Building biologically relevant in vitro models of the human gut microbiota: methodological considerations, current challenges, and future perspectives

The human gut microbiota (HGM) plays a pivotal role in human health by regulating metabolic, immune, and neurological processes and contributes to the susceptibility to various diseases. The increasing interest in microbiota-targeted therapies has highlighted the need for in vitro models capable of accurately reproducing the composition, function, and ecological dynamics of the intestinal microbial ecosystem. This review examines the methodological factors that determine the biological relevance of HGM in vitro models, emphasizing that reliable experimental outcomes depend not only on the choice of fermentation system (including batch and continuous fermentation systems), but also on the optimization of inoculum preparation, culture media, environmental control, and strategies that preserve microbial stability and reproducibility. We further discuss how these methodological decisions influence the interpretation of experimental results and determine the suitability of each model for different biological applications. The application of these models in dietary, prebiotic, probiotic, and pharmacological interventions is explored, demonstrating their potential to predict microbiota responses and elucidate mechanistic links between microbial activity and host responses. We propose that rigorous methodological standardization, rather than increasing technological complexity alone, represents the principal requirement for generating biologically relevant and reproducible in vitro gut microbiota models. Finally, we discuss emerging host-microbe co-culture systems, along with the integration of multi-omics and computational approaches, which are expected to expand the physiological relevance and translational applicability of these models. Unlike reviews primarily organized around individual fermentation technologies, this review adopts a workflow-based perspective and examines how methodological decisions interact to determine ecological fidelity, functional stability and reproducibility.