Novel In vitro Model Replicates Intestinal Microbiota Composition Without Requiring Anaerobic Incubation

The majority of the gut microbiota is unculturable using standard laboratory techniques, which has been well-documented since the advent of next generation sequencing in microbiome research. This limitation makes it difficult to characterise the phenotype and function of many gut bacteria, rendering the translation of microbiota research into clinical and well-being applications challenging.

While some protocols allow for the study of “unculturable” microbes in vitro, these are often complex and expensive. One such approach involves the assembly of “gut simulators”, which comprise a series of interconnected benchtop bioreactors. These systems effectively capture the complex dynamics of intestinal ecosystems; however, their cost and operational complexity make them more suitable for advanced research projects rather than early-stage, proof-of-concept in vitro studies.

More simple protocols used for early-stage studies involve inoculating liquid media with a microbe(s) of interest. The microbe(s) can then be screened and characterised before progressing onto more costly models. However, accomplishing effective culture of “unculturable” microbes within liquid media often requires strict anaerobic laboratory conditions presenting an operational barrier. Additionally, the translatability of data obtained from liquid-media studies into humans is limited.

For a probiotic developer, selecting which strains to move onto further development represents an important decision in the discovery/pre-clinical phase. Choosing strains which lack the desired traits to progress can lead to wasted capital, time and resource.

The microbiome field urgently needs reliable preclinical tools to de-risk clinical development and bridge the gap between early- and late-stage research.

In a groundbreaking study, Bac3Gel, a Portugal-based biotechnology start-up, presents a cost-effective, scalable, and accessible tool for early-stage gut microbiota research: Gut3Gel. This novel in vitro model offers a democratised platform for scientists to study gut microbiota without requiring anaerobic incubation, in a simple yet effective manner.

The research demonstrates that Gut3Gel can support the culture of complex microbial ecosystems, including strict anaerobes, without requiring an anaerobic chamber.

What is Gut3Gel?

Gut3Gel is a ready-to-use in vitro mucus model that recapitulates the complex 3D macrostructure of the intestinal mucosa, providing a physiologically relevant environment for studying gut microbiota. Composed primarily of mucin and sodium chloride, Gut3Gel provides a continuous supply of carbohydrates, an oxygen gradient, and offers a spatial scaffold for microbiota to proliferate in.

Gut3Gel’s specially designed structure provides distinct microenvironments that facilitate the growth of a diverse range of micro-organisms, including hard-to-culture obligate anaerobes, all without requiring anaerobic conditions.

Validating this innovative in vitro platform

To assess Gut3Gel’s capacity to support a stable, metabolically active, microbial community without requiring anaerobic incubation, Bac3Gel cultured faecal samples from five healthy donors and evaluated microbial composition and activity through sequencing, as well as measured production of exopolysaccharides and short chain fatty acids. As a control, the researchers used Brain Heart Infusion (BHI) medium, culturing samples under anaerobic or aerobic conditions to compare microbial growth and diversity.

Gut3Gel supports the stable growth of diverse microbial communities

Intestinal microbiota samples cultured in Gut3Gel maintained stable microbial richness and diversity throughout a 72-hour culturing period. At the phylum level, the microbial communities within Gut3Gel were predominantly composed of Firmicutes (Bacillota), Actinobacteriota (Actinomycetota), and Bacteroidetes (Bacteroidia). Notably, the abundance of Proteobacteria (mainly Escherichia and Pseudomonas, which are aerotolerant bacteria) was 3- to 9.5-fold lower than in samples cultured in BHI medium, showcasing Gut3Gel’s ability to sustain anaerobic conditions.

Mucins in Gut3Gel promote the growth of mucus-associated species

The mucin-based composition of Gut3Gel favoured the growth of mucus-associated bacteria, such as Bifidobacteriaceae, Lactobacillaceae, Lachnospiraceae, Ruminococcaceae, and Faecalibacterium, over bacteria typically more prominent in the lumen, such as Bacteroidaceae and Enterobacteriaceae. These findings closely mirrored communities cultured in more complex bioreactor systems and in vivo data1.

Gut3Gel fosters a unique fermentation profile with higher levels of SCFAs

Data analysis revealed that Gut3Gel cultures exhibited higher levels of acetate and butyrate compared to samples cultured in BHI medium, while propionate levels were comparable between the two. The difference in fermentation profiles might be attributed to the presence of a steady supply of carbohydrate sources from mucin metabolization in Gut3Gel2.

The missing link in gut microbiota research

In a discussion with The Microbiome Times, Bac3Gel’s CEO, Sebastião van Uden, emphasized the critical absence of mucus in preclinical models as a significant gap in research.

“We believe mucus holds the key to unlocking our full understanding of the host-microbiota interactions. The relationship between mucus and microbiota is bidirectional and dynamic, with mucus acting as a substrate for bacteria, while bacteria also shape its composition. Given the close relationship between mucus-lined surfaces and microbiota, we believe that omitting mucus from in vitro models compromises the translatability of research.”

“Our research shows that Gut3Gel is an accessible in vitro model for culturing faecal microbiota samples, with microbial compositions comparable to complex bioreactor systems, and enabling the growth of otherwise ‘unculturable’ microbes.”

Sebastião added,

“We’re excited to see how our clients will advance the development of their bacterial platforms by leveraging Gut3Gel”.

More than just the gut…

Expanding beyond their gut in vitro model, the Portuguese start-up offers a range of tailored mucus solutions designed to replicate the intricate architecture of mucosal surfaces across the body. Bac3Gel’s innovative in vitro models include cervicovaginal, airway, and gastric human mucus, providing advanced platforms for research and development in drug delivery, infection studies, microbiome interactions and microbiome mining.

To read the full research into Gut3Gel’s capabilities visit: https://www.biorxiv.org/content/10.1101/2025.02.21.639490v1


Citations:

  1. Lavelle, A. et al. (2015) ‘Spatial variation of the colonic microbiota in patients with ulcerative colitis and Control Volunteers’, Gut, 64(10), pp. 1553–1561. doi:10.1136/gutjnl-2014-307873.
  2. Paone, P. and Cani, P.D. (2020) ‘Mucus barrier, mucins and gut microbiota: The expected Slimy Partners?’, Gut, 69(12), pp. 2232–2243. doi:10.1136/gutjnl-2020-322260.