Multispecies Commensal Biofilm Reduces Glucose Transport in a Small Intestine on a Chip

Introduction: The small intestine is the primary organ for food digestion and absorption. The microbiome in the small intestinal lumen is important in assisting this process. Intestinal bacteria can metabolize indigestible carbohydrates and perform an essential role in vitamin synthesis [1]. The microbiome also promotes the adaptive immunity of the host [2]. Understanding microbiome and small intestinal epithelial interactions will advance the knowledge of small intestinal physiology. In the current work, a small intestine on a chip (SIOC) has been developed with intestinal epithelial cells and a mock community of the microbiome. The four species of bacteria used in the work are among the predominant organisms in the duodenum. Furthermore, the use of mechanical stimulation in the form of fluid shear stress generated an in vivo like environment on the chip. The device has been successfully used to study brush border enzyme activity and nutrient transport across the in vitro epithelial barrier.

Materials and

Methods: A bilayer microfluidic device was fabricated by polycarbonate machining, and a porous (0.2 µm) polyester membrane was used to separate the apical and basolateral chambers. The small intestinal epithelium was formed by seeding a mixture of Caco-2 and HT29-MTX-E12 cells in 3:1 ratio onto the apical chamber. The in vitro epithelium was allowed to differentiate in dynamic flow conditions (0.62 mPa) for 6 days before the introduction of the mock microbiome. A biofilm composed of four species of bacteria, specifically Bifidobacterium bifidum, Enterococcus faecalis, Lacticaseibacillus rhamnosus, and Streptococcus salivarius [3] was cultured for 6 days and the matured biofilm was collected and seeded (at 103 CFU/mL) on top of the differentiated in vitro epithelium. Co-culture of both the mammalian and bacterial cells continued for another 4 days in dynamic conditions and samples were collected at different intervals for brush border enzyme assays. A food model, mimicking the food intake of a normal US adult was in vitro digested and introduced to the apical chamber for glucose and protein transport assays. Samples of the basolateral chamber were collected, at various time points, to estimate the amount of nutrient transport from the food model to blood circulation.

Results, Conclusions, and Discussions: The in vitro small intestinal epithelium showed tight junction formation under a shear stress of 0.62 mPa as early as 5 days from the initiation of flow. Barrier differentiation was also confirmed by visualization of villi-like structure formation. The mucus layer thickness was 10.8 µm which is close to the thickness of the firmly adherent mucus layer in the small intestine [4]. Once the microbiome was introduced, the lucifer yellow permeability of the barrier remained comparable to the control. A distinct biofilm layer, mucus layer, and intestinal epithelial layer could be observed after 4 days of co-culture in the dynamic system (figure 1(a)). Enzyme assays revealed significantly higher aminopeptidase-N and alkaline phosphatase activity in the SIOC compared to static conditions (figure 1(b), (c)). Aminopeptidase-N activity was significantly higher for the mock microbiome alone, which shows the importance of gut bacteria in food digestion. The protein transport across the barrier remained comparable at various intervals with or without the presence of the microbiome (figure 1(d)). Glucose transport from the apical to the basolateral chamber was significantly reduced in the presence of the microbiota showing the importance of gut bacteria in small intestinal homeostasis (figure 1(e)).

The small intestine on a chip successfully replicates the human intestinal epithelium, microbiome, and dynamic conditions. A stable barrier integrity for a longer period enabled us to perform long-term studies on the chip. Nutrient transport studies and brush border enzyme activity on the chip show the significance of small intestinal microbiome. This SIOC can further be used to study food interaction and food-related toxicity to the intestinal wall. It can also be used as a model for oral drug transport studies of the small intestine.

Acknowledgements (Optional): This work was funded by the National Institutes of Health (R01ES028788)
References

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