infuse the cell suspension into the lumen side of the microchannel containing the "germ-free" 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.
   6. Perfuse the pre-warmed antibiotic-free culture medium into both upper and lower microchannels at 40 µl/hr with cyclic rhythmical deformations (10%, 0.15 Hz).
   7. 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 °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.
3. Image Cells^5,8-10
   1. Perform DIC, epifluorescence, or laser scanning confocal microscopy for recording microbial and epithelial morphology^5,8,10.
      1. 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.
      2. For fluorescence imaging of villus epithelium, flow PBS for washing the cells at 100 µl/hr for 10 min.
         1. Fix villi with 4% (w/v) paraformaldehyde for 15 min. Then flow PBS for washing the cells at 100 µl/hr for 10 min.
         2. 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 µl/hr for 10 min.
         3. Block cells with 2% (w/v) BSA solution in PBS for 1 hr. Then flow PBS for washing the cells at 100 µl/hr for 10 min.
         4. Add 300 nM of 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) solution diluted in PBS for the nuclear staining under light protection.
         5. Add 25 units/ml of fluorescent phalloidin (Phalloidin-CF647 conjugate) dissolved in PBS for F-actin staining under light protection.
         6. 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.

Results

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...

Discussion

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., organic ...

Materials

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