Virology research
Live-cell imaging
in virology research
Virology is the study of the biology of viruses and viral diseases. As obligate intracellular parasites, viruses hijack the molecular machinery of host cells and use it to produce new viral particles.
Although live-cell imaging cannot be used for direct visualization of viruses due to their small size (20 – 300 nm) and the limitations imposed by light microscopy itself, this technique greatly facilitates the understanding of complex virus-host interactions. Live-cell microscopy can be used for visualizing and quantifying morphological and functional changes caused by virus infection, known as viral cytopathic effects (CPE). Some viruses only cause subtle cellular changes, while other viruses lyse the cells and destroy the host cell monolayer. Continuous imaging of infected cells can detect the whole range of these virus-induced cellular changes in vitro, allowing to study the progression of viral infections as well as to screen new antiviral drugs and vaccines.
Live-cell imaging in BSL-3/4 laboratories
Biosafety level (BSL)-3 and 4 laboratories are high-containment facilities designed for the research of highly pathogenic viruses and bacteria. These include SARS-CoV-2, the causative agent of COVID-19, Mycobacterium tuberculosis, and Ebola virus, among many others. BSL-3 and 4 facilities play a central role in diagnostics and treatment development against world’s most dangerous microorganisms, however working in these facilities presents a number of challenges. Despite implemented health and safety measures, the researchers are still subjected to a substantial risk of contracting a life-threatening disease. In addition, the high cost of disposable protective equipment, and the time and labor associated with performing BSL-3/4-based experiments, impose further limitations on the researchers.
Remote monitoring of cell cultures reduces the time spent in the BSL-3/4 laboratories. It is also a much more cost-efficient alternative, as there is no need to use expensive protective equipment when analyzing the cells remotely. Once infected with the virus, the cell cultures can be placed inside the incubator and safely monitored via the CytoSMART live-cell imaging devices, minimizing the exposure of researchers to biological hazards. The images and time-lapse videos can be immediately accessed via the CytoSMART Cloud, providing real-time updates on the cell cultures and running experiments.
Non-endpoint analysis of viral cytopathic effects
Viral cytopathic effects (CPE) commonly include rounding, detaching, and clumping of adherent infected cells, leading to the formation of holes in a confluent cell monolayer. Conventional methods of CPE analysis (e.g. plaque and focus-forming assays) are typically the end-point virology assays that involve manual examination of CPE using brightfield microscopy, making it a labor-intensive, time-consuming, and user-dependent approach. The CytoSMART devices, including the CytoSMART Omni and Lux3 FL, can analyze CPE in real time, using both brightfield and fluorescence imaging. The occurrence of CPE can be monitored for days or weeks at a time, without missing any critical stages in the development of virus-induced CPE. In addition, the images and time-lapse movies are acquired and analyzed automatically. For example, the reduction in host cell count can be measured using the AI-based confluence algorithm, eliminating any subjectivity and bias in results interpretation.
Videos
Appnotes
Cell Viability Analysis
Cell Culture Optimization
Cell Culture Monitoring - Lux2
Cell Viability Analysis
Cell viability, growth and cytotoxicity studies can be performed using metabolic activity assays. The overall metabolic activity of the cell is indicated by the enzymatic cleavage of colorimetric or fluorescent substrates.
While these assays are relatively straightforward and cheap, they are dependent on culture conditions and intrinsic metabolic activity of the cell type that is being investigated. Furthermore, depletion of the metabolic substrate can lead to a plateau in the fluorescent signal, making assay output unreliable. To overcome these limitations cell viability could be determined optically using confluency measurements.
In the study described here the performance of confluency measurements to assess cell viability were compared to a metabolic activity assay: cell titer blue. Confluency was visualized using automated bright-field microscopy and subsequently analyzed using image analysis algorithms. Images were collected inside a CO2-incubator, keeping the culture at optimal conditions. For the cell titer blue assay resazurin was added to the medium and incubated for 3 hours. The fluorescent signal was normalized to the control to obtain the relative metabolic activity as a measure of cell viability. The comparison between the methods was performed for two pancreatic cancer cell lines, PACO7 and POCA43.
Cell Culture Optimization
Mammalian cells are being used in many research fields including tissue engineering, regenerative medicine and drug discovery. To ensure manageable research, cell cultures should be robust and experimentally reproducible. Therefor, optimization of culture conditions is an essential part of lab work. Media composition, temperature, O2 levels and seeding density influence the cell growth. To monitor the cell cultures, researchers regularly observe cell growth using bright-field microscopy.
In the study performed here, an alternative method for manual inspection of cell growth has been explored. Automated live-cell imaging was used to examine a 48-well plate containing CHO-K1 cells at varying seeding densities (n=6) from inside a CO2-incubator.
Cell Culture Monitoring - Lux2
Setting up cell cultures is easy enough. However monitoring your cultures and optimization is time-consuming and cumbersome. Waiting for the ideal confluency, quickly studying effects of various media means taking your cells in and out of the incubator more often than you would like.
Visualizing cell cultures from inside an incubator using a compact microscope that facilitates live cell imaging can overcome these issues. While live cell imaging has been restricted to costly, high-end devices, the CytoSMART Lux2 offers an affordable and easy-to-use alternative for virtually any lab. The CytoSMART Lux2 can be set up in minutes, enabling untrained users to quickly perform their own time-lapse recordings.
Images and videos can be easily accessed and retrieved from the CytoSMART cloud portal. Advanced functions, such as reporting of cell confluency, cell migration analysis and the option to use automatic confluency email alerts, can be applied to inform the user when certain culture conditions are reached (for example, once the cell culture has reached the desired confluency). Hence, the CytoSMART Lux2 can be used in many different ways to facilitate cell culture work and research.
In the following appnote several examples of applications of the CytoSMART Lux2 will be shown.
- Culturing cells in hypoxic conditions
- Standardizing cell culturing conditions
- The effect of confluency on transfection efficiency