An in vitro platform for study of the human gut microbiome under an oxygen gradient

Abstract

The complex, dynamic environment of the human lower gastrointestinal tract is colonized by hundreds of bacterial species that impact health and performance. Ex vivo study of the functional interactions between microbial community members in conditions representative of those in the gut is an ongoing challenge. We have developed an in vitro 40-plex platform that provides an oxygen gradient to support simultaneous maintenance of microaerobic and anaerobic microbes from the gut microbiome that can aid in rapid characterization of microbial interactions and direct comparison of individual microbiome samples. In this report, we demonstrate that the platform more closely maintained the microbial diversity and composition of human donor fecal microbiome samples than strict anaerobic conditions. The oxygen gradient established in the platform allowed the stratification and subsequent sampling of diverse microbial subpopulations that colonize microaerobic and anaerobic micro-environments. With the ability to run forty samples in parallel, the platform has the potential to be used as a rapid screening tool to understand how the gut microbiome responds to environmental perturbations such as toxic compound exposure, dietary changes, or pharmaceutical treatments.

Keywords: Gut; In vitro model; Microbial community; Microbiome; Oxygen.

Fig. 1

The 40-plex in vitro oxygen gradient platform. a Photograph of the 3D-printed platform containing 40 modified filter tube inserts for culture. b Photograph of the bottom of the culture layer showing the bottom of the filter tube inserts. c CAD cross-section of the system highlighting the 0% oxygen chamber (top; blue) and microoxygen chamber (bottom; red). The two optical oxygen probes, labeled 1 and 2, are inserted just off the bottom of a filter tube insert and the outflow of the 0% oxygen chamber respectively. d Complete setup including two convection heating fans for maintaining temperature in a custom Plexiglas box, gas humidification bubble jar, and tubing for gas delivery

Fig. 2

Oxygen traces of a PBS control sample in the in vitro oxygen gradient platform taken the bottom of a filter tube insert filled with reduced PBS (orange) and the 0% oxygen chamber (green) using fiber optic oxygen sensors. Numbers correspond to the position of the probe as indicated in Fig. 1c

Fig. 3

Comparison of initial microbial composition of human fecal microbiomes from three donors to that after 24-h oligotrophic incubation in the in vitro oxygen gradient platform or in anaerobic conditions. a Alpha-diversity (Shannon) index of OTUs identified in triplicate samples from the starting initial fecal slurry (blue) or after 24-h culture in the in vitro oxygen gradient platform device (green) or in anaerobic culture (red). The mean, standard deviation from the mean, and confidence intervals are indicated. b Beta-diversity comparison using principal component analysis (Bray-Curtis or Weighted UniFrac) of OTUs identified in triplicate samples from the starting initial fecal slurry (square) or after 24-h culture in the in vitro oxygen gradient platform (triangle) or in anaerobic culture (circle) of three human fecal microbiomes (indicated by color)

Fig. 4

Changes in the microbial compositions of human fecal microbiomes from three donors cultured for 24-h in anaerobic conditions or in the in vitro oxygen gradient platform. a Log₂ change in the average reads in triplicate samples from the initial fecal slurry after anaerobic (dark bars) or in vitro oxygen gradient platform (light bars). Data for the 5 most prevalent phyla are indicated for three different donor microbiomes (blue, gray, and green) and analysis of samples from three combined donors (red). A single asterisk (*) indicates a difference compared to initial fecal slurry with an adjusted p-value < 0.05. A dagger (†) indicates a difference between the anaerobic and in vitro oxygen gradient platform with an adjusted p-value < 0.05. b Log₂ difference in average reads at the genus level in triplicate samples of human gut microbiome cultured for 24-h under anaerobic conditions compared to the in vitro oxygen gradient platform. Data from three human donors and combined samples is indicated, with those with an adjusted p-value < 0.05 indicated (*). Only those genera with a significant difference in one donor or combined samples are indicated. Genera not found in a particular sample are displayed (n/a)

Fig. 5

Differences in the microbial compositions of samples of human fecal microbiome culture taken from the anaerobic (top) and microaerobic (bottom) regions of the in vitro oxygen gradient platform after 24-h. a Log₂ change in the average reads of triplicate samples from the anaerobic (top) sample compared to the microaerobic (bottom) sample. Data for the 5 most prevalent phyla are indicated for three different donor microbiomes (blue, gray, and green) and the combined samples (red). An asterisk (*) indicates a difference with an adjusted p-value < 0.05. b Log₂ difference in average reads in triplicate human gut microbiome culture samples between the anaerobic (top) and microaerobic (bottom) regions of the in vitro oxygen gradient platform. Data from three human donors and combined samples is indicated, with those with an adjusted p-value < 0.05 indicated (*). Only those genera with a significant difference in one donor or combined samples are indicated. Genera not found in a particular sample are displayed as (n/a)