Ordinarily, the microbial flora within the gut are amiable enough. That’s why they qualify as commensal. But when they are taken from their homes and brought to unnatural surroundings, they can quickly wear out their welcome. In fact, within a day, they can overgrow and kill the host human cells in culture dishes. Nonetheless, the gut’s microbes are capable of being good guests—and displaying ordinary host-microbiome behavior—if they are properly accommodated.

To show how hosts can help potentially disagreeable guests remember their etiquette, researchers at Harvard’s Wyss Institute have developed a microfluidic Intestine Chip, that is, an intestine-on-a-chip. It can culture a stable complex human microbiome in direct contact with a vascularized human intestinal epithelium for at least 5 days.

Key to the Intestine Chip’s success is the maintenance of low oxygen conditions. Many of the commensal microbes in the intestine are anaerobic, and so they require very low oxygen conditions to grow. But these conditions can injure human cells. The Intestine Chip’s design allowed it to accommodate both aerobic and anaerobic gut microbes as well as human cells.

Details appeared May 13 in the journal Nature Biomedical Engineering in an article titled, “A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip.”

“We [used] a microfluidic intestine-on-a-chip that permits the control and real-time assessment of physiologically relevant oxygen gradients,” the article’s authors wrote. “When compared to aerobic co-culture conditions, the establishment of a transluminal hypoxia gradient in the chip increased intestinal barrier function and sustained a physiologically relevant level of microbial diversity, consisting of over 200 unique operational taxonomic units from 11 different genera and an abundance of obligate anaerobic bacteria, with ratios of Firmicutes and Bacteroidetes similar to those observed in human feces.”

Most of what we know about human-microbiome interactions is based on correlational studies between disease state and bacterial DNA contained in stool samples using genomic or metagenomic analysis. This is because studying direct interactions between the microbiome and intestinal tissue outside the human body represents a formidable challenge.

This new Intestine Chip provides a way to study clinically relevant human host-microbiome interactions at the cellular and molecular levels under highly controlled conditions in vitro,” said study leader Donald Ingber, MD, PhD, the Wyss Institute’s founding director. “By providing direct access to the microbiome and differentiated intestinal tissue, this method can be used to discover specific microbes or their metabolites that cause disease or that might help prevent these conditions, and because we use cells isolated from patients, this approach could be used for personalized medicine as well.”

“Earlier tissue culture systems could not grow human microbiome and intestinal epithelial cells in direct contact to one another, and they did not mimic the gut’s low oxygen concentrations crucial for the survival of anaerobic bacteria. Further complicating things: traveling along the small intestine toward the colon, oxygen levels continuously fall, which also changes the local microbiome composition.

For their anaerobic Intestine Chip, the Wyss team leveraged its proven Intestine Chip containing two parallel microchannels separated by a porous membrane. They grew human intestinal epithelial cells on the top of the membrane in the upper channel and vascular endothelial cells from intestinal microvessels on the opposite side of the membrane in the lower channel. The intestinal cells used to line these Intestine Chips were either from a cell line or were obtained from human ileum biopsies and expanded through an intermediate organoid step in which they formed tiny, spherical intestinal tissue structures, which were broken up into fragments before being cultured in the chip.

To accommodate a complete microbiome, the team placed the Intestine Chips into a custom-engineered anaerobic chamber, that allowed them to drastically lower oxygen concentrations in the upper intestinal epithelial channel, while maintaining the lower endothelial channel at normal oxygen concentrations. “We generated an oxygen gradient across the two channels that still allows the intestinal epithelium to be supported with oxygen diffusing through the porous membrane,” said co-first author Elizabeth Calamari, a research assistant on Ingber’s team.

Complex gut microbiome samples either obtained from healthy human stool and stably cultured in germ-free (gnotobiotic) mice or freshly isolated from infant stool, were then injected into the upper epithelial channel, where they came into direct contact with the mucus layer naturally secreted by the underlying intestinal epithelium. More importantly, the diversity of the commensal bacterial populations, when grown under these low-oxygen conditions, maintained the richness observed in human intestine.

Being able to look at composition and changes of complete human microbiomes in direct contact with human intestinal tissue in vitro and over days opens up opportunities for personalized medicine and drug testing. “We can culture region-specific intestinal tissue and microbiomes from the same individual to find associations that cause sensitivity or tolerance to specific pathogenic, inflammatory, and systemic diseases,” said co-first author Francesca Gazzaniga, PhD, who is a postdoctoral fellow shared between Ingber’s group and that of co-author Dennis Kasper, MD, professor of immunology at Harvard Medical School. “With the anaerobic Intestine Chip, we can also test the direct effects of drugs on the human microbiome before giving them to people.”

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