Go beyond traditional wound healing assays with the Phasefocus Livecyte

• Directly measure cell motility

• Separate cell motility from cell proliferation

• Segment and track individual cells along the leading edge

• Characterise morphological and behavioural cell phenotypes during wound healing

Next Generation Wound Healing Assay | Application Note

Introduction

The Livecyte system can be used to image the

cells and analyse their behaviour as both an overall

population and as single individual cells. The non-

invasive nature of the measurement (label-free,

low illumination power) ensures that the cells

are unperturbed and behave in a more natural

manner over an extended period of time. Very

large field of views are available without stitching,

which allows for statistically significant population

metrics to be extracted (gap closure, proliferation),

while the quantitative, high contrast nature of

the images allows for single cell behaviour to be

characterised (morphological parameters and

motility). The Livecyte system generates a far

greater suite of information from such established

assays, compared to traditional methods such

as brightfield, fluorescence or phase contrast.

Methods

MDA MB231 cells were seeded at 5x105 cells/ml

in Ibidi culture-insert 2 well dishes (Cat. 81176) to

create a cell free gap by exclusion. 24 hours after

seeding the insert was removed and the cells were

treated either with a vehicle control (1% BSA\HBSS)

or with 50μM Lysophosphatidic acid (LPA). The

Phasefocus Livecyte system was used to perform

time-lapse imaging of the wound, every 10 minutes

for a total period of 48 hours. The Phasefocus

CAT (Cell Analysis Toolbox) software was used to

segment and measure wound area, from which

t1/2 gap (time for area of wound to reduce by half)

and Vmigration (cell migration rate) was calculated.

In addition, individual cells were segmented and

tracked to report motility and morphological data.

The wound healing assay is an established in vitro procedure that measures collective cell migration. The basis of this assay is to create a cell free region within a confluent layer of cells, either through the creation of a scratch by removing the cells post adherence, or through physical exclusion of cells during seeding. Once the cell free area has been created the closure of the wound is monitored through live-cell imaging. The measure of collective cell migration in this manner, replicates sheet migration that occurs in a range of biological processes such as; cancer metastasis, embryonic morphogenesis and tissue injury.

Figure 1a).

Figure 1d). Figure 1e).

Figure 1 - Measuring Gap closure using the Phasefocus CAT software a) Representative images of wound gap segmented using the CAT software (blue outline), at 0, 24 and 48 hours and for both control and LPA treated cells. b) Relative gap area and c) relative dry mass measured throughout the time-lapse sequence. d) t1/2 gap closure and e) collective migration calculated from the GAP closure.

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Results

The rate of gap closure is the most common metric that

is measured from a wound healing assay. Subsequently,

the t1/2 of the gap and Vmigration can be calculated. The

Livecyte system was utilised to accurately determine

the change in gap area over a 48-hour period (Fig.

1a). The addition of 50μM LPA caused the wound to

close at a faster rate (Fig. 1a, b) with a t1/2 gap of 21.4

hours and Vmigration of 5.3μM/hour when compared

to the control (41.9 hours and 2.5μM/hour, respectively.

Fig. 1 d, e.) However, the change in gap area can be

influenced by both cell migration and cell proliferation.

It is therefore important to account for any changes

induced as a result of cell proliferation. The quantitative

nature of images acquired allows for the determination

of optical volume of cells. Measuring total optical

volume of all cells over time, gives an indication of the

rate of cell growth and proliferation. In this experiment

cells treated with 50μM LPA increased the rate of cell

proliferation after 16 hours (Fig 1c). This indicates that

post 16 hours both cell migration and proliferation had

an influence on t1/2 gap and Vmigration. In addition to

modelling collective migration, the additional capabilities

of the Livecyte system allow for direct measurement

of single cells at the leading edge of the wound (Fig.

2a). Tracking such cells revealed an increase in mean

cell speed and instantaneous cell speed following

application of 50μM LPA. The LPA treatment also

induced a reduction in the meandering index, revealing

that the behaviour of migration was also influenced

(Fig. 2b). Further to motility parameters the virtue of

individual cell segmentation, allowed for quantification

of morphological parameters such as cell area,

thickness, sphericity and length/width ratio. Treatment

with 50μM LPA caused an increase in cell thickness

and sphericity, whilst reducing the length/width ratio.

Conclusion

In this application note we have demonstrated the advanced capabilities of the Phasefocus Livecyte system to image and analyse the wound healing assay. The non-invasive nature of the measurement allows for higher temporal intervals to be imaged and thus a more accurate measure of wound closure over time to be achieved. Further to information regarding collective migration, the high contrast, quantitative images produced, enable multi parametric analysis to be performed at a single cell level. This single-cell analysis directly measures cell migration, while the morphological information extracted, provides both a behavioural and morphological analysis of individual cells during the wound healing process.

Figure 2 - Advanced segmentation and tracking of single cells at the leading edge a) Representative images of cells at the leading edge of the wound segmented and tracked using the Phasefocus CAT software over 48 hours. b) Motility and c) Mean morphological metrics calculated from the tracked cells.

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Figure 2a).

Figure 2b). Figure 2c).

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T: +44 (0)114 286 6377

E: enquiries@phasefocus.com

Phase Focus Limited

Electric Works

Sheffield Digital Campus

Sheffield

S1 2BJ

UK

www.phasefocus.com

For more information on the benefits of the

Livecyte system, to access application notes

and for additional product information, please

visit: www.phasefocus.com/livecyte

A sample of time-lapse videos can be found at:

www.youtube.com/phasefocuslimited

© Phase Focus Limited. AN 001 | May 2016