Abstract
Most gut bacteria are obligate anaerobes and are important for human health. However, little mechanistic insight is available on the health benefits of specific anaerobic gut bacteria. A main obstacle in generating such knowledge is the lack of simple and robust coculturing methods for anaerobic bacteria and oxygen-requiring human cells. Here, we describe the development of a coculture system for intestinal Caco-2 cells and an anaerobic symbiont, Faecalibacterium prausnitzii, making use of 50 mL culture tubes. F. prausnitzii was grown in 40 mL YCFAG-agar with glass-adhered Caco-2 cells placed on top in 10 mL DMEM medium. Grown for 18–36 h in a humidified incubator at 37 °C and 5% CO2, coverslip-attached Caco-2 cells promoted growth and metabolism of F. prausnitzii, while F. prausnitzii suppressed inflammation and oxidative stress in Caco-2 cells. F. prausnitzii did not compromise Caco-2 cell viability. Exogenously added porcine mucin also promoted growth of F. prausnitzii, suggesting that it may be part of the mechanism of Caco-2-stimulated growth of F. prausnitzii. This ‘Human oxygen-Bacteria anaerobic‘ (HoxBan) coculturing system uniquely establishes host-microbe mutualism of a beneficial anaerobic gut microbe in vitro and principally allows the analysis of host-microbe interactions of pure and mixed cultures of bacteria and human cells.
Highlights
Most gut bacteria are obligate anaerobes and are important for human health
The main obstacle in a host-microbiome coculture system is that most (> 90%) gut bacteria are obligate anaerobes that die quickly when exposed to atmospheric conditions (21% O2), while human cells depend on oxygen
The ‘Human oxygen-Bacteria anaerobic’ (HoxBan) system developed in this study establishes coculturing of glass-adherent human cells in liquid medium and anaerobic bacteria in solid agar medium for over 24 h and allows the analysis of cell growth, transcriptome and exo-metabolome of cocultured cells
Summary
The ‘Human oxygen-Bacteria anaerobic’ (HoxBan) system developed in this study establishes coculturing of glass-adherent human cells in liquid medium and anaerobic bacteria in solid agar medium for over 24 h and allows the analysis of cell growth, transcriptome and exo-metabolome of cocultured cells. Together with adenine and inosine, concentrations of xanthosine and 5-methylthioadenosine were strongly reduced in medium of Caco-2-F. prausnitzii cocultures compared to the two monocultures (Fig. 5e,f) These compounds of purine metabolism are required for DNA (cell proliferation) and ATP (energy) synthesis. On PC2, a slight separation between the open and closed HoxBan Caco-2-F. prausnitzii cocultures was observed (Fig. 4b), this could not be attributed to significant changes in single metabolites This indicates that even in the closed condition, 10 mL of DMEM medium carries sufficient. Oxygen to support the growth of the coverslip-attached Caco-2 cells, which is further supported by the suppressed expression of hypoxia-sensitive HO-125 in both coculture conditions (Fig. 2c) These data show that coculturing Caco-2 cells with F. prausnitzii leads to a unique profile of excreted and consumed metabolites that is not the cumulative result of the individual cell types, indicating that these cells modify each other’s metabolism. Applications of the HoxBan coculture system for other cells than Caco-2 and/or F. prausnitzii may require specific optimization in culture conditions, but it holds the universal principle of coculturing oxygen-requiring human cells together with obligate anaerobic bacteria that will foster our understanding of the role of gut bacteria in human health and disease
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