Highlights

  • Examine cell proliferation and cell growth independently
  • High-contrast, label-free, imaging for robust single cell segmentation & tracking
  • Extract a wealth of single cell metrics in the form of cell count, mitosis events and dry mass

Introduction

Cell proliferation is the increase in the number of cells over time as a result of cell division. In contrast, cell growth is the process by which cells accumulate mass [1]. The interactions between cell proliferation and cell growth pathways are complex but these processes can operate independently under certain conditions. Cells can grow without dividing, in the example of muscle cell hypertrophy, whilst cells can proliferate without growing when an embryo undergoes cleavage.

Cell confluence and cell count measurements through traditional image analysis are common outputs for cell proliferation assays. The accuracy of the former is influenced by changes in cell morphology in response to compounds whilst the latter often requires the introduction of fluorescent labels to improve counting accuracy, which may alter normal cell behaviour. Notwithstanding these potential limitations, traditional imaging techniques do not reliably report changes in cell growth, and specifically biomass.

The ability to measure cell proliferation and cell growth independently could function as a tool for the development of drug treatments. For instance, abnormal accumulation of cell dry mass has been implicated in diseases, including cardiac hypertrophy [2] and tuberous sclerosis [3], whereas hyperproliferation is responsible for the development of cancers [4].

Cytochalasins inhibit actin polymersiation and, therefore, are frequently used to understand cytoskeletal mechanisms. However, these drugs can act via a diverse range of mechanisms which result in anticancer activity. For instance, cytochalasin D can inhibit the proliferation of cells by blocking cytoplasmic division (cytokinesis) and cause cell cycle arrest at the G1-S transition [5]. Our aim was to study the effects cytochalasin on the lung cancer A549 cell line by measuring cell proliferation and cell growth. The cell proliferation and cell growth measurements will be analysed independently using the Livecyte. This instrument utilises Ptychography, a quantitative phase imaging (QPI) technique, to produce high contrast images without the need for fluorescent labels. The enhanced contrast enables automatic segmentation and tracking of individual cells, as well as a quantitative measure of cellular dry mass.

Method

Cell Culture

A549 cells were routinely maintained in DMEM, supplemented with 10% FBS (complete medium hereafter) at 37C with 5% CO2/95% humidity prior to experiments.

Cell Proliferation and Growth Assay

Cells were harvested using standard techniques and cell count and viability determined by trypan blue exclusion (ViCell; Beckman Coulter®). Cells were seeded into the wells of a 96-well plate at 1600 cells/well and cultured overnight. Media was replaced with complete medium only or complete medium containing cytochalasin D (150 ng/mL- 9 ng/mL) and incubated for 1 hour prior to imaging.

Resources

  • A549 cell line
  • DMEM (Gibco)
  • Foetal Bovine Serum (FBS; Gibco)
  • Cytochalasin D (150 ng/mL- 9 ng/mL)
  • 96-well culture plate (Corning® 3603)
  • Livecyte Kinetic Cytometer (Phasefocus)
  • Livecyte Acquire & Analyse software (Phasefocus)

Time-Lapse Imaging

High-contrast quantitative phase images were automatically captured using the Livecyte Kinetic Cytometer. Cells were imaged with an Olympus PLN 10X (0.25NA) objective and 1 mm x 1 mm field of view (FOV) per well for 18 hours at 16-minute intervals. Cells were maintained inside an environmental chamber at 37°C with 5% CO2/95% humidity.

Analysis

Treatment of cells with cytochalasin D will affect cellular morphology and inhibit cellular processes such as cell division, and even induce apoptosis [5]. Metrics to measure these changes can be acquired using the Livecyte. This report will focus on single-cell metrics for proliferation and growth extracted from the Proliferation, Mitosis and Morphology Dashboards in Livecyte's Analyse Software

Results

Quantitative Phase Images & Cell Segmentation

The quantitative phase images generated by the Livecyte are label-free and high contrast. In this assay, they show that even at the low concentration of cytochalasin D (9 ng/mL) several cells were multinucleated which induced the formation of multiple large vacuoles after 18h (Figure 1). This is a consequence of cytochalasin D inhibiting cytoplasmic division (cytokinesis) leading to multinucleated cells with significantly perturbed cytoskeletons [5].

Cell Proliferation and Mitosis

Cell proliferation measurements such as cell count, confluence and cell doubling times are automatically determined and displayed on the Proliferation Dashboard. For example, these graphs show that an introduction of the cytotoxic agent, cytochalasin D, reduces cell proliferation rate and increase cell doubling time in a dose dependent manner (Figures 2 & 3). A mitotic event is easily identified using the Livecyte system from the rounding up of cells undergoing division. Corresponding data is clearly displayed on the Mitosis Dashboard. In this case, there is a reduction in the number mitotic events in groups treated with higher concentrations of cytochalasin D (Figure 4).

It is known that cytochalasin D inhibits cytokinesis at the late stages of mitosis. In addition, this drug is cell permeable and has been shown to activate p53-dependent pathways, causing arrest of the cell cycle at the G1-S transition [5, 6]. Both these affects will contribute to a reduction in cell proliferation and mitotic events, which is in line with our results from Livecyte's Analyse software.

Cell Growth – Dry Mass

Cellular dry mass is a metric unique to quantitative phase imaging and represents the total mass of all cellular components, including proteins, lipids, carbohydrates and DNA, amongst others, but excluding water [7, 8]. Through segmentation and tracking algorithms Livecyte automatically reports the total dry mass of individual cells and the population, as a whole, over time.

Cytochalasin D was shown to reduce the total dry mass accumulation per field of view in a dose-dependent manner (Figure 5). However, the differences between cytochalasin D groups and control for dry mass doubling time were less marked than that observed for cell doubling time (Figure 6). This indicates that although proliferation was reduced in the cytochalasin D groups, biomass synthesis was less affected.

Figure 5 and 6 shows cell growth at a population-level, however, Livecyte can segment and track individual cells which means single-cell measurements are also possible. For example, Figure 7 shows a line plot of median cell dry mass per cell over time. Interestingly, this reveals a significant increase in median cell dry mass over time for cells treated with higher concentrations of cytochalasin D (150 and 75 ng/mL). Whereas, lower concentrations of cytochalasin D (37.5, 18.8 and 9 ng/ml) showed a similar trend in median cell dry mass compared to the control group. This indicates that at the higher concentrations of cytochalasin D (150 and 75 ng/mL) individual cells are growing (increasing in biomass) but not proliferating. This may be a consequence of cell cycle arrest in G1 at these higher concentrations; however further studies would be needed to confirm this [9].

Figures

Figure 1

Figure 1: Illustration of Quantitative Phase Images at 18 hours a) treated with 9 ng/mL of cytochalasin D and b) untreated control.

Figure 2

Figure 2: Plot of cell count over time for cells
treated with cytochalasin D (150 ng/mL- 9 ng/mL) and untreated control.

Figure 3

Figure 3: Histogram plot illustrating median cell doubling time for cells treated with cytochalasin D (150 ng/mL- 9 ng/mL) and untreated control. Cells treated with 150 ng/mL of cytochalasin D had a cell doubling time of 388 hours.

Figure 4

Figure 4: Histogram plot illustrating total mitotic
events for cells treated with cytochalasin D (150
ng/mL- 9 ng/mL) and untreated control.

Figure 5

Figure 5: Line plot illustrating total dry mass in all fields of view, over time, for cells treated with cytochalasin D (150 ng/mL-9 ng/mL) and untreated control.

Figure 6

Figure 6: Histogram plot illustrating dry mass
doubling time for cells treated with cytochalasin D (150 ng/mL- 9 ng/mL) and untreated control.

Figure 7

Figure 7: Line plot illustrating median dry mass per cell, over time, for cells treated with cytochalasin D (150 ng/mL- 9 ng/mL) and untreated control.

Download the Application Note pdf

Summary

Cell proliferation and cell growth play key roles in development and regeneration but are also affected by drug treatments and disease states [3,4]. These pathways often work in parallel, however, they also operate individually under certain conditions.

Quantitative phase images are high contrast, enabling robust cell segmentation, and quantify cell mass. Coupled with time-lapse imaging and cell tracking algorithms, Livecyte provides users with the tools to monitor how cells alter their proliferation and growth states, independently, in response to external conditions.

In this short study we sought to examine the effects of cytochalasin D on cell proliferation and cell growth. Our Proliferation and Mitosis Dashboards showed a dose-dependent reduction in cell proliferation and mitotic events with cytochalasin D treatment. In addition, the Morphology Dashboard shows an increase in individual cell biomass over time in wells with higher concentrations of cytochalasin D (150 and 75 ng/mL). These results are in line with the mechanisms of action reported in literature as cytochalasin D is a cytokinesis inhibitor and can cause cell cycle arrest at the G1/S transition [5,9]

In summary, the various dashboards generated from Livecyte's Analyse software paint a full picture of cell behaviour by providing metrics at both a population and single-cell level. In this case, we have taken advantage of the Proliferation, Mitosis and Morphology Dashboards to understand the affect cytochalasin D has on cell proliferation and cell growth. Having the ability to measure these states independently can aid the identification or validation of a drug's mechanism of action.


References

  1. Lloyd A.C. et al., 2013. The Regulation of Cell Size. Cell., 12(154), pp 1194-205
  2. Loffredo F.S. et al., 2013. Growth differentiation factor 11 is a circulating factor that reverses agerelated cardiac hypertrophy. Cell., 153(4), pp 828-839.
  3. Crino P.B., 2001. mTOR signaling in epilepsy: insights from malformations of cortical development. Cold Spring Harb Perspect Med., 5(4). a022442
  4. Alvarado S.A., 2012. Cellular Hyperproliferation and Cancer Evolutionary Variables. Curr Biol., 22(17). R772-R778.
  5. Trendowski M., 2015. Using cytochalasins to improve current chemotherapeutics. Anticancer Agents Med Chem., 15(3). pp 327-33.
  6. Rubtsova S.N. et al., 1998. Disruption of actin microfilaments by cytochalasin D leads to activation of p53. FEBS Lett., 430(3). pp 353-7.
  7. Davies H.G. and Wilkinson M.H.F. 1952. Interference microscopy and mass determination. Nature. 169. 541.
  8. Aknoun S. et al., 2015. Living cell dry mass measurement using quantitative phase imaging with quadiwave lateral shearing interferometry: an accuracy and sensitivity discussion. J Biomed Opt. 20(12). 126009.
  9. Uetake Y. and Sluder G., 2004. Cell cycle progression after cleavage failure. J Cell Biol., 165(5), pp 609–615.