Live cell imaging allows researchers to observe and measure cellular changes over extended periods of time. It complements classical endpoint methodologies such as slide- or plate-based immunocytochemical staining of fixed cells by providing unique insight to dynamic processes within living systems. A major challenge of live cell imaging lies in creating an environment that enables precise control and manipulation of critical cell culture parameters to maintain cell health and viability. This article describes how microfluidics technology has achieved this to improve the accuracy and reproducibility of live-cell visualization studies.
Real-time analysis of mammalian cells is typically performed at 5% CO2 and 37oC, yet these parameters can fluctuate significantly according to environmental conditions and the level of manipulation required during an experiment. Such variation not only impacts cell growth, leading to questionable results, but may even cause cell death. To address this issue, culturing units that combine an environmental regulation system and a microfluidic plate within a single device minimize fluctuations by allowing reliable control of multiple key cell culture parameters. Moreover, by providing an automated hands-free approach to live cell culture experiments, these platforms enhance reproducibility and greatly reduce the risk of contamination.
Live cell imaging usually involves at least one media exchange. For instance, long time courses require that growth media is refreshed to supply cells with essential nutrients and remove waste, while studies designed to investigate the effect of a specific media component such as a drug or a stimulant invariably involve replacing the growth media with media containing the molecule of interest. During an experiment that is performed manually, media exchange can cause considerable cellular stress as temperatures fall, intercellular interactions are disturbed, and changing CO2 levels alter the pH of the growth medium. Instead, by using a pneumatic manifold to pump media through microfluidic channels, culturing units optimized for live cell imaging avoid these problems by ensuring rapid yet gentle media addition under consistent, undisturbed environmental conditions.
Image: Modern microfluidic plates for a live culture system are engineered for hands-free delivery of environmental parameters with waste outlets alongside a culture chamber optimized for adherent or suspension culture.
A major advantage of using microfluidics technology to perform live cell imaging is that it permits the cell culture environment to be altered extremely quickly. This makes it ideal to investigate cellular responses to conditions such as hypoxia, enabling researchers to study the mechanisms used by cancer cells to evade apoptosis within the hypoxic tumor micro-environment. Standard cell culture is limited for studying hypoxia because the media overlying the cells presents substantial and nonuniform resistance to oxygen diffusion. Not only does this prevent rapid switching between different gas conditions, but it also contributes to significant inter-experiment and user-to-user variability. In contrast, microfluidics technology allows pre-conditioned media to be added to the cells without incurring a lag in cell-level gas changes relative to the gas phase, meaning it provides more robust and reliable results during the study of hypoxic environments.
Many conventional cell culture control devices are unsuitable for live cell analysis because they are not designed expressly for integration with microscopy systems. However, this gap has recently been bridged by combining a low-profile manifold and a high-quality optical-bottom microfluidic plate into a compact platform compatible with most inverted microscopes. Using a small controller placed next to the microscope to regulate multiple key cell culture parameters, researchers can now perform continuous, high magnification imaging of live cells as they react to their environment in real time.
Image: Microfluidic plates for suspension cells enable uninterrupted culture and real-time observation of mammalian cells in the 12 μm diameter range using flexible traps that maintain cells in a single focal plane.
Fluorescent detection methods are widely used to identify distinct cell types and monitor changes in protein expression levels, and it is now common practice for researchers to measure several parameters in parallel within a multiplexed experiment. Indeed, with many live cell fluorescent dyes, probes, and biosensors developed specifically for real-time cellular tracking and analysis, multiplexing is by no means restricted to the study of fixed cells. Since microfluidics technology can easily be integrated with both brightfield and fluorescent microscopy, it is well placed to support multiplexed live cell imaging. A microfluidics system enables real-time visualization of dynamic response from fluorescent markers under different environmental conditions, all within an intuitive and hands-free walkaway system.
Image: Cell division of S. cerevisiae imaged over 15 hours with continuous flow in a microfluidic plate optimized for yeast culture. Uniformity of focal plane is maintained throughout continuous imaging with 40x phase objective.
The CellASIC® ONIX2 platform is a compact, easy-to-use system with microfluidic plates designed for live microscopy imaging of adherent, suspension, or bacterial cells. More information can be found here.