Development of a simple cultured cell–anaerobic microbial co-culture system using liquid paraffin

Conventional co-culture systems are complicated, lack versatility, and do not adequately replicate the intestinal lumen environment. This study aimed to devise a system that allows for (i) arbitrary sampling of the culture medium, (ii) monitoring the growth of co-cultured cells, (iii) aerobic-anaerobic co-culture, (iv) simple operation, and (v) evaluation of multiple samples. We built a simple cell-anaerobic microorganism co-culture system using liquid paraffin to separate growth spaces for aerobic cells and anaerobic bacteria. Mineral oil was added to the top of the anaerobic bacterial cultivation space to seal the space and reduce gas exchange. Co-culture of anaerobic, Bifidobacterium bifidum and aerobic, epithelial Madin-Darby canine kidney (MDCK) cells demonstrated that the barrier function and viability of co-cultured MDCK cells were comparable to those of a pure MDCK culture after 24 h, and the growth curve of co-cultured B. bifidum was similar to that of pure B. bifidum. Furthermore, the growth of B. bifidum pure culture under sealed conditions was approximately 1.5 times greater than that under non-sealed conditions at 24 h. Glucose consumption at 24 h of co-culture under sealed conditions was 10%-15% higher than that under non-sealed conditions. This highly versatile culture method enabled the quantitative characterisation of B. bifidum and MDCK cells upon co-culture. The newly established co-culture system could be applied to various aerobic cell-anaerobic bacteria co-culture which will provide a strategy for basic and applied research on host-microbe interactions.