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Studying Dose Response Relationship on MDCK II Cells
PhoxyCube enables online 96 well O2 detection for toxicity studies
Sandra Friedrich1, Tobias Naber1, Barbara Goricnik1, Robert Johannes Meier2, Joachim Wegener1
1 Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Regensburg, Germany
2 PreSens Precision Sensing GmbH, Regensburg, Germany
The measurement of dose-response relationships is essential for understanding the effects of pharmacological agents on cellular functions. This report focuses on the exemplary evaluation of Antimycin A and Malonoben on the respiration of MDCK II cells, a widely used model for kidney epithelial cells. Antimycin A, a well-known inhibitor of the mitochondrial electron transport chain, and Malonoben that acts as an inhibitor of succinate dehydrogenase in the citric acid cycle, are both used to disrupt mitochondrial function and thus modulate cellular respiration. Due to change in energy metabolism of the cells by agent alternations in the oxygen uptake rate can be observed by monitoring the oxygen content in the wells of a 96 well plate which helps to quantify the dose-response effect.
The experiments were conducted using the PreSens PhoxyCube device with PhoxyPlate 96 O, 96-well microplates equipped with oxygen sensors, allowing precise monitoring of oxygen consumption in all 96 wells in parallel. This method provides valuable insights into the mitochondrial activity and overall cellular health under the influence of these modulators. By determining the dose-response relationships, the study aims to better understand the mitochondrial toxicity and potential cellular adaptations to these compounds. The findings could have implications for drug screening and the study of mitochondrial dysfunction in various diseases.
Materials & Methods
The oxygenation status was monitored during culture in the incubator at 37°C using the PhoxyCube device (PreSens, Regensburg, Germany) with a PhoxyPlate 96 O, a 96-well plate with O2 sensors in each well (see Fig. 1). PhoxyPlate was pre-equilibrated in 95 % air saturated medium at 37 °C in the incubator (5% CO2) for 1 hour followed by a one-point-adjustment at 95 % a.s.. MDCK II cells were seeded in each well of columns 2 to 12 in 500 µL Phenol red free growth MEM medium (Gibco) + 5 % FCS + Glucose with a seeding density of 450 T/cm² forming a consistent 2D cell layer at the bottom of each well. Row 1 in Fig. 2 is a blank control without cells. Cells were allowed to attach and proliferate for a total of 48 hours. After 24 hours fresh growth medium was exchanged in all wells.
48 hours after initial cell seeding and proliferation, growth medium was exchanged with Phenol red free Leibovitz´s L15-Medium with glucose (Gibco, short L15) and agents were added to the respective wells. The doses of the test substances were decreased by a dilution factor of 10 in each row with N=5 replicates each. The starting concentration of Antimycin A in row A was 20 µM and finally reached 0.02 nM in row H. For Malonoben, the concentration in row A was 1 µM and reached 1 pM in row H.
The cell adhesion during growth phase was recorded with a measurement interval of 20 minutes, the experimental phase (starting with medium/substance exchange after 48 hours) was recorded with an interval of 1 minute.
Results
In the initial 48 hours of cell seeding, attachment and proliferation, all cells containing samples depict a drop of O2 (see Fig. 3) to below 10 % a. s. due to the strong natural respiration of MDCK II cells. Subsequent to the attachment and growth phase (48 hours), the actual dose-response experiment takes place. The air saturation of the blank is higher than in the previous attachment phase, since 5 % CO2 supply of the incubator was switched off.
The control group shows a decrease of oxygen reaching a low of 18.5 % a.s. The Antimycin A addition shows a distinct and clear dose dependent effect with a extensive blocking of the MDCK II respiration and thereby quickly rising O2 levels in the cultures. The lower the concentration of the added Antimycin A the more delayed and the less pronounced this effect shows. The effect with Malonoben addition is opposite since it is a respiration and ATP uncoupling agent, that boosts the cellular respiration and thereby O2 decrease in the cultures. Depending on the added dose of Malonoben, the oxygen depletion by respiration is quicker and more pronounced leading to more hypoxic O2 levels.
Conclusion
The PhoxyCube device along with PhoxyPlate 96 O enables high-throughput tests on the effects of pharmaceutically active compounds, such as dose-response-relationship studies on adherent cell cultures. Here the cells formed a confluent 2D cell layer and the monitoring during the cultivation phase allowed to specify cell activity input in the subsequent assay for each well separately. As shown in this report the parallel measurements in 96 wells allowed quantifying the dose response of the cells. As PhoxyCube is designed to be applied directly in the incubator, it has the potential to enhance many more applications in biopharmaceutical and cell culture research.