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

Published: August 30th, 2016



1Department of Biomedical Engineering, The University of Texas at Austin, 2Wyss Institute for Biologically Inspired Engineering at Harvard University, 3Vascular Biology Program, Boston Children's Hospital, Harvard Medical School, 4John A. Paulson School of Engineering and Applied Sciences, Harvard University

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 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.

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) he....

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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 membrane5,9. Fabricate the gut-on-a-chip (Figure 1A, left) following the steps provided.

  1. Micr.......

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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.......

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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 .......

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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....

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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
Trypsin/EDTA solution (0.05%) Gibco 25300-054 Warm it up at 37℃ in a water bath.
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
25G5/8 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

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