Establishment and Metaproteomic Characterization of Stable and Scalable Human Fecal-Derived In Vitro Gut Microbial Community

Abstract

The human gut microbial community plays a fundamental role in maintaining health and contributes to essential biological processes including immune regulation and metabolism. Currently, fecal microbiota transplantation (FMT) is a regulatory-approved therapeutic to prevent the recurrence of Clostridioides difficile infection and is being explored for additional indications, including cancer and inflammatory bowel disease. However, donor-derived fecal material is highly complex and variable, posing challenges for standardization, reproducibility, and safety assessment. While synthetic microbial communities have been developed as an alternative, they do not well capture the ecological complexity and functional capacity of the native gut microbiome. A stable fecal-derived in vitro gut microbial community represents another alternative to FMT, offering balanced microbial diversity and reproducibility, though the dynamics of the community structure establishment remain largely unknown. To address these limitations, this study generated in vitro human fecal-derived gut microbial communities through serial passaging of ex vivo cultures of biobanked human stools and used metaproteomics to characterize the community dynamics. The results demonstrate that serial passaging leads to the emergence of stable and reproducible microbial communities with compositions dependent on the donor microbiomes and culture medium. Although compositional shifts occur and newly detectable genomes emerge during culturing, the community complexity is preserved rather than being simplified. Culture medium was identified as the primary determinant of community composition and function after early adaptation, with donor effects being secondary. Among the tested culture media, RapidAIM resulted in the most native microbiome-like composition and functional profiles. Overall, this study establishes and characterizes a stable, scalable, and reproducible in vitro gut microbiome from human stools. This framework provides a controlled platform to study microbe-microbe and microbe-phage interactions, systematically dissect drivers of gut ecosystem dynamics, and supports the development of microbiome-based therapies through a standardized, controllable model of the human gut microbiome.