REAL-TIME MONITORING OF SPECIFIC OXYGEN UPTAKE RATES OF ADHERENT CELLS IN A MICROFLUIDIC DEVICE
University College London, UK
Microfluidic cell culture devices offer precise control over the soluble, physical and mechanical microenvironment of the cells, and cells can be perfused with a variety of different compounds; yet monitoring in these devices is often limited to end-point analyses only. Oxygen plays a key role for cell growth and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs) provides invaluable insight. Combining the precise control over the cellular microenvironment afforded by microfluidic culture devices with real-time monitoring of oxygen tensions and cell culture confluency, will thus help to unravel kinetics of cell cultures, and provide new insight into the impact of natural bio-active compounds.
Monitoring in the microfluidic culture device consisted of two alternating sequences: For the imaging sequence, a motorized stage of an inverted fluorescence microscope scanned the entire culture chamber. Sequentially acquired phase-contrast microscopy (PCM) images were then stitched together and processed to determine global and local cell confluency. For the oxygen monitoring, an optical fibre (attached to the microscope objective with a custom-built adapter) aligned with a planar oxygen sensor recessed in the bottom of the culture chamber (for peri-cellular oxygen measurement). Additionally, oxygen was monitored at the inlet and outlet of the device using optical flow-through oxygen sensors. Stage positioning and data acquisition routines were controlled via LabVIEW. Using a bespoke pressure pump to create gas-driven flow (5% CO2, 21% O2), embryonic stem cells and Chinese Hamster Ovary cells were cultured under continuous perfusion (300 μL/h) and oxygen concentrations and cell density (cell number) monitored. Image processing further enabled the quantification of local growth and cell migration patterns in the culture chamber. The device was sterilized by autoclaving.
Mouse emryonic stem cells and Chinese Hamster Ovary cells were successfully cultured until they reached confluency, i.e. 6 days and 2 days, respectively. Our image processing algorithm enabled the real-time estimation of the number of cells in the microfluidic culture chamber without any disruption of the culture. The oxygen sensors enabled real-time detection of oxygen uptake rates (OUR) of the culture. By dividing the OUR with the cell density, we estimated the specific oxygen uptake rate. All data was generated non-invasively, i.e. did not require the disruption of the culture, and only phase-contrast microscopy images were required.
We demonstrate a label-free, non-invasive approach to monitor cell density, oxygen levels, specific oxygen uptake rates from a tiny cell culture. Therefore, we can determine cellular oxygen kinetics, and we can quantify the kinetics without disrupting the culture. Furthermore, the cell culture can be continuously perfused. Whilst we demonstrated perfusion with growth media for the expansion of two cell lines (mouse embryonic stem cells and Chinese Hamster Ovary cells), the same technology is applicable to culture other adherent cells and to perfuse them with a variety of compounds. Such compounds could be nutritional or bio-active substances. Currently we are working on expanding our multiplexed system for 3 cultures in parallel to a system with 6 or 12 culture wells.
Keywords: Microfluidic cell culture, Cell growth kinetics, Specific oxygen uptake rate, Cell energy metabolism, Real-time monitoring, Probiotics
Szita N. (2016). Real-time monitoring of specific oxygen uptake rates of adherent cells in a microfluidic device. Conference Proceedings of IPC2016. Paper presented at the International Scientific Conference on Probiotics and Prebiotics, Budapest (p. 73.). IPC2016