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) he....

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 age....

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
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