Objective: 

M1-cells originate from the mouse collecting duct and are used as a model to study transepithelial ion transport and ion channel regulation. Incidental observations indicated that growth and development of a tight and confluent monolayer of M1-cells on permeable supports depended systematically on ambient CO₂ concentrations. In the context of this observation, we analyzed the development of an M1-cell line growing in a 2% (A) and 5% (B) CO₂ atmosphere for several passages with respect to ENaC function, transepithelial resistance (R_te), luminal pH and osmolality.

Methods: 

M-1 cells of each passage were grown on permeable supports (filters) in PC-1 medium (Lonza) plus supplement enriched with 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. Filters were investigated daily for transepithelial resistance (R_te). The luminal pH value of the culture medium was assessed daily by pH photometry before the luminal and basolateral change of cell culture media. At a threshold value of 1000 Wcm² in type (A) culture, 50% of both, (A) and (B) filters were analyzed in Ussing chamber experiments for the electrophysiological parameters R_te, transepithelial voltage (V_te) and the amiloride-dependent short circuit current (DI'_sc). The remaining filters were analyzed when (B) cultures reached the threshold or at least at day 10.

Results: 

Within the first days of cell culture the increase in R_te was accompanied by a substantial acidification (>1pH unit/d) of the luminal cell culture compartment. This acidification was not observed in the basolateral compartment. Filters grown under (A) conditions reached the resistance threshold after 3.7 days, cells grown under (B) conditions were delayed and reached the threshold after 6 days. At the first time point, (A) cells showed a higher R_te, V_te and DI'_sc than (B) cells. When (B) cells reached the threshold, there was no significant difference concerning R_te, V_te and DI'_sc between A and B cells. The analysis of luminal pH showed an acidification on the luminal side of the epithelium in both cell lines. A quantitative assessment of H⁺ secretory capacity will show if there is a difference between M1 cells propagated under different CO₂ culture conditions.

Conclusion: 

M1-cells grown under a 2% CO₂ atmosphere build a confluent epithelium on filters faster than under standard conditions. Our observations suggest that M1-cells, in addition to Na⁺ transport, express substantial H⁺ secretory capacity. The cellular origin of this transport and its CO₂ dependence is under current investigation.