gms | German Medical Science

Objective: Nitric oxide (NO) plays a crucial role in multiple cancer signalling pathways and its antitumor effects are studied in various cancers including malignant gliomas. To assess pericellular NO levels after exogenous NO administration in glioma cell cultures, we developed an electrochemical NO sensing on-chip cell culture well (SCCW). This permits monitoring the amount of pericellular NO and its release kinetics under different experimental conditions and correlating these findings with various biological effects induced by NO.

Method: The NO sensing chip, based on thin-film technology, consists of platinum working- and counter-electrodes and Ag/AgCl reference-electrodes. Coating electrodes with O-Phenylenediamine and a charged perfluorinated polymeric membrane minimizes undesirable electrochemical influences. The sensor chip serves as the bottom of a culture well. Functionality of the sensing unit was assessed using NO gas saturated medium (233 µM), the pH sensitive NO donor Spermine NONOate (SPER/NO, 50 µM) and the GST-activated JS-K (50 µM) under cell-free conditions and in U87 and LN229 glioma cell monolayers under normoxic and hypoxic conditions.

Results: Addition of gaseous NO under cell-free conditions was rapidly detected by the sensor and produced a steep rise in NO followed by a rapid decline according to the short half life of the gas. The influence of nitrite ions was less than 8%. SPER/NO caused a gradual increase in current with a maximum after 45 min which returned to baseline after 7 h. Cultivation of U87 and LN229 glioma cells directly on the sensor surface had no influence on cell viability or NO sensing. Reproducible NO release curves were obtained under baseline conditions and after treatment with SPER/NO or JS-K for up to 65 h. O₂ saturation had no influence on NO sensing. According to half life of the NO donor, NO release from SPER/NO could be detected in LN229 cells over a time period of 32 h under normoxia. In U87 glioma cells a constant NO signal was measured after metabolization of JS-K over 4 h under hypoxic conditions.

Conclusions: The microchip-based NO sensing platform permits the reproducible, real time sensing of NO without interference of nitrite ions over extended periods in a modified cell culture well. As the cells grow on the sensor surface, this system allows measurement of pericelluar NO. This is the prerequisite for studying the impact of NO changes in the pericellular microenvironment on intracellular pathways.