Fig. 1

Development of an asymmetric gas coculture system utilizing an immortalized gingival epithelial cell line as a barrier. a A schematic illustration depicting the gas composition within the apical and basolateral chambers of an asymmetric coculture setup housed in a vinyl anaerobic chamber, as well as the overall composition of the entire asymmetric coculture system. (Left) Input gas containing 10% oxygen enters the basolateral chamber to support cell monolayer respiration, progressively depleting the oxygen. Concurrently, the apical chamber maintains an anaerobic environment that closely replicates the surrounding anaerobic conditions, thereby ensuring the optimal growth of anaerobic bacteria. (Right) The plate cap ① covers the gas-permeable plate ④, preventing contamination and leakage. The apical chamber of the Transwell inserts ② is filled with coculture medium (CCM, see Table S1), which supports the stable growth of the cell monolayer ③ and oral anaerobes. The lower compartment (i.e., the basolateral compartment) of the Transwell insert is filled with cell culture medium. The gas-permeable membrane ⑤ at the bottom ensures unidirectional oxygen diffusion. Basolateral gas flow containing 10% oxygen enters through the gas inlet ⑨, spreading evenly through the asymmetric coculture chamber ⑧ with a magnetic stirrer ⑦. Exhaust gas is discharged through the gas outlet ⑥, completing the system’s airflow.⁶ The figure was created using BioRender. b A physical picture of the asymmetric coculture chamber. c Comparison of fluorescence intensities of FITC-dextran in the basolateral chamber at 24 h after adding FITC-dextran to the apical chamber of Transwells containing TIGK monolayers. Undifferentiated (negative control) and differentiated TIGKs under the normoxic culture conditions (referred to as “normoxic”) were compared to differentiated TIGKs under the asymmetric culture condition (referred to as “asymmetric”). The background fluorescence intensity of the blank culture medium was subtracted for each condition. Undifferentiated TIGK monolayer cultured under the normoxic condition before switching to a differentiation medium containing Ca²⁺ serves as the negative control (N.S., P > 0.05, *** P < 0.001, n = 2 technical replicates, N = 3 biological repeats). d The morphology of TIGK monolayers in Transwell inserts maintained in a cell culture incubator under normoxic conditions or cultured in an asymmetric coculture chamber for 24 h. The collagen coating is known to affect bright field imaging due to the presence of the collagen fibers, which can obscure fine details of the cells or structures being observed compared to uncoated surface.⁶² e Comparison of cell viability in TIGK monolayers cultured under normoxic and asymmetric culture conditions. Heat-treated cells are the negative control (N.S., P > 0.05, ** P < 0.01, n = 3, N = 3)