Expanding Live-Cell Imaging Applications in Drug Discovery

Advances in microscopy are helping scientists discover more than ever before at the cellular and molecular levels. One important “dimension” in modern microscopy is time. Being able to observe cell behavior over a continuous time frame can be useful, for example when observing responses to novel drug candidates. By contrast, getting a “snapshot” view at a single endpoint does not always convey the whole story in terms of cellular response and kinetics. That’s where live-cell imaging comes in.

Live-cell imaging tools have evolved significantly and, combined with environmental control chambers and widefield, confocal, light-sheet, or super-resolution fluorescence microscopy, are becoming more accessible than ever before. This article will focus on the use of live-cell imaging in drug discovery including applications, technologies, and expert advice.

Filling an important gap in drug discovery

“It’s important to have reliable predictors for how drug behavior in vitro will translate to in vivo. As the median cost of getting a new drug to market is around $1 billion, fully understanding cellular behavior, processes, and responses to drug pharmacology is essential to preventing failure of drug launch to market,” says Ryan Raver, Senior Product Manager at Agilent Technologies.

It's no surprise, therefore, that these technologies are of great interest to scientists working in the pharmaceutical and biotech industries and can be of great value when applied to drug discovery and development. Raver continues: “In biopharma, the search starts with understanding the functional level of a gene and its behavior (expression) in a cell, for example by manipulating gene expression. Live-cell imaging can also help uncover new genetic pathways or drug mechanisms of action, and measure drug cytotoxicity, efficacy, dosage, and on/off-target effects.”

Live-cell imaging can have a positive impact on the drug discovery workflow, comments Inge Thijssen, Senior Application Scientist at CytoSMART: “Without this technology, a fixed-point assay might show that a drug has little to no effect on cells. With live-cell imaging, cell growth might seem inhibited at first but then the cells might recover after a certain period. This could lead to further investigation of the drug, whereas in the past it might have been rejected at that earlier stage.”

The types of cell behaviors and activities that can be measured following exposure to a drug are broad, including cell migration, invasion, and morphology changes. “Visual proteomics can also facilitate downstream analysis, incorporating spatial omics methodologies for a more comprehensive look,” advises Renée M. Dalrymple, Life Science Business Sector Marketing Manager at ZEISS Research Microscopy Solutions.

Despite these advantages, ensuring scalability and reproducibility remains critical, states Lindy O’Clair, Head of BioA Applications, Reagents and Consumables at Sartorius: “Live-cell imaging offers the opportunity to generate predictive and pathophysiological data by resolving spatial and long-term, temporal detail. For live-cell imaging to truly enable drug discovery, it should be able to track biology continuously and must be efficient, scalable, and, most importantly, reproducible.”

With regards to scale-up, Raver points out that large-scale manufacturing of cell therapies like CAR-T can be costly and risky due to safety and efficacy. Measuring a cell therapy or drug’s potency pre-administration to patients would therefore be of great value, helping to mitigate risk via an expanded and complete workflow. These deeper insights allow scientists to address important questions before advancing into the next stages of drug discovery and/or early clinical trials.

O’Clair acknowledges the potential clinical impact of “four-dimensional” live-cell imaging data: “Due to the high attrition rates of drug candidates, strategies that can quantify biological complexity with multiplexed, objective analysis are sought after, with the long-term potential to increase clinical predictivity.”

Watching kinetics’ athletics in vitro

Drug discovery scientists are aware of how important kinetics can be in cell-based experiments. Thijssen shares her insights: “Drug kinetic information from cell culture experiments is increasingly important. End-point data about cell structure and morphology is no longer sufficient. Live-cell imaging provides much more information about drugs’ mechanisms of action, including their effect on cellular behavior and processes.” Raver agrees, stating that live-cell, real-time imaging produces long-term kinetic measurements that are highly sensitive and reproducible.

Several live-cell analysis technologies are useful for drug discovery. These include 3D imaging as well as electrical impedance. “In the case of live-cell impedance, a lot of work over the past decade has advanced drug discovery research, including the identification of small molecule mitosis inhibitors, measuring drug cytotoxicity, and GPCR screening,” says Raver, “with imaging providing an additional form of validation.”

Dalrymple points out that protein trafficking in cells is often studied by monitoring specific target movement in live samples. “Super-resolution microscopy is advantageous for locating proteins within small organelles in cells, but historically has been challenging to use with live samples.” However recent advances combining lattice illumination with super-resolution structured illumination microscopy (SR-SIM) can provide the combination of speed and gentleness needed for live-cell imaging, with resolutions down to the 60 nm sub-organelle level.

Maximizing the use of live-cell imaging tools

When it comes to imaging techniques, says Dalrymple, be sure to choose one that exposes samples to as little light as possible to minimize phototoxic effects. Lattice light sheet technology provides the gentlest fluorescence microscopy by minimizing the amount of light the sample is exposed to—potentially extending live-cell observations from hours to days.

For many applications, fluorescent microscopy is still preferred due to its high contrast and multiplexing options. “Technologies like spinning disc confocal microscopy also reduce phototoxic stress on cells, providing excellent image quality while removing background,” says Jonas Schwirz, Field Application Scientist for High Content Screening Revvity.

O’Clair thinks that 3D imaging is of increasing importance, especially for more complex constructs such as organoids and spheroids. “The most important challenge in live-cell imaging for drug discovery is the need to maximize data obtained from advanced cell models, specifically 3D structures, to exploit as much information as possible.” Schwirz advises that the best confocal systems for 3D cultures use microlens-enhanced spinning discs. Their efficient use of excitation light makes them faster, and for thicker samples they also provide better confocal performance compared to traditional spinning disc designs.

No matter which system is used, however, consistent cell handling is imperative. “For truly biologically relevant drug discovery data, cells must be healthy and have reliable cell seeding and distribution,” adds O’Clair. Thijssen additionally recommends using an in-incubator imaging system to ensure optimal conditions.

Automation has also had an impact on live-cell imaging. “Automation involving artificial intelligence means that the microscope can now do the majority of the heavy lifting,” says Dalrymple. And once the reams of continuously gathered data have been collected, having capable and sophisticated analysis tools is fundamental to unlocking their insights. O’Clair concludes: “As we move into more complex models and interrogating deeper biological questions, there is a need to mine data from live-cell analysis systems and to integrate it with other technologies. This will help us achieve our goal of discovering novel treatments that could positively impact human health.”

Image: Automation and artificial intelligence allow for automatic detection and calibration of multiwell plates and easy experimental setup for cell segmentation and counting to facilitate drug discovery research. Sample courtesy of P. Denner, Core Research Facilities, German Center of Neurodegenerative Diseases (DZNE), Bonn, Germany. Image collected with ZEISS Celldiscoverer-7. Courtesy of ZEISS Research Microscopy Solutions.