A flexible high-throughput cultivation protocol to assess the response of individuals' gut microbiota to diet-, drug-, and host-related factors.

The anaerobic cultivation of fecal microbiota is a promising approach to investigating how gut microbial communities respond to specific intestinal conditions and perturbations. Here, we describe a flexible protocol using 96-deepwell plates to cultivate stool-derived gut microbiota. Our protocol aims to address gaps in high-throughput culturing in an anaerobic chamber. We characterized the influence of the gas phase on the medium chemistry and microbial physiology and introduced a modular medium preparation process to enable the testing of several conditions simultaneously. Furthermore, we identified a medium formulation that maximized the compositional similarity of ex vivo cultures and donor microbiota while limiting the bloom of Enterobacteriaceae. Lastly, we validated the protocol by demonstrating that cultivated fecal microbiota responded similarly to dietary fibers (resistant dextrin, soluble starch) and drugs (ciprofloxacin, 5-fluorouracil) as reported in vivo. This high-throughput cultivation protocol has the potential to facilitate culture-dependent studies, accelerate the discovery of gut microbiota-diet-drug-host interactions, and pave the way to personalized microbiota-centered interventions.