Construction of a simplified co-culture system using MDCK cells as surrogate intestinal epithelium with intestinal anaerobic bacterial mixtures

In vitro co-culture systems that mimic the intestinal environment are important tools for analysing interactions among intestinal bacterial communities. Conventional systems face challenges in co-culturing microorganisms with various oxygen requirements and aerobic intestinal epithelial cells, particularly in distinguishing and quantifying multiple bacterial species. To address these challenges, as a first step, we established a simplified co-culture system using a partially oil-sealed co-culture system (POS-CCS). Using Lactobacillus paragasseri and Bifidobacterium longum subsp. longum as model microorganisms, we successfully co-cultured them with MDCK cells and analysed their growth using multiplex quantitative polymerase chain reaction targeting the V6 region of the 16S rRNA gene. Our system allowed the individual measurement of growth curves of both bacterial species, and changes in the transepithelial electrical resistance of epithelial cells, cell viability, pH, and concentration of bacterial cells during co-culture. Notably, co-culture with MDCK cells led to a rapid decrease in pH, which coincided with an increase in transepithelial electrical resistance, suggesting a potential link between bacterial metabolism and epithelial barrier function. Furthermore, MDCK cells promoted the growth of both L. paragasseri and B. longum subsp. longum. This simplified and adaptable co-culture system offers a valuable tool for investigating host–microbe interactions in the gut and is expected to contribute to studies on probiotics, prebiotics, and control of the intestinal environment.