Host-microbe interplay analysis using “apical anaerobicchamber”, a device that enables co-culture of anaerobic gut
microbes and host epithelial cells
Takane KATAYAMA
Division of Integrated Life Science,
Graduate School of Biostudies, Kyoto University
Report: The apical anaerobic chamber developed in this study was found to be effective in
keeping the dissolved oxygen concentrations of the apical side and the basal side of theCaco-2 monolayer at < 0.3% and > 60%, respectively, for at least 5 days. The transepithelial
electrical resistance value of the cells was comparable to that of the cells maintained in theCO2 incubator. Fluorescence immunostaining of claudin-2 revealed clear tight-junction
formation of the cells. When obligate anaerobes belonging to the Bacteroides andBifidobacterium genera were added to the apical medium of the Caco-2 cells in the apicalanaerobic chamber, the growth of these bacteria was stimulated by 100- to 10,000-folds. The
transepithelial electrical resistance both prior to and post co-culturing was comparable.
These results indicate that the device serves as a useful tool for the analysis of symbiosis
between intestinal epithelial cells and gut microbes.
Research objective
Gut microbes have significant impact on host health and disease. Animal experiments
have greatly contributed to the understanding of the mechanism underlying the crosstalk
between them; however, our community needs to develop alternate methodologies to address
the increasing attention to animal welfare. In addition, recent studies have pointed out thedifference in the microbiota composition between mice and humans. I recently commenced
the research to develop a specific device (apical anaerobic chamber) that enables
co-culturing obligate anaerobic gut microbes with human intestinal cell lines, by keeping theapical side of the epithelial cells anaerobic while keeping the basal side of the cells aerobic.
Here I describe the usefulness of the apical anaerobic chamber in analyzing the molecularbasis of symbiosis between gut microbes and humans.
Methods
The apical anaerobic chamber was designed and constructed by WakenBtech (Fig. 1). The
Caco-2 cell line was grown in DMEM containing 10% FBS in a CO2 incubator to form amonolayer in the culture insert. The insert with the layered cells was placed in the chamber

and transferred to the anaerobic station. The dissolved oxygen concentration was monitored
using Micro Fiber Optic Oxygen Transmitter (PreSens). The nucleus was stained with DAPI,
while claudin-2 was stained with the anti-claudin-2 monoclonal antibody, which wasvisualized via fluorescence. The bacterial strains were obtained from the RIKEN
Bioresource Center. The GAM medium was routinely used for bacterial growth, while
DMEM (FBS) was used for co-culture with Caco-2 cells.
Results
When the Caco-2 cells were incubated in the apical anaerobic chamber, the dissolved
oxygen concentrations of the apical side and the basal side were kept < 0.3% and > 65%,
respectively, for at least 5 days. When the culture insert was directly placed in the anaerobic
station, the dissolved oxygen concentrations of both the sides dropped to 0% within a day.
As shown in Fig. 2, the transepithelial electrical resistance values were quite similar between
the cells incubated in the apical anaerobic chamber and the cells maintained in the CO2
incubator. The values became significantly low when the culture insert was directly placed in
the anaerobic station. Lactate dehydrogenase activity in the supernatant significantly
elevated when the cells were incubated anaerobically. The clear tight-junction was visible inthe cells incubated in the CO2 incubator as well as in the apical anaerobic chamber, as
revealed via fluorescence staining of claudin-2. On the other hand, when the cells wereexposed to anaerobic conditions, the tight-junction collapsed (Fig. 3).
When Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides caccae,
Parabacteroides merdae, Dorea longicatena, Lactococcues lactis, Bifidobacterium longum,
and Bifidobacterium bifidum were added to the apical side of the Caco-2 cells incubated in
the apical anaerobic chamber, the growth of these bacteria enhanced by 100- to 10,000-folds.
The transepithelial electrical resistance values both prior to and post co-cultivation werecomparable.
Conclusion
These results revealed the usefulness of the device in co-culturing the anaerobic gut
microbes with human cell lines, enabling us to decipher the mechanism underlying thesymbiosis between them. The device also serves as a tool to analyze the drug and foodmetabolism in intestines, on which gut microbiota should have significant influence.

Fig. 1. Apical anaerobic chamber（11 cm x 11 cm x 5 cm）
The device is transferred into the anaerobic station once the transwell is placed into
the center hole of the chamber.

Fig. 2. Transepithelial electrical resistanceCaco-2 monolayers were incubated under the respective conditions. Transepithelial
electrical resistance was monitored each day.

Fig. 3. Tight junction formation, evaluated via fluorescent immunostaining
Caco-2 cells at day 5 were used for staining. Nucleus (blue), claudin-2 (green).
Scale bar: 20 μm.