Studying cells under optimal conditions for temperature, humidity, and CO2 levels is better for your cells—and for your data. Live-cell assays benefit from being conducted within the cozy interior of a cell culture incubator. As such, live-cell analysis systems are now available that capture data and monitor cell health from within an incubator. This article provides a glimpse into today’s tools for conducting live-cell analysis within cell culture incubators, and some examples of how these tools are being applied to disease research.
Keeping a sharp eye on culture conditions
One of the benefits of live-cell analysis is the ability to keep tabs on the health of cell cultures. CytoSMART offers the Lux2 for online cell culture video monitoring, and the Omni for high-throughput live-cell imaging. The CytoSMART Lux2 is a single position live-cell imager that is commonly used for assessing cell proliferation, morphology, and migration, and evaluating angiogenesis and spheroid-based model systems. The Lux2 generates time-lapse videos that can be accessed on a local PC or remotely via an online environment, preventing unwanted disturbances to the samples.
The compact CytoSMART Lux2 fits inside most standard CO2 incubators and hypoxia chambers. Its integrated confluence detection algorithm helps researchers to time subculturing more precisely, which is important for setting up reproducible assays and preventing undesirable effects that arise from using over-confluent cultures, such as differentiation and gene expression changes. “Users can set confluency targets and receive notifications when the target is reached,” says Joffry Maltha, CEO at CytoSMART.
The CytoSMART Omni, while larger than the Lux2, fits within 150 L CO2 incubators, and also stores results online for easy access. The CytoSMART Omni can image a wide range of tissue culture vessels and provides whole-well data for multi-well culture plates. “Users can generate time-lapses, which can be analyzed by their own image analysis algorithms, or they can choose from several label-free assays provided by CytoSMART, such as proliferation, wound healing, and colony analysis,” says Maltha. For example, at the Heidelberg Institute for Stem Cell Technology and Experimental Medicine, the research group headed by Martin Sprick uses the CytoSMART Omni to investigate the proliferation of pancreatic cancer cell lines, with an aim to overcome drug resistance and improve treatment efficiency of pancreatic cancer subtypes, according to Maltha.
Compact microscopes to fit any incubator
Etaluma’s Lumascopes—compact inverted microscopes—are sized to perform live-cell imaging within cell culture incubators and hypoxia chambers. They offer three manual Lumascope models, as well as one automated model. The automated LS720 is a 3-channel fluorescence microscope that “performs extended, multiple position timelapse acquisition including tiling and z-stacks,” says Chris Shumate, president and CEO of Etaluma. It can analyze samples in microplates from 6 to 1536 wells, as well as in microfluidic chambers. Etaluma’s microscopes can use objectives with a range of magnifications, from 1.25x up to 100x. “Our optics have a short and simple light path for extraordinary sensitivity and widefield resolution,” says Shumate. “We also offer open-source control for custom applications using Matlab or Python.”
Researchers use Etaluma’s microscopes for a range of applications besides standard assays for cell health. “We have customers doing cardiomyocyte beat rate assays over days, tumor spheroid invasion and sprouting assays, stem cell differentiation, perfused nematode model research, and even tumor margin imaging in mice,” says Shumate. “Our open architecture also allows microfluidic applications where tubing or injectors need access to the sample.”
Automated live-cell microscopy
BioTek Instruments and Zeiss both offer automated systems for live-cell analysis. BioTek Instruments’s BioSpa™ 8 and Cytation™ comprise a fully automated system for live-cell imaging. Samples reside in the benchtop BioSpa Automated Incubator, and are transferred automatically via robotic arm to the Cytation Cell Imaging Multi-Mode Reader. “The Cytation is both a digital microscope and multi-mode plate reader with customizable live-cell environmental control, thus offering solutions for a multitude of kinetic live-cell assays,” says Rebecca Mongeon, principal scientist at BioTek Instruments. BioTek’s software coordinates the BioSpa and the Cytation for automated real-time analysis, from “cell counts or confluency to multiplexed phenotypic analysis of subpopulations and kinetic subcellular signal compartmentation,” she says.
Many disease research programs use the BioTek system’s live-cell applications, including label-free cell proliferation, fluorescent cell death imaging assays, and 3D tumoroid invasion assays. The BioSpa 8 and Cytation system was recently used to study the role of tumor-associated non-cancer cells that can contribute to the spread of cancerous cells. “Researchers employed several complimentary types of assays that analyze cancer cell invasion over time, from 2D mobility assays to 3D tumoroid assays,” says Mongeon. “They used the live-cell imaging results to then identify molecules that might underlie this interaction between the normal and cancerous cells, and ultimately identified potential targets for drug treatments to control metastatic breast cancer progression.”
Another automated system is Zeiss’s Celldiscoverer 7 (CD7), which offers a flexible range of features, depending on what the researcher needs. The CD7 is an automated microcopy system surrounded by an incorporated incubator, which provides a humidified CO2 environment, and heating or cooling as needed. “Essentially we create this microenvironment right around the plate, so the sample can be imaged in a very stable way for a long period of time,” says Scott Olenych, product marketing manager at Zeiss. The automated system samples the well plate to ascertain whether it’s plastic or glass, adjusting objective settings to obtain the best image possible. The CD7 also automatically minimizes spherical aberration to optimize resolution and brightness.
The CD7’s optical features are valuable to researchers needing particular magnifications or high resolution. Users can select from among 12 different magnification possibilities for more flexibility. In addition, the CD7 offers the choice of using a dry objective, or a water objective for greater resolution. “Using a 25x 1.2 water objective, we flood the space beneath the sample with water in an automated way, and you can remove the water and switch to a dry objective as needed,” says Olenych. Zeiss recently added the option of a confocal attachment called the Airyscan, which images more gently with less laser power, but greater speed and super resolution.
Several pharma and biotech companies are using the CD7, and researchers use it in core facilities at universities. “Although used in a wide variety of applications, it’s generally geared toward drug discovery work, where you’re examining the effect of a drug or treatment on a cell or a spheroid,” says Olenych. “A number of pharmaceutical companies are using CD7 for drug discovery in disease models, but it can also be used for automated screening of sample plates.”
Live-cell imaging is thriving within the snug environs of cell culture incubators, and the technological options available to researchers will continue evolving to meet their needs. Stay tuned for more developments in this quickly moving field.