Real-time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device.

Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real-time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time-course data for bulk and peri-cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non-invasive and label-free approach. Additionally, we confirmed non-invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell(-1) s(-1) , and 5 and 35 amol cell(-1) s(-1) were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non-invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell-based therapies.

Development of a multiplexed microfluidic platform for the automated cultivation of embryonic stem cells.

We present a multiplexed platform for a microfabricated stem cell culture device. The modular platform contains all the components to control stem cell culture conditions in an automated fashion. It does not require an incubator during perfusion culture and can be mounted on the stage of an inverted fluorescence microscope for high-frequency imaging of stem cell cultures. A pressure-driven pump provides control over the medium flow rate and offers switching of the flow rates. Flow rates of the pump are characterized for different pressure settings, and a linear correlation between the applied pressure and the flow rate in the cell culture devices is shown. In addition, the pump operates with two culture medium reservoirs, thus enabling the switching of the culture medium on-the-fly during a cell culture experiment. Also, with our platform, the culture medium reservoirs are cooled to prevent medium degradation during long-term experiments. Media temperature is then adjusted to a higher controlled temperature before entering the microfabricated cell culture device. Furthermore, the temperature is regulated in the microfabricated culture devices themselves. Preliminary culture experiments are demonstrated using mouse embryonic stem cells.