Optimized Protocol for the Collection, Cryopreservation, and In Vitro Cultivation of Human Gut Microbiota for Toxicomicrobiomics Applications.

Xenobiotics, including environmental pollutants such as bisphenols, phthalates, and parabens, are widely present in food, cosmetics, packaging, and water. These compounds can reach the gastrointestinal tract and interact with the gut microbiota (GM), a complex microbial community that plays a key role in host immunity, metabolism, and barrier function. The GM engages in bidirectional communication with the host via the production of bioactive metabolites, including short-chain fatty acids, neurotransmitter precursors, and bile acid derivatives. Dysbiosis induced by xenobiotics can disrupt microbial metabolite production, impair gut barrier integrity, and contribute to the development of systemic disorders affecting distant organs such as the liver or brain. On the other hand, the GM can biotransform xenobiotics into metabolites with altered bioactivity or toxicity. In vitro models of the human GM offer a valuable tool to complement population-based and in vivo studies, enabling controlled investigation of causative effects and underlying mechanisms. Here, we present an optimized protocol for the collection, cryopreservation, and cultivation of human GM under strictly anaerobic conditions for toxicomicrobiomics applications. The method allows the assessment of xenobiotic-GM interactions in a cost-effective and ethically sustainable way. It is compatible with a wide range of downstream applications, including 16S rDNA sequencing, metabolomics, and endocrine activity assays. The protocol has been optimized to minimize oxygen exposure to less than 2 min, ensuring the viability of obligate anaerobes that dominate the gut ecosystem. This approach facilitates reproducible, mechanistic studies on the impact of environmental xenobiotics on human GM. Key features • Strict anaerobic handling of human fecal samples: The protocol maintains anaerobic conditions from collection to cultivation, with oxygen exposure limited to less than 2 min. • Pooled-sample inoculum for reproducibility: Cryopreserved inoculum derived from pooled donor samples reduces inter-individual variability and ensures high reproducibility across experiments. • Compatibility with diverse downstream applications: The protocol supports a wide range of analyses, including 16S rDNA sequencing, untargeted metabolomics, SCFA profiling, and host-GM interaction studies. • High-throughput capacity: Up to 192 samples can be cultured simultaneously, enabling efficient large-scale experiments.