Oxygen Consumption Rate Measurements of MDCKII Cells

This application note details two distinct methodologies for calculating the Oxygen Consumption Rate (short: OCR) in adherent MDCKII cells grown in a 96-well PhoxyPlate 96 O with the PreSens PhoxyCube reader. In the initial kinetic window of any measurement, a rapid drop in dissolved oxygen occurs immediately following the start of the experiment, a phase where cellular respiration significantly outweighs oxygen transport from the air-liquid interface by diffusion. Conversely, the steady-state diffusion method utilizes the thermodynamic equilibrium, where oxygen intake from the air balances out the cells' metabolic usage. This report explains the mathematical foundations and physicochemical parameters, such as the volumetric mass transfer coefficient k_La, required for these calculations. By comparing both strategies, it is highlighted how researchers can assess both oxygen consumption rate and long-term physiological stability.

The oxygenation status of adherent MDCKII cells was monitored in a 20 min measurement interval during culture in an incubator at 37°C with 5% CO₂ using the PhoxyCube device with a 96-well sensor plates, which has O₂ sensors in each well (PhoxyPlate 96 O), and a well plate lid. The lid enables sufficient gas exchange with the incubator air, so it is defined as an open system. The PhoxyPlate was pre-equilibrated in 95 % air saturated (5% CO₂) media at 37 °C in the incubator for 1 hour followed by a one-point-adjustment at 95% a.s.. MDCKII cells were seeded in 300µL Phenol Red free growth MEM media (Gibco) + 5 % FCS + Glucose with a seeding density of 450 T/cm² forming a consistent 2D cell layer with ~144000 cells per well. 8 wells were a blank control without cells. After ~26 hours fresh growth media was exchanged in all wells and measurement was continued.

Eight separate wells were grouped to one replicate group each. In the initial phase over the first 1-2 hours after seeding or media exchange all groups containing samples depict a steep drop of O₂ due to the strong respiration of MDCKII cells in non-physiologically regulated conditions versus the re-diffusion through the media air interface (see Fig. 2). This drop is followed by a more moderate decline in the physiologically relevant cell state (2-16 hours from start or media exchange). In this report we focus on the OCR in this physiologically relevant cell state. This drop produces a quasi-linear decline curve where diffusion is still negligible. After around 16 hours the consumption of O₂ reaches the thermodynamic equilibrium state and the O₂ saturation gets constant to below 2.9 to 7 % a.s. in all wells balancing O₂ consumption and re-diffusion.

There are three steps needed to calculate OCR_kin:

1. Find the slope and calculate the rate of O₂ decline per minute,
2. Convert to concentration change to absolute amount per well,
3. Calculate OCR per one thousand cells in (mol/min · 1000 cells)

The kinetic window was determined from 2 to 5 hours after media exchange. The slope of the decline Δc(O₂ in % a.s.) / Δt of the groups was determined with a Δt of 120 minutes and a moving average of 5 consecutive measurement points (see Fig.3). The calculated slope is converted to the unit µmol/L/min, which is more common for use in OCR calculations.  The media volume of 300µL and the number of seeded cells (~144.000 cells) per well are then used to determine the OCR_kin of each group. The determined average OCR_kin was found to be 0,173 +- 0,012 pmol/min/1000 cells.

The calculation of the OCR_eq requires

1. The volumetric mass transfer coefficient k_La 
2. The O₂ level in the equilibrium between ingress and consumption, measured by the PhoxyCube
3. Calculating the diffusion flux at equilibrium and thereby the OCR_eq per cell.

k_La was simply determined by exploiting the kinetic phase data from Method 1. Oxygen consumption remains constant throughout the whole experiment in Fig. 3., Therefore, we calculated k_La by rearranging the steady-state diffusion equation and using the measured mean equilibrium O₂ concentration (3,8% a.s.) and the OCR_kin (derived from the kinetic method) to k_La = 0,00042 min^-1. This calibration approach requires no external assumptions about well geometry or surface properties. It enables reliable calculation of respiration rates from equilibrium measurements in subsequent time experiments as long as the used volume is kept constant.

The calculated average OCR_eq of all groups was determined to be 0,168 +- 0,001 pmol/min/1000 cells and matched the kinetically determined OCR to within 3%.

PhoxyCube determines the OCR of cell cultures in 2 easy ways. Either using the kinetic method or simply from the equilibrium if k_La is known from one pre-measurement or from literature. This equilibrium-based OCR calculation eliminates the need for time-critical kinetic measurements, making it ideal for high-throughput screening of respiration dynamics over extended culture periods.
The PhoxyCube device along with PhoxyPlate 96 O sensor equipped well plates opens up the potential of high throughput test for kinetic and thermodynamic equilibrium OCR on the effects of pharmaceutically active compounds on cellular respiration such as dose-response-relationship studies on adherent cell cultures cytotoxicity studies.