Seeing is believing: label-free, time-lapse microscopy for quantifying cell proliferation

A quick introduction to cell proliferation

Cell proliferation describes an increase in cell number through the linked processes of cell growth and division and is counteracted by cell death and differentiation¹. In this process, cell division and growth are tightly regulated by a complex network of signaling molecules, which ensures that cells in the same state exhibit uniform cell size. Proliferation is crucial for normal biological events, such as embryogenesis and tissue repair. Although proliferation is carefully regulated, defects in these regulatory measures can lead to pathologies, the main example being tumor formation.

The rate of cell proliferation is dependent on a number of factors, including the cell type being examined, its environmental and metabolic conditions, its state (e.g. senescence), and many other factors. While cell proliferation describes the rate and characteristics of cell division, cell viability indicates the overall health and functionality of cells. Indeed, cell proliferation is one indicator that is used to assess cell viability, although not all viable cells undergo cell division.

 

The importance of proliferation assays

Cell proliferation assays are used in a wide variety of contexts. For example, proliferation can be used to gauge the health of new transgenic cell lines or to evaluate the success of tissue regeneration experiments¹. Quantitative cell proliferation assays are also essential for evaluating the dose-response of new pharmaceutical agents, such as in the context of anti-cancer drug development.

 

Methods for quantifying cell proliferation

There are many different methods for quantifying cell proliferation that take advantage of the unique properties of growing and dividing cells. These assays are either (1) endpoint assays that involve chemical incubation and typically feature colorimetric- or fluorescent-based outputs or (2) label-free, image analysis-based methods. While the former assay type is amenable to high-throughput analysis, the batch of cells being examined typically cannot be used for further downstream applications. In contrast, label-free live-cell imaging allows proliferation to be assessed over a long period of time and does not prevent further processing of cells in downstream assays. In addition, this method is applicable to both low- and high-throughput applications through the use of novel automation strategies.

 

Chemical analyses versus label-free, image-based analyses

While chemical-based proliferation assays are well established in the literature and can provide detailed information about the molecular characteristics of a cell population, these methods do have some drawbacks (Figure 1). Many of these methods are endpoint assays, and as a result, are very resource-heavy and time-consuming to perform. In the case of the MTT assay, for example, the cells for each individual experimental condition and time point over a time course must be separately processed, which can introduce experimental error. In addition, although many of the popular proliferation assays discussed in this article use reagents that do not overtly exhibit cytotoxicity, the addition of these probes can still perturb physiological cell behaviour^2,3. Further, many of these assays are highly sensitive to the culture conditions, such as the length of reagent incubation, the cell confluency at the beginning and end of the assay (which will influence whether the cells are in log phase growth), and the metabolic activity of the cells, and therefore must be thoroughly optimized prior to data interpretation. Finally, because these assays are indirect readouts of cell proliferation, they may not accurately represent cell growth and division, and instead may be reflective of the specific processes being measured (e.g. changes in metabolic status that occur independently of proliferation).

Although the use of label-free, time-lapse microscopy cannot reveal the same extent of molecular-level information as chemical-based methods, its main advantage is the ability to produce robust quantitative measurements of proliferation in a non-invasive way that better preserves the native physiology of the cells. Indeed, label-free assays eliminate the uncertainty associated with exogenous probes and the unexpected side effects they may have on cell behavior, including direct effects on cell proliferation rates^2,3. Further, live-cell imaging allows the processes of cell growth and division to be directly visually evaluated, which can corroborate quantitative assessments. However, a drawback of this technique is the difficulty of automating cell segmentation and counting. While there is a wide literature on segmentation techniques, creating a universal method that is applicable to many cell types in many distinct contexts remains a challenging area of research.

Methods for Assessing Cell Proliferation
Assay Features	Chemical Assays	Label-free, Image-based Assays
Reagents	Well established probes and commercial kits available; many reagents are cytotoxic or can cause abnomal cell behavior	Standard bright-feld mirosoopy, image analysis packages commercialy available
Molecular-evel intormation on cell status?	Yes	No
End-point assay?	Yes, most assays	No; long-term imaging possible (days-weeks)
Requirement for optimization	Yes; sensitve to environmental factors	Optimization of image analysis
Type of readout	Plate reader/spectrophotometer measurement of absorbance; ICC/IHC in some assays	Direct visualization of cell divisions
Data colection and anaysis	Resource-heavy and time-consuming data colection; data analysis straightorward	Image analysis can be complex

Figure 1 | Differences between chemical and label-free, image-based assays for measuring cell proliferation.

 

Chemical assays for measuring cell proliferation

The four major classes of cell proliferation assay measure (1) the rate of DNA synthesis, (2) the metabolic activity, (3) the presence of immunomarkers of proliferation, and (4) non-toxic, long-term labeling and dilution (Figure 2). These methods produce either colorimetric or fluorometric signals that are usually detected using a spectrophotometer or fluorescence microscope^1,4-6. If analyzed using a spectrophotometer/plate reader, and depending on the specific assay, the data may be displayed as absorbance over time or absorbance per increasing drug dose.

Category	Assay	Overview	Detection	Application notes
DNA Synthesis	BrdU	Thymidine nucleoside analogue incorporated into DNA: detection with anti-BrdU antibody	ICC/IHC FACS ELISA	In vivo and in vitro Single cell resolution Lengthy detection step
	EdU	Thymidine nucleoside analogue incorporated into DNA: detection by fluorescent dye through click chemistry reaction	ICC/IHC FACS ELISA	In vivo and in vitro Single cell resolution Faster detection
Metabolic Activity	MTT Assay	Yellow tetrazolium sat, MTT, is reduced by metabolic enzymes to purple formazan crystals	Plate Reader	Solubilization required Cytotoxicity End-point assay
	XTT Assay	Yellow tetrazolium sait, XTT, is reduced by metabolic enzymes to soluble orange formazan product	Plate Reader	Soluble Less toxic End-point Assay
	Resazurin Assay	Non-fluorescent blue resazurin reduced by metabolic enzymes to red fluorescent resorufin	Plate Reader	Cell permeatble, Less toxic Fluorescent and colorimetric End-point assay
	Luciferase ATP Assay	ATP-dependent activity of luciferase produces a bioluminescent signal from luciferin	Plate Reader	Fast detection End-point assay
Immunomarkers	Ki-67,phospho-Histone H3, PCNA, etc.	Antigens that are only present during cell division can be detected with antibodles	ICC/IHC WB	In vivo and in vitro Single cell resolution Lengthy detection step
Labeling and dilution	CFSE	Fluorescent dye CFSE labels intracellular proteins; cell division dilutes the fluorescent signal	ICC FACS	Live cell imaging Observe cutures over time Cytotoxicity

Figure 2 | Chemical assays for measuring cell proliferation.

DNA synthesis

Prior to dividing, the DNA in a cell must first be replicated. Therefore, the rate of DNA synthesis can be used as a proxy for cell proliferation. DNA synthesis can be measured using the incorporation of labeled nucleotides or nucleoside analogs, the concentration of which can then be measured. A classical method that is now less commonly used involves radioactive ³H-thymidine incorporation, followed by measurement with a scintillation counter¹.

The modern alternative instead uses the nucleoside analog of thymidine, 5-bromo-2’-deoxyuridine (BrdU), as a marker for DNA synthesis. BrdU can be detected using monoclonal antibodies, and its incorporation can, therefore, be evaluated using in vivo or in vitro immunofluorescence-based assays coupled with microscopy, as well as a colorimetric or chemiluminescent enzyme-linked immunosorbent assay (ELISA). An updated version of this assay uses the incorporation of 5-ethynyl-2′-deoxyuridine (EdU) and rapid detection with a fluorescent dye through a “click” chemistry reaction⁷.

Metabolic activity

Cell growth, and therefore metabolism, is essential for cell proliferation and can be used as another means of assessing the proliferation rate. The production of metabolic intermediates, including ATP, NADPH, NADH, FADH, and FMNH, increases in response to elevated cell proliferation. A number of mitochondrial oxidoreductase and dehydrogenase enzymes that are dependent on these molecules are then activated, and chemical sensors can be used to indicate this activation. Tetrazolium salts, such as MTT, XTT, MTS, and WST, are reduced to colored formazan products by these enzymes, while resazurin (7-hydroxy-10-oxidophenoxazin-10-ium-3-one, sodium) is reduced to a red fluorescent compound called resorufin¹.

In the case of the MTT assay, the resultant formazan crystals must be solubilized in DMSO, SDS, or other reagents under acidic conditions prior to measuring the absorbance, precluding further downstream applications. In contrast, XTT, MTS, and WST substrates are soluble in water, but require additional electron acceptor reagents, such as phenazine methyl sulphate, to permeabilize the cell membrane.

WST, MTS, and XTT are less cytotoxic than MTT, and can, therefore, be used for kinetic assays. The resazurin assay is particularly advantageous in this regard, as the substrate is non-toxic, cell-permeable, and amenable to more sensitive fluorometric measurement methods¹.

In addition to these metabolic intermediates, the relative ATP concentration can also be used as a readout of proliferation. Assays that measure ATP levels rely on the ATP-dependent activity of luciferase to produce a bioluminescent signal from the substrate luciferin.

Immunomarkers of cell proliferation

Many proteins are specifically activated during cell division, resulting in the presence of a unique set of antigens that can be detected using antibody staining, coupled with microscopy or flow cytometry. For example, the Ki-67 protein is absent in non-cycling cells but accumulates during the S, G2, and M phases of the cell cycle⁸. Additional markers of cell proliferation include proliferating cell nuclear antigen (PCNA), phospho-histone H3, and topoisomerase IIB¹. If analyzed using microscopy, proliferation can be quantified using this technique by manually counting positively stained cells or using a more automated image analysis-based method⁵.

Long-term labeling and dilution

During cell division, the contents of the cytoplasm in the mother cell are divided between the two daughter cells. The cell-permeable, non-toxic fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) can be used to uniformly label-free amines on intracellular proteins. With each successive division cycle, the fluorescent signal from the labeled proteins will be diluted. Thus, the proliferation rate will be linked to the decreasing fluorescent signal in this assay, which can be assessed using flow cytometry or spectrophotometery⁹.

 

Label-free methods for measuring cell proliferation

Live-cell imaging

Kinetic growth curves, which typically describe the cell count or percent confluency of a culture over time, provide a quantitative evaluation of cell proliferation rates¹⁰. This method of analyzing proliferation requires only brightfield, phase contrast, or differential interference contrast (DIC) microscopy, coupled with subsequent image analysis, and is appropriate for both low-throughput – such as a single culture dish – and high-throughput – using, for instance, a 96-well plate – approaches. Because the cultures can be visualized in real-time using live-cell imaging, remain in their incubators under their optimal culture conditions, and are not exposed to potentially behavior-altering chemicals, this method can be performed over a long period of time, from days to weeks depending on the lifetime of the culture. Label-free imaging is especially useful for long-term lineage tracing over multiple cell division cycles without perturbing cell health. For example, this technique was used to show that cultured, karyotypically normal human embryonic stem cells (hESCs), in contrast with karyotypically abnormal hESCs, exhibit “bottlenecks” in proliferation that include initial survival upon plating, entry into mitosis, and death after mitosis¹¹.

Image analysis

Various techniques have been published for automating cell segmentation, tracking, counting, and lineage tracing, from simple algorithms that can be imported as plug-ins in ImageJ^12-14, to implementations that are bundled into the high-end image analysis software. Cell segmentation algorithms usually involve feature detection, morphological filtering, intensity thresholding, and deformable model fitting, among other image analysis methodologies¹⁵. As mentioned earlier, a major limitation in automated cell segmentation is developing an algorithm that is applicable to many different cell types, which exhibit disparate morphologies and optical features. However, new and highly sophisticated methods using deep neural networks will likely help resolve this issue¹⁶.

 

Image-based proliferation assays using CytoSMART Omni

The CytoSMART Omni, which can sit inside an incubator with cell cultures and perform continuous live-cell imaging, can be used for monitoring each well of a multi-well plate in full. With this device, a user can follow cell proliferation over time for many wells at once, the analysis of which can be automated using some of the methods described above. This type of high-throughput technique is useful in many contexts, including dose-response assays, cell line development, organoid culture, and stem cell culture. For example, this methodology can be used to monitor mitosis versus differentiation events in stem cell populations under many distinct culture conditions, which can be used to optimize regenerative medicine processes^17-19.