Live-cell imaging has transformed the study of cell behaviour and function. The technique allows researchers to monitor internal cell structures, interactions and processes in real-time, and over time.

In drug discovery, understanding the dynamic processes of cells provides an enhanced picture of drug pharmacology, enabling more reliable predictions of clinical responses earlier in the process. In this article, we highlight some of the key commercial products helping to drive the adoption of this valuable approach and how it could help improve efficiencies and advance the development of medicines.

Traditional imaging experiments in drug discovery involve endpoint measurement approaches that typically use fixed cells requiring staining or artificial labelling (for example, using genetically-encoded fluorescent proteins). These artificial conditions mean that the cells do not sufficiently mimic the complex in vivo cellular responses to pharmacological or molecular perturbations that would be seen in the patient.

“To increase the chance that a drug’s effects in vitro translate to the clinic, cell models must be as physiologically relevant as possible, comments Tim Dale, Head of BioAnalytics Applications, Sartorius Corporation. “We also need to implement new methods to gather information over time. As biology is incredibly dynamic and complex, single snapshots using endpoint measurements cannot provide the full picture.”

Fortunately, a solution has emerged with the advent of novel time-lapse microscopy technologies. These enable powerful imaging analysis of live cells, fresh tissue or live in vivo models, performed in a time-resolved manner. Live-cell imaging enables the monitoring of internal cellular structures, processes and interactions to reliably determine the safety and efficacy of a drug, such as whether it selectively binds to the intended target or whether it has any off-target effects. These insights enable better predictions about which drugs will be successful in clinical development to reduce late-stage attrition.

A more complete picture of cellular processes

As well as offering a better understanding of cell behaviour and function, live-cell imaging offers drug discovery several other advantages over the traditional endpoint approach. This includes the ability to study dynamic cellular processes, behaviour and function from both spatial and temporal perspectives to get an enhanced picture of the pharmacological or molecular effect on the cell. Additionally, as live-cell imaging allows cellular structures to be studied in their native environment, it is less prone to experimental artefacts. This means the technique typically provides more reliable and statistically-relevant information than fixed-cell microscopy.

Live-cell imaging can also reduce workload and streamline laboratory operations: “Live-cell imaging allows both controls and treated samples to be placed in the same plate, with the same sample followed over an extended period. This can dramatically reduce the experimental set-up time and complexity, as well as the number of samples required compared to endpoint experiments,” comments Joffry Maltha, CEO, CytoSMART Technologies BV. “On top of that, live-cell imaging typically doesn’t require the calibration or normalisation that endpoint assays need, which can save a lot of pre-experiment preparation time.”

Live-cell imaging is already facilitating a wide range of drug discovery applications, including early target discovery and validation, informational screens, secondary cell-based assays and ADMETox (Absorption, Distribution, Metabolism, Excretion and Toxicity) studies for prioritising primary hits. Prominent examples where live-cell assays have been particularly valuable include the assessment of fast signalling responses, and experiments where multiple cell types interact to affect the disease model, such as in complex cellular models such as organoids and immune cell killing assays, which require both spatial and temporal insight.

Overcoming the challenges of live-cell imaging

To harness the power of live-cell imaging and the many benefits it offers drug discovery research, it is vital to overcome the challenges that commonly arise throughout the workflow, from image acquisition to analysis. One major issue is to sufficiently mimic the in vivo environment and avoid disrupting the cells throughout the entire duration of the experiment, which can last days, weeks or even months.

“Controlling environmental parameters, such as temperature, humidity and CO2 is critical for reducing the variability between observations and guaranteeing the relevance of the results in live-cell imaging assays,” notes Karin Boettcher, Associate Product Manager for High-Content Screening and Applications, PerkinElmer. “The use of an onstage incubator can help manage the environmental conditions to keep the cells alive and functioning normally for extended periods.”

As cells often move during a time-lapse experiment, it is beneficial for live-cell imaging systems to be capable of keeping the cells of interest within the image. Furthermore, because there tends to be inhomogeneous growth and response of biological wells in culture plates, modern livecell imaging systems must be able to take a full scan of the entire plate to produce statisticallyrelevant data.

There are further issues associated with data storage and processing power. Time-lapse imaging at high resolution and high magnification generates large numbers of images, particularly when data is collected over extended time periods, such as for long-term cultures like 3D spheroids. This means that modern live-cell imaging systems need enough data storage for potentially hundreds of thousands of large images, as well as a high levels of processing power for image stitching and running image analysis software.

“It’s also crucial to minimise photobleaching and phototoxicity caused by the fluorescent illumination in live-cell imaging, especially in the UV range, says Chris Shumate, CEO, Etaluma. “Repetitive exposure to illumination and excitation sources can bleach the fluorescent labels and create reactive oxygen species that cause cell damage. Often, this effect isn’t recognised until an extended time lapse has been performed.”

Commercially-available live-cell imaging products

Many commercially-available products overcome the challenges associated with live-cell imaging. Here we provide a summary of some of the key products available on the market:

CytoSMART™ – imaging with cloud-based storage and analysis

The CytoSMART™ Lux2 is a cell-culture monitor that operates at low-voltage and can be safely used in any regular CO2 incubator. The system can run for extended periods (days or weeks) and will image cells in a wide range of cell-culture vessels, including slides, petri dishes, T-flasks and microplates. The monitor can be used across a wide range of applications, including cell migration, wound healing and scratch assays analysis, stem cell behaviour studies and chemotaxis studies.

The CytoSMART™ Omni is an automated livecell imager small enough to fit in any regular cellculture incubator. The system is capable of imaging a complete T-flask or well plate by taking 6,500 high magnification digital phase contrast images at predefined time intervals. The system comes with a CytoSMART™ Connect Cloud, which is a suite of image analysis software for high volume confluency analysis, colony detection and scratch assays.

The CytoSMART™ Connect Cloud runs on Microsoft Azure, which provides cost-efficient, large storage capacity and high levels of processing power for analysing and storing images. The platform is accessible from any computer, tablet or mobile phone to enable remote, real-time monitoring. It can also be set up to send automatic notifications to users once the cell-culture has reached the confluency threshold (Figure 1).

Etaluma Lumascopes – real microscopy simplified

Etaluma’s technology centres on a compact and simple optics path allowing lower power fluorescence excitation for the avoidance of photo damage. The solid state filter design eliminates pixel shift between channels and real phase contrast provides high quality transmitted light images. An open design allows fluidics and micro injectors to be integrated. Magnifications from 2.5x to 100x are available for research quality images. All four Lumascope models can be placed in environmental chambers, including common cellculture incubators, as well as hypoxia chambers and refrigerators. Additionally, tiling and z-stacking capabilities help to keep the cells of interest within the image during time-lapse experiments (Figure 2).

PerkinElmer – application flexibility for all throughputs

The new PerkinElmer MuviCyte™ live-cell imaging system is designed to operate inside an incubator to enable long-term observations under controlled conditions. The system can scan a broad range of vessel types and is compatible with labelfree transmitted light readout as well as threechannel (red, green, blue) fluorescent imaging to enable a wide range of commonly-used live-cell assays (Figure 3).

The PerkinElmer Opera Phenix™ and Operetta CLS™ high-content screening systems can run a wide-variety of assays in a live-cell format. When used with automation, both systems enable high throughput live-cell assays and can be configured with the integrated environmental control chamber and spinning disk optical technology to minimise photobleaching and phototoxicity of samples. Additionally, these systems enable gentle live-cell analysis (ie, without using a fluorescent probe) by using the digital-phase contrast imaging mode and brightfield imaging function (Figure 4).

Sartorius Corporation – accessible automated image acquisition and analysis

The Sartorius IncuCyte® is a live-cell analysis system that sits inside standard tissue culture incubators and automatically analyses cells for extended periods. The cells stay stationary during image acquisition and are not shuttled between instrument and incubator, minimising disruption. The system has also been designed to make it accessible for even first-time users by automating as many of the decision points as possible. The images are organised alongside experimental details and realtime data analysis is provided by a guided user interface tailored for different experimental paradigms (Figure 5).

Picturing the future of live-cell imaging

The role of live-cell imaging is likely to grow across drug discovery and development. Enhanced data capture and analysis features will improve the accessibility of the technique, allowing researchers to keep pace with the ever-growing need for more physiologically-relevant and complex models of disease. This will likely lead to the generation of better statistically-relevant and reproducible results compared to traditional endpoint measurements.

Moreover, as a quicker and easier alternative to endpoint experiments, kinetic (time-based) data such as that generated from live-cell imaging is predicted to become increasingly vital in drug discovery. This type of data enables a better understanding of cell behaviour as a result of different stress factors, meaning that a wider variation of cell types can be studied, including minor and less frequent effects as well as long-term effects.

Time-lapse microscopy such as that used in livecell imaging could also help improve the efficiency and speed in which hit compounds can be identified, by applying the technique to large compound screens in primary high-throughput screening.

As technologies such as CRISPR, CAR-T and other immuno-oncology techniques continue to expand the biology that can be measured in cells, it is predicted that the scope of applications for livecell screening will keep growing. As 3D biology and mixed cultures yield more physiologically-relevant measurements, live-cell imaging of these cultures will complement the replication of the in vivo environment.

Conclusion

Live-cell imaging provides greater insights into cellular behaviour and function to enhance drug discovery. Modern technologies offer numerous benefits, including improved cost-efficiency and streamlined laboratory operations, as well as the generation of more reliable and relevant data that could potentially expedite the successful progression of drugs through discovery. As live-cell imaging becomes progressively important in the development of new medicines, it is vital that technologies continue to adapt to the needs of researchers to fully unlock the benefits of this ground-breaking technique. DDW

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This article originally featured in the DDW Fall 2019 Issue

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Helen Stewart-Miller is Director of PR Services and Dr Kate Tointon is a Science Writer at BioStrata, a life science specialist marketing agency. The company’s growing team in Cambridge (UK) and Boston (US) includes a significant number of people with deep scientific experience and knowledge. The agency offers a range of services from strategy, branding and message development through to content creation, creative design, digital marketing and public relations.