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

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 milieu^1,2. The composition and functionality of the gut microbiota change spatially along the GIT, forming region specific communities, with the most diversity found distally^2,3,4,5. 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.......

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.......

Materials

Name	Company	Catalog Number	Comments
TWINSHIME	Prodigest	NA
defined medium (Adult M-SHIME growth medium with starch)	Prodigest	NA
Masterflex tubing	cole Parmer	NA
Urine Drainage bag	Bard	NA
Labsorb	Sigma-Aldrich	NA
Fecal sample	Openbiome	NA
Syringes	Becton Dickson	NA
Defined medium	Prodigest	NA
Oxgall Bile	Becton Dickson	NA
Pancreatin	Sigma-Aldrich	NA
Glass ware	Ace Glass	NA
Porcine mucin	Sigma-Aldrich	NA
Bacteriological agar	Sigma-Aldrich	NA
Sterilization pouches	VWR	NA
BeadBug	Benchmark Scientific	NA
Triple-Pure High Impact Zirconium 0.1mm Bead beater tube	Benchmark Scientific	NA
RNAse free, DNAse free, sterile water	Roche	NA
Shimadzu QP2010 Ultra GC/MS	Shimadzu	NA
Stabilwax-DA column, 30m, 0.25mm ID, 0.25µm	Restek	NA
plastic mucin carriers	Prodigest	NA