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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its complex nature, and its diverse association with mammalian components. The goal of this protocol is to culture the gut microbiota in vitro, which allows for the study of the gut microbiota dynamics, without having to consider the contribution of the mammalian milieu. Using in vitro culturing technology, the physiological conditions of the gastro intestinal tract are simulated, including parameters such as pH, temperature, anaerobiosis, and transit time. The intestinal surface of the colon is simulated by adding mucin-coated carriers, creating a mucosal phase, and adding further dimension. The gut microbiota is introduced by inoculating with the human fecal material. Upon inoculation with this complex mixture of bacteria, specific microbes are enriched in the different longitudinal (ascending, transverse and descending colons) and transversal (luminal and mucosal) environments of the in vitro model. It is crucial to allow the system to reach a steady state, in which the community and the metabolites produced remain stable. The experimental results in this manuscript demonstrate how the inoculated gut microbiota community develops into a stable community over time. Once steady state is achieved, the system can be used to analyze bacterial interactions and community functions or to test the effects of any additives on the gut microbiota, such as food, food components, or pharmaceuticals.

Introduction

The gut microbiota is a community of micro-organisms that reside in the human gastrointestinal tract (GIT).  This community reaches maximum concentration in the colon, which is estimated to hold 1013-1014 bacteria, from 500-1,000 species, that live in symbiosis with the colon milieu1,2. The composition and functionality of the gut microbiota change spatially along the GIT, forming region specific communities, with the most diversity found distally2,3,4,5. F....

Protocol

1. Materials and Preparations

NOTE: The defined medium is purchased as a powder (see Table of Materials). The composition of the defined medium in g/L is the following: Arabinogalactan (1.2), Pectin (2.0), Xylan (0.5), Glucose (0.4), Yeast extract (3.0), Special peptone (1.0), Mucin (2.0), L-cysteine-HCl (0.2).

  1. Prepare the defined medium
    1. Fill a 4 L flask with 2 L of double distilled, deionized water.
    2. Add 29.2.......

Representative Results

The above protocol describes set up, inoculation, and running of a 5-stage in vitro system to study the gut microbiota of the colon. To generate the data presented below, following DNA extraction, 16S rRNA marker gene DNA sequencing of the V1V2 region was performed using the high throughput sequencing (e.g., MiSeq Illumina platform) by the Microbiome Center at the Children’s Hospital of Philadelphia27. QIIME (Quantitative Insight into Microbial Ecolo.......

Discussion

In vitro culturing systems have been developed to study the gut microbiota of the large intestine. They use apparatuses designed to simulate the physiological conditions of the gastro intestinal tract, promoting the growth of a mature gut microbial community for each region of the colon33. While the concept is logical and comprehensible, the actual running of in vitro culturing systems to study the gut microbiota requires precision and an understanding of what is required and expected to produce r.......

Acknowledgements

Ms. Audrey Thomas-Gahring is acknowledged for her GC/MS work. We would also like to thank Massimo Marzorati for editing the manuscript.

....

Materials

NameCompanyCatalog NumberComments
TWINSHIMEProdigestNA
defined medium (Adult M-SHIME growth medium with starch)ProdigestNA
Masterflex tubingcole ParmerNA
Urine Drainage bagBardNA
LabsorbSigma-AldrichNA
Fecal sampleOpenbiomeNA
SyringesBecton DicksonNA
Defined mediumProdigestNA
Oxgall BileBecton DicksonNA
PancreatinSigma-AldrichNA
Glass wareAce GlassNA
Porcine mucinSigma-AldrichNA
Bacteriological agarSigma-AldrichNA
Sterilization pouchesVWRNA
BeadBugBenchmark ScientificNA
Triple-Pure High Impact Zirconium 0.1mm Bead beater tubeBenchmark ScientificNA
RNAse free, DNAse free, sterile waterRocheNA
Shimadzu QP2010 Ultra GC/MSShimadzuNA
Stabilwax-DA column, 30m, 0.25mm ID, 0.25µmRestekNA
plastic mucin carriersProdigestNA

References

  1. Johansson, M., Larsson, J., Hansson, G. The two mucus layers of colon are organized by the MUC2 mucin, whereas the outer layer is a legislator of host-microbial interactions. Proceedings of the National Academy of Sciences of the United States of America. 108 (1), 4659-4665 (2011).
  2. Xu, J., Gordon, J.

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In Vitro CulturingGut MicrobiotaColonDefined MediumMucinFecal HomogenateBioreactorPH ControlLuminal SampleMucosal SampleDNA AnalysisShort chain Fatty Acids

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