The human gut microbiota is a diverse community of prokaryotic and eukaryotic microorganisms closely associated with the host (Renwick et al., 2021). Increasing evidence reveals a strong connection between gut microbiota and various diseases, including chronic inflammatory, metabolic, neurological disorders, and cancer (Rackaityte and Lynch, 2020). To explore the relationship between these pathologies and gut microbiota, experimental strategies have relied on clinical and animal models. While human studies offer the most realistic results, they face challenges in controlling variables independently and identifying disease-causing factors (Adelfio and Ghezzi, 2022). Additionally, monitoring real-time microbial community composition and behavior in the colon is difficult, as data primarily relies on stool samples (Li and Zhang, 2022). Animal models, while useful, often fail to accurately replicate human gut conditions due to differences in microbiota composition and tissue structures, complicating efforts to model the human gut environment fully (Li and Kong, 2022; Park and Im, 2020). One of the most fundamental challenges in both clinical and in vivo studies is the inability to continuously collect samples (Renwick et al., 2021). Ethical and technical barriers make real-time, continuous sampling in vivo impractical (Biagini et al., 2023).

To address these limitations, in vitro fermentation models have emerged as a promising experimental strategy (Renwick et al., 2021). These models simplify the replication of the human gut environment, enabling cost-effective studies of microbial behavior under controlled conditions (Adelfio and Ghezzi, 2022; Li et al., 2022). The ultimate goal of in vitro models is to replicate the complex human gut microbiota ecosystem outside the body (Biagini et al., 2023; Renwick et al., 2021). To achieve this, a cultivation system capable of reproducing the structural and functional characteristics, along with the dynamic physiological conditions of human organs, is essential. In vitro fermentation systems designed to mimic the gut microbiota ecosystem typically consist of several key components: a habitat (scaffolds), nutrients (culture media), environmental conditions (fermenters), and analytical techniques (Fig. 1). Recent innovations in cultivation materials, techniques, and detection methods have accelerated progress in bringing in vitro models closer to their ultimate goal (Biagini et al., 2023). Scaffolds offer mechanical and physiological benefits, allowing for the movement of nutrients and gases while facilitating interactions with microbes (Nikolova and Chavali, 2019). Culture media are designed to support the entire gut microbiota community by providing essential nutrients such as water, carbon, nitrogen sources, and minerals, alongside factors like amino acids, vitamins, and purines (Biagini et al., 2023). Fermentation systems aim to replicate the distinct regions of the colon, gut peristalsis, maintaining oxygen levels, pH, and flow conditions that mirror human gut environments (Nikolova and Chavali, 2019; Li and Kong, 2022). Analytical techniques, including culture-based tests, instrumentation, and spectroscopy-based technologies, enable the identification and quantitative analysis of microbial communities (Gopinath et al., 2014; Mazur et al., 2023).

For human gut microbiota research to advance, it is crucial to develop improved methodologies based on current in vitro human gut microbiota platforms and their components. This review examines the current state of knowledge surrounding these systems to develop experimental strategies for improving human gut microbiota research methods. By analyzing both widely used components and the recent innovations in human gut microbiota fermentation systems, this review aims to provide a foundation for gaining deeper insights into microbial behavior, interactions, and environmental adaptation mechanisms.