4.3. Determination of Cell Viability and Toxicity

For impedance-based cell monitoring, the XCelligence Real Time Cell Analyzer Dual Plate (ACEA Biosciences Inc., San Diego, CA, USA) was used. This system is able to measure the electrical impedance across interdigitated microelectrodes which are situated at the bottom of cell culture wells [20]. By cell cultivation onto electrodes, impedance increases as the number of cells increases. The impedance measurement is given as a dimensionless Cell Index (CI), and reflects cell status, morphology, adherence, and viability. A high CI means less cytotoxicity because of a high number of adherent cells and a low CI means a loss of cell adherence and cell number.

To monitor the influence of CoCr28Mo6 and AMC particles, 10,000 cells per well were seeded in 16-well E-plates. After 30 min at room temperature, the E-plates were placed in an incubator at 37 °C and 5% CO₂. After 24 h, the medium was replaced with particle-containing medium, and impedance was measured over 24 h. The cell growth, expressed as CI curve, was monitored for each well over the whole 48 h period. Particle concentrations of 0.05 mg/mL and 0.01 mg/mL were used. Cells cultured in DMEM alone and DMEM containing 70% EtOH in the respective dilutions (1:20, 1:100) served as controls. To assess the impedance of particles in the medium, complete DMEM with particles (0.05 mg/mL and 0.01 mg/mL) was used. The measurements were done under standard culture conditions.

The CI values of the control group (EtOH) were compensated for by the CI values of medium only, while the particle-exposed groups were adjusted for the CI values of medium plus particles. The following subtractions at each time-point were performed:

The compensated CIs were then normalized by the CI at 25 h which was the time-point of cell exposure to particles. The normalized CI was calculated as follows:

Besides impedance measurements, the cell viability of human fibroblasts was determined by the end-point assay WST-1 (Roche, Penzberg, Germany), using 30,000 human fibroblasts per well in 24-well plates. After 24 h under standard cell culture conditions, cells were exposed to CoCr28Mo6 and AMC particles at 0.05 mg/mL and 0.01 mg/mL. Cells treated with the respective concentration of 70% EtOH served as controls. After 48 h of incubation, the particle/EtOH solutions were removed and cells were incubated with a defined volume of WST-1/medium reagent (ratio 1:10) at 37 °C and 5% CO₂ for 30 min. Afterwards, supernatants were transferred into 96-well cell culture plates (in duplicates) to determine the absorption at 450 nm (reference wave length: 630 nm) in a microplate reader (Dynex Technologies, Denkendorf, Germany).