Continuous live-cell monitoring techniques have witnessed a significant development in recent years, transforming the way cells are used in the laboratory.
These technologies are able to extract valuable cellular information that was not possible before. Their features are also closely aligned with the key changes in the realm of cellular research.
In an effort for greater significance, biomedical researchers are moving from basic recombinant cell systems to stem-cell derived cells and primary cells that are usually human and specific to patients.
Sophisticated tissue organoid models and in vitro co-cultures are becoming increasingly common. Though target-specific assays will continue to be significant, cell-based, phenotypic assays are becoming more popular because they enable holistic measurement of integrated cell functions.
Although these advancements present opportunities for novel studies, they also bring new requirements for cellular workflows and technical complications. This article shows how these changing requirements are addressed by advancements in continuous live-cell monitoring and analysis techniques.
Immortalized cell lines
HEK, CHO, HeLa cells, and other classic cell lines are simple and routine. Through advanced molecular approaches, researchers are able to manipulate or express genes in these cell lines. Kit-based, turnkey methods can also be used to induce stable gene expression for various cell passages.
Recombinant cells are predictable, robust, and grow quickly, reliably, and homogeneously for a long duration of time without any major complexities induced by loss of expression. These cells are low-cost and high-yield, allowing investigators to discard unused ‘spare’ cells frequently.
Primary cells, for example neurons, can accurately show the in-vivo function of biological systems when compared to immortalized cell lines. However, they cannot be easily acquired in large numbers, and many also not divide, meaning they cannot be scaled-up further.
There are certain primary cell types that do not survive for an extended period of time in culture. The genetic manipulation of primary cells is also more complicated when compared to immortalized cells. Transfection procedures and special constructs may also be required.
Compared to immortalized and primary cells, stem cells provide better flexibility. With next-generation technology, fully differentiated mature cells can be derived from replicating stem cell pre-cursors.
Such cells provide several therapeutic and diagnostic applications, for instance, genetic pre-disposition and disease etiology can be studied by comparing the properties of cells obtained from different patients.
Differentiated cells and mesechymal stem cells can also be thought of as potential therapies. These methods combined with the recent developments in precision gene editing bring us closer to the concept of truly personalized medicine.
However, the procedures involved for differentiating, reprogramming, and scaling stem cells are quite lengthy, complicated, and poorly defined. In order to resolve these challenges, several commercial suppliers provide differentiated, cryopreserved cells. These cells are expensive and make it unfeasible for laboratories to use on a routine basis.
As stem cells mature and differentiate, their properties change over time and in many situations only a low yield of healthy, fully differentiated cells is obtained. Due to the dynamic nature of primary and stem-cell derived cultures, more emphasis is placed on the requirement for real-time decision making and active management by researchers, particularly in comparison to immortalized cells.
The role of the microenvironment
Another emerging factor is the extent to which the microenvironment influences cell behavior. In the quest to simulate true pathology and physiology, scientists are developing more comprehensive multi-culture and co-culture cell systems.
Growing cells in scaffolds and bio-matrices to form 3D microtissues and organoids is becoming popular and so are organ-on-a-chip systems (Figure 1). However, these experiments can be complicated, need a large amount of optimization, and place greater demands on the detection systems employed for analysis and quality control of target cells.
Figure 1. Researchers are combining more relevant cell types with more relevant organizational structures to create advanced and more translational model systems.
Cellular models are changing, the new commercially available cell systems may prove to be more translational and applicable but they are more expensive and complicated.
When using advanced cellular models, increased time and effort has to be devoted to the experimental studies. The cells are dynamic, requiring careful observation and they are valuable so shouldn’t be wasted.
Benefits and challenges of different cell model systems
Table 1 shows the benefits and challenges of various cell model systems.
Table 1. Comparison of the typical attributes of cell model systems – relative cost (1=low), ease of use, availability and perceived translational relevance
Defining the need - continuous live cell analysis
The added dynamic complexity has to be addressed through greater real time experimental techniques, which convey the long-term changes in the properties of living cells.
This data would be normally collected across the cell life and in terms of other cells and their respective microenvironment. This method also has to detect and report heterogeneity in cells.
For the sake of time and cost, methods which acquire the most decision-making data from the smallest number of cells are preferred. This can be obtained either directly such as assay miniaturization or indirectly for example reducing the number of studies and required repeat assays. Reducing the amount of initial assay development work and the number of assay failures can also boost efficiency.
Scientists can gain a better insight into the timeline of biological processes, and thus make informed decisions about their analysis workflows and cell husbandry using reporter, phenotypic, and cell-health assays.
This is because such assays offer real-time information and do not disrupt the cells. Most importantly, addressing these needs should not be at the cost of simplicity and ease-of-use to investigators. Fortuitously, these increasing requirements can now be met with several new methods.
Non-invasive, live-cell analysis
Live-cell imaging and electrical monitoring are two new technologies developed for non-invasive cell analysis over the long term. While neither method is particularly new, both have developed in ways that suit the dynamic requirements of cell biologists.
In the case of electrical monitoring, cells are initially plated on electrodes integrated in plastic, followed by recording extracellular field potentials or cellular impedance. Sampling can be done and high frequency (kHz) data acquisition can be obtained.
The system can be placed in a cell incubator to record signals for long durations without disrupting the cells. The analysis of excitable cells is considered to have the most convincing applications.
For instance, with the use of impedance recordings, the changes in the way human stem-cell derived cardiomyocytes contract and beat can be determined over days and weeks. This enables scientists to investigate both chronic and acute cardio-toxicity during the development of drugs.
With regard to neurons, multi-electrode array chips have been shown to be useful for monitoring the action potential spike patterns over the long term.
As cells spread, migrate, proliferate, or bind to the electrodes, impedance values change. This forms the basis for other phenotypic assays, which have easy work flows and do not disturb the cells.
However, a major drawback is that large numbers of cells and specialist consumables have to be used. Further, the systems do not provide much spatial context, making it difficult to collect data about the location of cells and how they interact.
Impedance can also be regarded as a relatively blunt tool for studying non-excitable cells; in these cells, signal changes can occur from various cellular functions, making it difficult to understand the data.
For the research community, live-cell imaging would typically involve a complicated inverted microscope system, along with a confusing range of heated stages, complex image analysis software, light-source options, etc.
A small number of cells are normally observed in a plate/dish and are viewed for several minutes or hours at the most.
For most researchers, the concept of using a microscope to monitor cells over many days or weeks is implausible, particularly if the imaging system is based in a facility which is shared by other researchers.
As a result, the scale and throughput rendered by most live-cell imaging workflows are limited by these factors.
Different systems have emerged over the past few years to resolve these issues, mostly by removing the strain from live-cell imaging and simplifying the workflows. Keeping the ease and affordability in mind, several compact, low-cost, single-plate microscopes have been designed to be integrated into traditional cell incubators.
Though the functionality and optics will be slightly limited as opposed to high-end imagers, they provide novice users with entry level access to live-cell analysis.
All users have to do is to place their cells on the stage and press ‘go’. The software tools for these systems are designed more for cell tracking than elaborate phenotypic analysis. For instance, remote viewing can be set up and the system can be configured to activate automated e-mail alerts when cells reach a specified confluence.
There are certain systems that allow fundamental fluorescence measurements. However, in the majority of cases, only one single plate can be examined at a time and the software also lacks the ability to execute advanced image analysis.
In contrast, many micro-plate imagers and high-content imaging devices are available that come with heated chambers or stages to house the living cells. The fluorescence capabilities of these devices are added with bright field optics for ‘label-free’ data capture, which further extends the available applications. These devices offer high throughput and enable miniaturization of assays for low usage of cells.
Although the analysis packages are not particularly configured for handling temporal data, they have robust functionality for fluorescence image quantification. If considered individually, the bright-field optics in these systems are limited and the systems also lack the preferred long-term humidity and temperature control, which is important for preserving cell health.
To further increase throughput, robotic plate handlers have been integrated to move the plates back and forth between the plate readers/microscopes and cell incubators. While this method had some amount of success, it is quite expensive.
Between these two methods, lie medium to high-throughput solutions such as microscope workstations and live-cell imagers that are aimed at functionality/cost/throughput/ease-of-use sweet-spot for kinetic assays and cell monitoring. These systems offer the following features:
- Fully automated image capture and analysis
- Flexibility to work with a variety of cell culture plates and flasks
- Stable humidity and temperature control
- User-friendly software tools for real-time, on-the-fly visualization of cell behavior
Continuous live-cell analysis: imaging solution types
Table 2 shows the comparison between different imaging methods for continuous live-cell analysis.
Table 2. High level comparison of different imaging approaches for continuous live-cell analysis. *the relative scoring for ‘capacity, throughput’ and ‘automated analysis’ are made with reference to use for continuous live cell analysis rather than in end-point read mode
Live cell imaging and automated analysis set up
Figure 2 shows the IncuCyte® Live Cell Imaging set up for continuous live cell analysis.
Figure 2. IncuCyte® Live Cell Imaging set up for continuous live cell analysis. The IncuCyte® system resides within a standard cell incubator for optimal environmental control, and captures images in situ from standard cell culture vessels (plates, flasks etc.). Images are automatically analyzed in real time with user-trainable algorithms and visualized to report changes in biological parameters over time. Data shown is a cell migration scratch wound assay – images are analyzed over time for migration into the wounded zone and displayed as a full time course for each well in a micro-plate assay.
Extended cell monitoring and temporal measurements
The learning opportunity begins the moment cells are placed in culture, whether de-frosted from a cryo-vial or freshly harvested. Most laboratories may lack the formal procedures required for cell monitoring before analysis.
No images or measurements are recorded and key questions regarding the appearance, growth, and differentiation of cells are often tackled subjectively.
A solution is provided by real-time, non-invasive monitoring that enables capturing of images from the moment the cells enter the incubator, with the data employed for monitoring the morphology and growth of cells.
It is also possible to store the images for future analysis. When using proliferating cells, researchers can use real-time tracking to observe the growth profiles and estimate the best possible time for call passaging, while observing any sudden changes in morphology or growth rate.
There are certain systems that allow users to do all this from the comfort for their homes, without entering the laboratory or disrupting the cells.
When cells have to be prepared for analysis or feeding, the process outcome can be affected by several unknown factors. The effect of such experimental variables can be evaluated more accurately with non-invasive, real-time monitoring.
This way, such variables can be optimized for upcoming studies. For example, optimization can be made simple and reliable by performing matrix experiments and tracking the cells before and after modifying a selected variable.
Exhaust cells or individual time point measures no longer have to be set up as part of the analysis procedure. Optimal parameters can be used again for later experiments, and the cells employed in the matrix study can be used directly in downstream assays.
In order to ensure optimal performance of the assay, healthy cells should be uniformly plated and at optimal densities throughput the micro-assay plates. However, this procedure is not always simple, especially for miniaturized formats or when plating co-cultures or low numbers of cells.
In the majority of cases, plating of the cells is done for a certain period before the assay, so that they have time to recover prior to the measurements. During this recovery time, tracking the plates will enable researchers to locate the sources of across- and within-plate assay variance, such as poor cell health, uneven cell plating, and so on.
Even if a small number of assay plates are used, a clear understanding of the association between assay signal and treatments, plate coatings, cell plating densities, and other protocol parameters can be obtained over time.
Capturing rare events
Capturing stochastic, rare, biological events is where continuous tracking really comes into its own. As an example, let us consider stem cell workflows. While reprogramming adult cells into pluripotent stem cells, various transcription factors are introduced which results in gradual formation of stem cell colonies.
However, just a small portion of the cells are effectively reprogrammed and colonies can emerge at any point of time during the procedure. It is also laborious and time-consuming to check the plates each day for several weeks to locate new colonies, while cell health is affected by the non-stop movement of cells into and out of the incubator.
Cell cultures can be tracked over time and remotely monitored for emerging colonies through continuous monitoring methods. Fluorescent gene expression reporters can also be used to locate where and when the target genes are turned on during the process of reprogramming and differentiation.
Here, the ability to observe the cells’ morphological history is very useful when learning how to detect the best colonies and knowing the best time to pick them. Likewise, cancer stem cell assays and clonal cell lines can be created by applying similar principles to limiting dilution experiments, where it is important to know if colonies emerged from a single cell or not.
Regardless of the type of cellular workflow, cell monitoring has little or no disadvantage if the overhead is low. Researchers do not have to examine each and every image that is captured; they can do so only when something unexpected happens and check the image library to see what exactly had happened.
Thanks to quantitative, real-time analysis of the images, the summary data can emphasize any changes that occur in the relevant target cell parameters.
Workflow applications of continuous live-cell analysis
Figure 3 shows an interconnected cellular work flows backed by continuous live-cell analysis, and Figure 4 shows the comparison of cell workflows using standard end-point and continuous live-cell analysis methods.
Figure 3. Interconnected cellular work flows supported by continuous live-cell analysis.
Figure 4. Comparison of typical cell workflows using traditional end-point (e.g. plate reader) and continuous live-cell analysis methods (e.g. IncuCyte®). In the continuous live-cell analysis paradigm, observations and measurements from cells are taken throughout the work flow to inform of changes in cell health & morphology, the impact of manipulations (e.g. transfection, plating steps) as well as the phenotypic parameters in the assay per se.
Blurring the boundaries of phenotypic assays and cell husbandry
Researchers who are interested in the measurement of higher order phenotypic cells will find that the edges between the assay and preparing cells for the assay may not be clear.
For example, before adding any test agents, non-invasive imaging techniques can be used to determine the time-course of neurite outgrowth from neurons in culture, and the same can be employed as a pre-treatment baseline against which the treatment effects are calculated.
If there are unstable or inappropriate baseline parameters for the assay for any reason, treatments can be postponed or withheld until a suitable time. In another example, if there is a requirement for transfection of a marker or reporter such as GFP, the cells can be tracked across the transfection period before judging the assay and the treatment timing to coincide with the optimal expression of a reporter.
These workflows are in complete contrast with standard end-point imaging studies, where the target parameter is only determined at a random time point towards the end of the experiment.
Better statistical precision than cross-well comparisons is obtained by measuring changes over time and non-invasively in each of the wells. This enables researchers to design more effective washout experiments and drug association.
Furthermore, real-time monitoring means that the relevant signal is never overlooked by researchers, who can then obtain meaningful data regarding the kinetics of their biological system without any extra overhead or cost.
Example application of continuous live-cell analysis
Figure 5 shows A549 cells grown in culture and tracked for growth and morphology.
Figure 5. A549 cells were grown in culture and monitored for morphology and growth, prior to transfection with NucLight Red fluorescent protein. Stably transfected cells were then plated on 96-well round bottomed ULA plates at 5K cells per well to form 3D spheroids. The transfection, spheroid formation and growth process was monitored using IncuCyte® live-cell analysis, prior to the assay on the effects of cytotoxic test drugs (e.g. staurosporine, SSP).
In the quest for more translational and relevant test systems, researchers are increasingly shifting towards sophisticated cellular models. However, these models have complicated, long-term dynamic biology, placing more burden on the detection and readouts techniques used for analyzing them.
These parameter changes are not illuminated by standard end-point assays over time. The latest cell models are associated with higher cost and consumption considerations, promoting the notion that cells are precious and should not be thrown away.
These factors collectively create a demand for cell monitoring solutions that allow long-term and temporal biological measurements, while reducing the usage of cells. These requirements are being addressed with advances in non-invasive detection technologies, especially live-cell imaging, which closes the gap between cell manipulation, cell culture¸ and cell-based assay workflows.
Since continuous live-cell monitoring and analysis provides several benefits, it could well become the new gold-standard for cellular studies, particularly when compared to more conventional, end-point measures. By comparison with other fields of research like deep sequencing, the challenge will shift to extracting knowledge from the data, instead of collecting data as such.
Trends and anomalies, which could be lost in the possible data overload, can be detected using ‘Big and Smart Data’ tools. It is hoped that the additional insight afforded by continuous monitoring will align with the complexity of state-of-the-art cell culture and analysis systems to bring our understanding of translational science and cell biology to a whole new level.
Produced from materials originally authored by Del Trezise, Ph.D, Co-founder and Director of Essen Bioscience, Ltd.
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