Figure 2: In vitro co-culture of human and microbial cells inside the HuMiX device.

(a) Characterisation of epithelial cell monolayer formation in HuMiX in comparison with the standard Transwell system. In both cases, the transepithelial electrical resistance (TEER) was determined on 7-day-old Caco-2 cell layers using standard chopstick electrodes. The error bars indicate the s.e.m. (n=3). * Indicates a statistically significant difference (paired Student’s t-test, P<0.05). (b) Immunofluorescent microscopic observation of the tight junction protein occludin (green) in Caco-2 cells following 24 h of co-culture with LGG grown under anaerobic conditions. The cell nuclei are stained with 4,6-diamidino-2-phenylindole and appear in blue. (c,d) Viability assessment of Caco-2 cells and LGG at 24 h post co-culture, respectively. The cells were stained using a live–dead stain and observed using a fluorescence microscope. The live cells appear in green, whereas the dead cells appear in red. The collagen-coated microporous membrane does support the attachment and proliferation of the Caco-2 cells, whereas the mucin-coated nanoporous membrane provides a surface for the attachment and subsequent proliferation of the bacteria. (e) Representative electropherogram of an RNA fraction obtained from the Caco-2 cells co-cultured in HuMiX. The RNA Integrity Number (RIN) is provided. (f) Sampled eluates from the HuMiX device following a 24 h co-culture with LGG. (g) Oxygen concentration profiles within the perfusion and microbial microchambers upon initiation of the co-culture with LGG. indicates the pre-inoculation oxygen concentration of 2.6% in the microbial microchamber. (h) The relative abundances (in %) of Lactobacillus spp. and Bacteroides spp. following 24 h of co-culture with Caco-2 cells determined by 16S rRNA gene amplicon sequencing (n=4). Scale bars, 10 μm (b–d).